Identification of a thermogenic target in the dorsal raphe nucleus for weight management in mice

Domain: biology
Published: 2026-01-13
Editorial outcome: minor_revision

Abstract
Obesity emerges from a complex interplay of factors, including imbalanced interoception, genetic predisposition, and environmental cues, which ultimately disrupt body weight homeostasis1. While much research has concentrated on strategies to suppress appetite for sustained weight loss, insufficient attention has been given to counter regulatory mechanisms that promote energy expenditure. Here, we show that chronic inhibition of GABAergic neurons in the dorsal raphe nucleus and ventrolateral periaqueductal gray (DRN/vlPAGVGAT) reduces body weight in diet-induced obese (DIO) male mice. In this study, molecular profiling and in-situ hybridization in rodent and human brains revealed that the constitutively activated orphan receptor GPR6 is selectively enriched in DRN/vlPAGVGAT neurons. We next developed and administered a potent and highly selective GPR6 inverse agonist, which significantly prevented weight gain in male mice exposed to a high-fat diet by stimulating brown adipose tissue thermogenesis without affecting appetite. Altogether, this study integrates transcriptomic profiling, high-throughput drug screening and metabolic phenotyping to successfully identify a candidate to treat obesity.

Introduction
Overweight and obesity are highly prevalent conditions, currently affecting an estimated 38% of the global population2. Particularly alarming is the surge in childhood obesity, which may have lasting effects on neurodevelopment3,4. The intricate brain circuits orchestrating feeding behaviors and energy expenditure are particularly affected in this context5. Readily available calorie-dense food exacerbates the imbalance between energy supply and demand, imprinting neural circuits and resetting homeostatic baselines6,7. While recent pharmacological interventions to suppress appetite have been clinically promising, they often have undesirable side effects and tend to plateau in efficacy8–13.

Consumption of a high-fat diet (HFD) rewires homeostatic and hedonic neural systems involved in feeding and body weight regulation. For instance, agouti-related peptide (AgRP)-expressing neurons in the hypothalamus are pivotal in regulating feeding behavior and are profoundly affected by HFD intake14. Diet-induced obesity (DIO) blunts the inhibition of AgRP neurons by exteroceptive cues, such as food presentation, and interoceptive signals, such as intragastric fat infusion15.

Here, we describe that both short- and long-term exposure to a HFD diet activate GABAergic neurons located in the dorsal raphe nucleus and nearby ventrolateral periaqueductal gray (DRN/vlPAGVGAT). Chemogenetic inhibition of these neurons prevents excess weight gain in mice fed an HFD. Molecular profiling of DRN/vlPAGVGAT neurons identified GPR6, a constitutively active GPCR highly enriched in this population, as a potential druggable target. Pharmacological inhibition of GPR6 attenuated DRN/vlPAGVGAT neuronal activity and decreased body weight by enhancing brown fat thermogenesis. Collectively, this work presents a framework for identifying neural targets that regulate energy expenditure and may be modulated to treat obesity.

Results
Inhibition of chronically active DRN/vlPAG neurons ameliorates obesity in mice fed an HFD
Building on studies demonstrating that neural systems involved in feeding are remodeled by HFD consumption, we hypothesized that a relatively short exposure to HFD could alter the activity patterns of distinct brain regions before the onset of obesity in mice. We employed an unbiased screen of whole-brain Fos expression using iDISCO+ and ClearMap16. Among the areas significantly more active in mice fed a HFD for 7 days compared to chow-fed mice, two clusters of regions drew our attention: (1) hypothalamic nuclei, such as the paraventricular hypothalamus and lateral hypothalamus and (2) brainstem nuclei, predominantly the DRN/vlPAG (Fig. 1A and Supplementary Fig. 1A). Given our prior work in the DRN with regards to energy balance regulation17–19 and recent findings indicating vlPAG’s role on food consumption20, we next tested whether DRN/vlPAGVGAT neurons are persistently activated in DIO mice, which, if true, would support our hypothesis that inhibiting them would reduce body weight in obese mice. Toward this end, we examined cFOS expression in the DRN/vlPAG of mice fed an HFD for 3 days (acute exposure), 2 weeks (prolonged exposure), and 16 weeks (chronic exposure) and found a significant increase in cFOS-positive neurons in the DRN/vlPAG at all time points, indicating heightened activity induced by HFD consumption (Fig. 1B). Importantly, the responsiveness of DRN/vlPAGVGAT neurons to HFD is independent of elevated leptin levels, as these neurons lack leptin receptors. In DRN/vlPAG brain sections from mice fed a HFD, we found that ~75% of VGAT neurons overlap with cFOS, compared to 60% in mice fed a regular chow diet (Supplementary Fig. 1B). To identify upstream brain regions tuned to metabolic states and limbic behaviors that are connected to DRN/vlPAGVGAT neurons and may drive their persistent activation, we performed rabies-based monosynaptic tracing (Supplementary Fig. 2A). We found that DRN/vlPAGVGAT neurons receive direct input from many brain loci known to regulate feeding, including the ventral tegmental area (VTA), lateral hypothalamus (LH) and bed nucleus of the stria terminalis (BNST) (Supplementary Fig. 2B, C). These observations together suggest that DRN/vlPAGVGAT neurons constitute a responsive hub to caloric surplus.

