Pharmacological targeting of glutamatergic neurons within the brainstem for weight reduction

Abstract

Food intake and body weight are tightly regulated by neurons within specific brain regions, including the brainstem, where acute activation of dorsal raphe nucleus (DRN) glutamatergic neurons expressing the glutamate transporter Vglut3 (DRN^Vglut3) drive a robust suppression of food intake and enhance locomotion. Activating Vglut3 neurons in DRN suppresses food intake and increases locomotion, suggesting that modulating the activity of these neurons might alter body weight. Here, we show that DRN^Vglut3 neurons project to the lateral hypothalamus (LHA), a canonical feeding center that also reduces food intake. Moreover, chronic DRN^Vglut3 activation reduces weight in both leptin-deficient (ob/ob) and leptin-resistant diet-induced obese (DIO) male mice. Molecular profiling revealed that the orexin 1 receptor (Hcrtr1) is highly enriched in DRN Vglut3 neurons, with limited expression elsewhere in the brain. Finally, an orally bioavailable, highly selective Hcrtr1 antagonist (CVN45502) significantly reduces feeding and body weight in DIO. Hcrtr1 is also co-expressed with Vglut3 in the human DRN, suggesting that there might be a similar effect in human. These results identify a potential therapy for obesity by targeting DRN^Vglut3 neurons while also establishing a general strategy for developing drugs for central nervous system disorders.

Fig. 1. DRN^Vglut3 neurons are integrated to the broader feeding circuitry through ascending projections.

a, Schematic of whole-brain projection mapping of DRN^Vglut3 neurons (IDISCO⁺/ClearMap). b, Allen Brain Atlas annotation and localization of the Vglut3 cell bodies expressing GFP in the DRN (n = 3 mice). c, Axonal projections from DRN^Vglut3 neurons into numerous hypothalamic loci, such as dorsomedial hypothalamus (DMH), lateral hypothalamic area (LHA), arcuate nucleus (ARC), ventromedial hypothalamus (VMH) and paraventricular hypothalamus (PVH), following the Allen Brain Atlas annotation. d,e, Quantitative projection mapping of hypothalamic projections using TRAILMAP modified code. f, Left, Schematic of axonal nerve endings in the LHA, created using a synaptophysin–GFP adeno-associated virus (AAV9-DIO-Synp-Venus-GFP). Right, representative IHC validation of DRN^Vglut3 projections to ARC and LHA. g, Left, Schematic of DRN^Vglut3 terminal stimulation in the LHA to assess energy balance. Right, optogenetic (AAV5-DIO-ChR2-eYFP or AAV5-DIO-eYFP (control)) terminal stimulation from DRN^Vglut3 to the LHA. Mice expressing ChR2 exhibited suppressed food intake when ChR2 was stimulated in the laser-on phase of a test in which mice were subjected to laser stimulation (n = 5–6 mice per group) (degrees of freedom (2), F statistics 20.31, P < 0.001) h,i, Activation of DRN^Vglut3 terminals in the LHA does not impair thermoregulation, assessed as core temperature (h) or BAT thermogenesis (i) (n = 5-6 mice per group). j, Left, optogenetic photoactivation of DRN^Vglut3 neuron terminals to the LHA enhances acute locomotion (2 degrees of freedom, F statistic 10.98) in an OFT. Middle, no differences are observed in the transitions or time spent in the center (measure of anxiety) of the OFT. Right, representative locomotor activity traces for optogenetic activation of DRN^Vglut3 terminals in the LHA. Traces were taken from a stimulation (laser-on) epoch. Data are represented as mean ± s.e.m. P values were calculated using a two-way analysis of variance (ANOVA) with a multiple-comparisons test (Tukey post-hoc). In a–e, the number of projections of each sample in the considered regions or annotated brain areas was analyzed using independent two-sample Student’s t-test, assuming unequal variances, using ClearMap/TrailMap. Multiple-comparison corrections were applied to P values to obtain q values (for false-discovery rate). In g–j, a two-way repeated-measures ANOVA was used, comparing control and treated groups (n = 5 mice per group). The blue region in g–j highlights the laser-on epoch. Scale bars, 200 µm. P < 0.05 is considered significant and are indicated above the bar graphs. Brain regions indicated in Fig. 1e are as follows: bla (basolateral amygdala), cea (central amygdala), pvh (paraventricular hypothalamus), arh (arcuate nucleus), dmh (dorsomedial hypothalamus), lha (lateral hypothalamic area), ndb (nucleus of the diagonal band), so (supraoptic nucleus) and ot (optic tract). Source data