Since activating these neurons increases food intake and reduces energy expenditure—physiological changes that could lead to increased body weight if sustained over the long term—we reasoned that blunting the overactivation of DRN/vlPAGVGAT neurons would prevent mice from gaining excess body weight when fed a HFD. To test this, we first used chemogenetics and injected an AAV5-hSyn-DIO-hM4D(Gi) virus into the DRN/vlPAG of vGat-Cre mice, and treated both experimental and control mice (AAV5-hSyn-DIO-mCherry) with clozapine-N-oxide (CNO) at the same time as they were switched from chow to HFD. We found that chemogenetic inhibition of DRN/vlPAGVGAT neurons effectively suppressed body weight gain compared to control littermates (Fig. 1C). Similarly, treatment with CNO also significantly reduced body weight and adiposity in obese mice fed an HFD for 16 weeks (Fig. 1C—right panel, D). This inhibition also led to a significant decrease in food intake (Fig. 1E) and an increase in energy expenditure, evidenced by elevated BAT thermogenesis (Fig. 1F), core body temperature (Fig. 1G), indirect calorimetry assessment (Fig. 1H, I, K and Supplementary Fig. 1C, D) and locomotor activity (Fig. 1J). Notably, after the removal of CNO treatment, we observed that mice started to regain body weight when injected with vehicle, suggesting that chronic inhibition of DRN/vlPAGVGAT neurons is necessary to maintain weight loss (Fig. 1C—right panel). A recent study has shown that DRNVGAT neurons are activated during feeding-related behaviors, such as food contact, and their responsiveness to food cues is increased by hunger and palatability21. Behavioral screening experiments also demonstrated that DRNVGAT neurons’ activation increased contact duration with an inedible object, reduced walking duration, and decreased chamber exploration, suggesting a possible role for DRNVGAT neurons in maintaining food consumption21.

The heightened activation of DRNVGAT neurons observed during the consumption of highly palatable food and the significant real-time place preference for the light-paired chamber elicited by optogenetic activation of these neurons22 also raise intriguing possibilities about their potential involvement in reward-promoting behaviors. However, the specific reward circuitry by which DRNVGAT neurons operate remains to be determined.

Transcriptomics analyses of DRN/vlPAGVGAT neurons reveal enrichment of GPR6
Our results show that chemogenetic inhibition of DRN/vlPAGVGAT neurons reduces body weight of DIO mice suggest that small molecules inhibiting their activity would also reduce weight. We thus set out to identify druggable targets enriched in DRN/vlPAGVGAT neurons that could be pharmacologically targeted to inhibit these neurons. We profiled these neurons by introducing a virus expressing a Cre-dependent GFPL10a fusion protein into the DRN/vlPAGVGAT of vGat-Cre mice. We then used translating ribosomal affinity purification (TRAP) to molecularly profile DRN/vlPAGVGAT neurons (Fig. 2A), followed by differential expression analysis of genes enriched in the immunoprecipitated (IP) RNA over RNA input (Fig. 2B). These data identified 24,100 genes, of which 2257 genes were significantly enriched (1.5-fold-enrichment with an adjusted P value (p < 0.05) (Supplementary Table 1). The molecular phenotyping of DRN/vlPAGVGAT neurons was subsequently compared with neuronal transcriptomes available in the GENSAT database23. About 8519 genes were enriched in DRN/vlPAGVGAT compared with other neural populations cataloged in the GENSAT database (Supplementary Table 2). To identify druggable targets, we used the Gene Ontology browser24 database to identify genes enriched in DRN/vlPAGVGAT that were also listed in both the signaling receptor activity (GO:0038023) and plasma membrane (GO:0005886) categories (MGI, informatics.jax.org). After applying these filters, 259 druggable receptors were identified with more than 1.5-fold enrichment in DRNVGAT neurons compared to the GENSAT database (Supplementary Table 3 and Supplementary Fig. 3). Next, we evaluated the expression patterns of the enriched genes using the Allen Brain Atlas database25 to identify the most differentially expressed receptors in the DRN/vlPAGVGAT neurons compared with other cell types. Three orphan G-protein-coupled receptors (gpr3, gpr6, and gpr39) showed significantly higher expression in the DRN/vlPAG (Fig. 2C). Consistent with this, aggregate differential expression analysis and the relative expression of these selected receptors, compared with molecular profiles of other cell types in the GENSAT database, reconfirmed the cell-specific enrichment of these genes in DRN/vlPAGVGAT neurons (Fig. 2D). (TRAP IP vs input: gpr3: 2.2-fold enrichment, gpr6: 5.7-fold enrichment and gpr39: 2.5-fold enrichment; relative expression to GENSAT: gpr3: 2.6-fold enrichment, gpr6: 21-fold enrichment and gpr39: 200-fold enrichment). Of further interest, sphingosine 1-phosphate is a potential ligand for GPR3 and GPR6 and has a cannabinoid-like structure26. Moreover, its plasma levels are elevated in obesity27. GPR39, in turn, is a Zinc signaling receptor28. We decided to focus on GPR6 because it is almost exclusively expressed in the central nervous system and is more highly enriched in DRN/vlPAG than GPR3 and GPR39.