Fig. 2. Chronic neuromodulation of DRN^Vglut3 neurons can reduce body weight in obese mice (DIO and ob/ob).

a, Schematic of the curative approach using DREADDs in Vglut-Cre ob/ob mice injected with the hM3(Gq) DREADD into the DRN (Vglut3-Cre^{ob/ob-hM3(Gq)} mice; n = 5). b, Weight curve after stimulating DRN^Vglut3 neurons in ob/ob mice (P = 0.003). c, Food intake after chronic stimulation of Vglut3-Cre^{ob/ob-hM3(Gq)} mice. d, Adaptive thermogenesis in DRN-Vglut3-Creo^{ob/ob-hM3(Gq)} mice. In a–c, littermate DRN-Vglut3-Cre^{ob/ob-mCherry} mice were used as controls. e–h, Indirect calorimetry in DRN-Vglut3-Creo^{ob/ob-hM3(Gq)} or DRN-Vglut3-Cre^{ob/ob-mCherry} mice using metabolic cages. The parameters measured in a day–night cycle were locomotor activity (e) (n = 7–8 mice per group), oxygen consumption (f) (n = 7–8 per group), CO₂ production (g) (n = 7–8 per group) and energy expenditure (h) (n = 7–8 per group). i, Schematic of the curative approach using DREADDs in DRN-Vglut3-Cre^HM3Dq mice with DIO (DRN-Vglut3-Cre^DIO-HM3Dq mice). The representative image shows the injection site. j, Body weight of mice with a DIO background after stimulation DRN^Vglut3 neurons (n = 6–7 per group, P < 0.0001). k, Food intake during the chronic stimulation of DRN^Vglut3 neurons, demonstrating hypophagia (n = 6–7 per group). l, Adaptive thermogenesis in DRN-Vglut3-Cre^DIO-HM3Dq mice. In i–l, littermate Vglut3-Cre⁺ DIO mice injected with AAV2/5-DIO-mCherry were used as controls. All mice received saline for 7 days, CNO for 14 days and saline for 7 days in a sequential manner. m–p, Indirect calorimetry assessment in DRN-Vglut3-Cre^DIO-HM3Dq and DRN-Vglut3-Cre^DIO-mcherry mice. The parameters measured in a day–night cycle were locomotor activity (m) (n = 4–5 per group), oxygen consumption (n) (n = 4–5 per group), CO₂ production (o) (n = 4–5 per group) and energy expenditure (p) (n = 4–5 per group). Data are represented as mean ± s.e.m. P values were calculated using a two-way ANOVA with a multiple-comparisons test using a Tukey post-hoc approach (b,j) or an unpaired two-tailed Student’s t-test (c,d,k,l), or using CalR software and a two-sided ANCOVA regression analysis taking body weight into account (e–h,m–p). P < 0.05 is considered significant. The CNO dose used was 1 mg/kg. Source data

Fig. 3. DRN^Vglut3 neuronal profiling together with GENSAT comparison reveals three unique targets for drug discovery.

a, Schematic illustrating the approach for AAV-mediated TRAP (vTRAP) studies using immunoprecipitation for ribosomal protein L-10 tagged with GFP (GFP-L10). b, Heat map of the three pairs of samples used for the identification of valuable druggable targets in DRN^Vglut3 neurons, comparing immunoprecipitated RNA samples (IP) to each sample’s input RNA. c, Volcano plot showcasing the genetic markers enriched in the three IP samples in comparison to all the previously immunoprecipitated samples found in the TRAP-based GENSAT database. Additionally, two Gene Ontology filters were applied to screen for plasma-membrane-bound signaling receptors, narrowing down the list of interesting targets. Red dots highlight the differentially enriched markers of interest for small-molecule-based drug discovery in DRN^Vglut3 cells (CalcR, Hcrtr1 and GPR4), after contrasting the data with the existing literature and in situ hybridization studies. d, Heatmap representing the expression levels of the three genes of interest (calcR, hcrtr1 and gar4) in comparison to each individual immunoprecipitated sample from the TRAP experiments available in the GENSAT database, to highlight their higher expression in DRN Vglut3 cells. The legend is color coded for each TRAP driver line, cell type and brain region. The first column represents the IP data from DRN-Vglut3 neurons. Data are represented as enrichment in IP over input. n = 3 for IP/input TRAP experiments. The number of cell lines in GENSAT database contrasted (n = 50). Red indicates genetic overexpression, and blue indicates downregulation. GOI gsva refers to gene of interest gene set variation analysis. A.U., arbitrary units.

Fig. 4. Local infusion (intra-DRN) of drug compounds to CalcR and Hcrtr1 ameliorates energy-balance defects after development of DIO.

a, Left, schematic of profiling-based drug targeting. Middle, local intra-DRN drug-infusion schematic. Right, cannula placement validation. b, Acute chow-diet feeding assessment after a single dose of s-CT (30 ng) into the DRN (n = 7 per group). c, Weight assessment after 3 days of s-CT (30 ng) infusion in chow diet (n = 7 per group). d, Adaptive thermogenesis after DRN s-CT (30 ng) injection (n = 7 per group). e, Acute chow-diet feeding assessment after a single dose of SB-334867 (250 ng) into the DRN (n = 6 per group). f, Weight assessment after 3 days of SB-334867 (250 ng) infusion in chow diet (n = 6 per group). g, Adaptive thermogenesis after DRN SB-334867 (250 ng) injection (n = 6 per group). In b–g, saline-injected controls were used. h, Left, schematic of profiling-based drug targeting in mice fed a HFD. Right, schematic of drug infusion into the DRN of mice implanted with a cannula after 16 weeks of HFD feeding. i, Body weight after agonism of CalcR or antagonism of Hcrtr1 with s-CT (30 ng) or SB-334867 (250 ng) after intra-DRN infusion. Baseline measurements and post-treatment measurements were taken in vehicle-infused mice (7 days) (n = 5 per group, 2 degrees of freedom, F statistic 91.02, P = 0.0001). j, Weight gain after CalcR agonism or Hcrtr1 antagonism with s-CT (30 ng) or SB-334867 (250 ng) in mice in the pre-treatment, treatment and post-treatment phases (n = 5 per group). k, Food intake during the treatment window (14 days) of s-CT (30 ng) or SB-334867 (250 ng) (n = 6 per group). l, Schematic of profiling-based drug infusion in the DRN of s-CT/SB-334867 with or without DREADD inhibition of DRN Vglut3 cells. m,n, Cumulative food intake (n = 7 per group) of vehicle and s-CT (30 ng)- and SB-334867 (250 ng)-treated mice with and without CNO (n = 6 per group). Data are represented as mean ± s.e.m. P values were calculated using a two-way ANOVA with a multiple-comparisons test (Tukey post-hoc) (i), one-way ANOVA (Bonferroni post-hoc multiple comparisons) (b,e,j–k,m,n) or a two-tailed unpaired t-test (c,d,f,g). P < 0.05 was considered significant. Source data

Fig. 5. Intracerebroventricular brain-wide infusion of drug compounds to CalcR and Hcrtr1 ameliorates energy-balance defects upon DIO.

a, Left, Schematic of profiling-based drug targeting. Middle, drug-infusion schematic for i.c.v. treatments. b, Dipsogenic angiotensin II infusion (10 ng) was used to verify cannula placement (n = 14). c, Acute chow-diet feeding assessment after a single i.c.v. dose of s-CT (300 ng) (n = 7). d, Adaptive thermogenesis after i.c.v. s-CT (300 ng) infusion (n = 7). e, Weight assessment after i.c.v. s-CT (300 ng) infusion in mice fed a chow diet (n = 7). f, Acute chow-diet feeding assessment after a single i.c.v. dose of SB-334867 (2.5 µg) (n = 7). g, Adaptive thermogenesis after i.c.v. SB-334867 (2.5 µg) infusion (n = 7). h, Weight assessment after i.c.v. SB-334867 (2.5 µg) infusion in mice fed a chow diet (n = 7). In c–h, saline-injected mice were used as controls. i, Drug-infusion schematic for i.c.v. treatments in mice after 16 weeks of HFD feeding. j, Dipsogenic angiotensin II infusion (10 ng) to control cannula placement for evaluating drinking response (n = 13). k, Twenty-four-hour food-intake assessment from a leptin-sensitivity assay (5 mg/kg) after 16 weeks of exposure to HFD (DIO) (n = 7). Chow-diet-fed mice were used as controls (n = 13). l, Weight after i.c.v. CalcR agonism or Hcrtr1 antagonism with s-CT (300 ng) or SB-334867 (2.5 µg), respectively. Pre-treatment baseline measurements and post-treatment measurements were taken in ACSF vehicle-infused mice for 7 days (2 degrees of freedom, F statistic 97.79, P = 0.0001) (n = 4). m, Weight gain after i.c.v. CalcR agonism or Hcrtr1 antagonism with s-CT (300 ng) or SB-334867 (2.5 µg), respectively (n = 4). n, Food-intake assessment during the i.c.v. treatment window (14 days) with s-CT (300 ng) or SB-334867 (2.5 µg) (n = 4). Data are represented as mean ± s.e.m. P values were calculated using a two-way ANOVA with a multiple-comparisons test (Tukey post-hoc) (l), one-way ANOVA with Bonferroni post-hoc multiple comparison analysis (m,n) or two-tailed unpaired t-test (b–h,j–k). P < 0.05 was considered significant. Source data

Fig. 6. Pursuing Hcrtr1 antagonism through a specific Hcrt1 antagonist, CVN45502, is a potential strategy for weight management.

a, Strategy and chemical structure, synthesized by Cerevance, from a highly selective orexin 1 receptor (Hcrtr1) antagonist CVN45502 using a combination of structure-activity relationships and a FLIPR Ca screen. b, Pharmacokinetic assessment of brain penetration for compound CVN45502 when given orally to mice, demonstrating a high brain penetrance in a 60-minute window (n = 3 mice). c, In vitro pharmacokinetic percentage of receptor inhibition for test compound CVN45502 when using in vitro assays expressing murine or human Hcrtr1 as well as human Hcrtr2. The pharmacokinetic panel shows binding to the murine and human Hcrtr1 receptor without binding to Hcrtr2 at therapeutic doses. Pharmacokinetics assay pinpoints to concentrations from 0.1 µM or greater to inhibit 100% of cells expressing Hcrt1, thus validating the dosing used of 30 mg/kg (n = 4 mice). Data are represented as mean ± s.e.m. P values were calculated using an unpaired t-test (b). P < 0.05 was considered significant. Figure 6a was performed with Biorender. Source data

Fig. 7. Chronic oral treatment with CVN45502 ameliorates energy-balance defects after DIO.

a, Schematic of oral delivery of CVN45502 (30 mg/kg) in peanut butter in chow-diet-fed or HFD-fed mice. b, Acute chow diet feeding after CVN45502 (30 mg/kg) treatment (n = 7). c, Adaptive thermogenesis after CVN45502 (30 mg/kg) treatment in mice fed a chow diet (n = 7). d, Acute HFD feeding assessment after CVN45502 treatment (30 mg/kg) (n = 7). e, Adaptive thermogenesis after oral delivery of CVN45502 (30 mg/kg) to mice fed a HFD (n = 7). f, Schema for oral delivery of CVN45502 (30 mg/kg) after induction of DIO (16 weeks in HFD). Chow-diet-fed mice were used as controls. g, Feeding assessment from a leptin-sensitivity assay (5 mg/kg) in DIO and chow-diet controls (n = 10). h, Sleep assessment after CVN45502 treatment (30 mg/kg) (n = 10). i, Weight measurement following CVN45502 treatment (30 mg/kg) in chow-diet-fed mice. Pre-treatment and post-treatment measurements were taken in vehicle-treated mice (n = 5). j, Weight gain after Hcrtr1 antagonism with CVN45502 (30 mg/kg) compared with controls in chow diet (n = 5). k, Food intake after CVN45502 (30 mg/kg) treatment in chow-diet-fed mice (n = 5). l, Plasma levels of CVN45502 12 hours after treatment with CVN45502 (30 mg/kg) (n = 5). m–p, Indirect calorimetry assessment in chow-diet-fed mice (n = 7) using metabolic cages. The parameters measured are oxygen consumption (m), CO₂ production (n), locomotor activity (o) and energy expenditure (p). q, Weight curve after Hcrtr1 antagonism using CVN45502 (30 mg/kg) in DIO mice (n = 5). r, Weight gain after Hcrtr1 antagonism using CVN45502 (30 mg/kg) compared with vehicle-treated controls in DIO mice (n = 5). s, Food intake after Hcrtr1 antagonism using CVN45502 (30 mg/kg) in DIO mice (n = 5). t, Plasma levels of CVN45502 12 hours after treatment with CVN45502 (30 mg/kg) (n = 5). u–x, Indirect calorimetry assessment in DIO mice using metabolic cages. Parameters measured are oxygen consumption (u) (n = 6), CO₂ production (v) (n = 6), locomotor activity (w) (n = 7) and energy expenditure (x) (n = 6). Data are represented as mean ± s.e.m. P values were calculated using a two-way ANOVA with a multiple-comparisons test (Tukey post hoc) (j,k,r,s), one-way ANOVA (b,d,g) or two-tailed unpaired t-test (c,e,h,l,t), or using CalR software and ANCOVA regression analysis taking body weight into account (i–l,q–t). P < 0.05 was considered significant. Source data

Fig. 8. In situ hybridization in human donor samples reveals colocalization of both Hcrtr1 and Vglut3 in the DRN.

Left, illustration representing human brain donors for ISH testing. Brain samples from three human donors were used. Middle, multiplex ISH studies using RNAscope of Hcrtr1 and Vglut3 (Slc17a8) in DRN sections of human donors, confirming co-labeling of Hcrtr1 and Vglut3 in the DRN and recapitulating our mouse studies in humans. Right, quantification of ISH RNAscope studies demonstrating a 48% overlap between Slc17A8 and Hcrtr1 (orange). 37% of the neurons counted were only Slc17A8-expressing (yellow), and 15% only expressed Hcrtr1 (red). Scale bar, 50 µm. Source data

Extended Data Fig. 1. Ascending projections from DRN^Vglut3 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^Vglut3 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^Vglut3 projections throughout the brain from multiple viewpoints. Scale bars 200um. (b) Schema of the most prominent site of projection from DRN^Vglut3 neurons to the LHA. See also Extended Data Video 1 for animated view of projection patterns from DRN^Vglut3 neurons and Supplementary Table 1 for quantification of the projection targets identified in the entire brain.

Extended Data Fig. 2. DRN^Vglut3 Neurons Anatomic Analysis Suggests the Blockade of Energy Homeostasis occurs through LHA^Vglut2 neurons.

. (a) Schema for DRN^Vglut3 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^Vglut3 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^Vglut2 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^Vglut2 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). Source data

Extended Data Fig. 3. Chemogenetic Stimulation of DRN^Vglut3Neurons fed HFD Exhibits a Significant Reduction of Energy Balance in a Preventive Setting.

. (a) Schematic for chronic stimulation of DRN^Vglut3 neurons in HFD fed mice injected with an AAV5-DIO-hm3Dq-mcherry in DRN^Vglut3 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^Vglut3 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^Vglut3 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) C ore 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. Source data

Extended Data Fig. 4. DRN^Vglut3 Neurons Drive Reduces Both Food Intake and Body Weight in DIO Female Mice.

(a) Schema illustrating chronic stimulation of DRN^Vglut3 neurons in DIO female mice by injecting an AAV5-DIO-hm3Dq-mcherry in DRN^Vglut3 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^Vglut3 neurons upon DIO in female mice (n = 6 per group). (c) Food intake during the chronic stimulation of DRN^Vglut3 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. Source data

Extended Data Fig. 5. Translational strategy comparing DRN^Vglut3 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^Vglut3 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 Hypothalmus and Lateral Hypothalamic Area. Scale bars 25um (d) 100um (e). (F-Left) Schema DRN infusion of drug ligands to CalcR and Hcrtr1 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. Source data

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. Source data

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. Source data

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. Source data

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. Source data

Extended Data Fig. 10. Hcrt1 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 Hcrt1 demonstrating antibody specificity to move forward with human anatomy studies with the selected antibody. The cells used in the bottom image are Hcrt1-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. Source data