We next confirmed that DRN/vlPAG neurons expressing GPR6 also express vGat using fluorescent in situ hybridization (RNAscope) and found colocalization of GPR6 with vGat (slc32a1) in ~50% of slc32a1-positive neurons (Fig. 2E). Since our goal was to develop druggable strategies for weight loss in humans, we also obtained human brain sections from donors and found GPR6 expression in the human DRN (Fig. 2F). Quantitative assessment was performed using the Paxinos Atlas of the human brainstem following the ventral extension of the midline, revealing a 33% overlap between SLC32A1 and GPR6 (Fig. 2G). Together, these results demonstrate co-expression of these genes in human DRNVGAT neurons, recapitulating our in situ experiments in mice. GPR6 is a Gs-coupled receptor, suggesting that blocking it could inhibit DRN/vlPAGVGAT neurons and potentially provide a therapeutic approach for treating obesity.

Development and validation of CVN527 as an inverse agonist of GPR6
While some data suggest that S1P may act as a ligand for GPR6, this has not been confirmed. As an alternative, an inverse agonist screen was used to identify CVN527, a potent and selective GPR6 inverse agonist developed by structure-activity analysis following the identification of a lead compound in a high-throughput screen29 (Fig. 3A).

To determine the binding affinity of CVN527 against native GPR6 receptors, mouse coronal striatal sections were isolated and incubated with 2.5 nM [3H]-RL338 along with CVN527 over a concentration range (10−11–10−4 M). CVN527 displaced [H3]-RL338 with a Ki of 4.31 nM, showing potent binding to the native mouse receptor. To determine the potency of CVN527 to inhibit mouse GPR6-mediated cAMP signaling, a functional GPR6 cAMP HTRF cell-based assay was developed using CHO-K1 cells. Under these conditions, CVN527 fully inhibited GPR6 with an EC50 of 39.5 ± 17.4 nM, similar to the previously reported EC50 of 34 nM at human GPR629 (Fig. 3B). These data show that CVN527 has similar activity at human and mouse GPR6.

To correlate ex vivo receptor occupancy and exposure in vivo, wild-type mice received a single oral dose of vehicle or CVN527 (1, 3, 10, or 30 mg/kg). Plasma and striatal tissue samples were processed 1 h after each dose. Receptor occupancy of CVN527 at GPR6 increased in a dose-dependent manner, reaching 46% at 10 mg/kg and 66% at 30 mg/kg. Plasma and brain tissue concentrations of CVN527 were quantified by LC-MS/MS bioanalysis and correlated to receptor occupancy. This revealed that the plasma or total brain concentration associated with 50% receptor occupancy were 7.2 ng/ml of CVN527 and 16.6 ng/mg, respectively (Fig. 3C). The bioanalysis also showed a dose-dependent increase in exposure with similar brain-to-plasma ratios across doses ranging from 2.0 at 1 mg/kg to 2.6 at 30 mg/kg (Fig. 3D). These data show that CVN527 is highly brain penetrant in mice.

We next performed slice electrophysiology to test whether CVN527 directly affects the cell excitability of DRNVGAT neurons. We reasoned that demonstrating a direct inhibitory effect of CVN527 on DRNVGAT neurons in lean mice would provide a clear mechanistic basis to further evaluate this drug in vivo in DIO mice. vGat-IRES-Cre driver mice were crossed to Cre-dependent EGFP-L10a reporter mice to selectively label vGat-expressing cells for recordings (Fig. 3E). As almost no DRN cells were spontaneously active30, we measured changes in intrinsic action potential firing properties in response to current step injections before and after CVN527 (100 nM) application. Neurons were classified based on their baseline firing frequency at any current injection step as either low-firing (<50 Hz) or high-firing (>50 Hz), with the majority of recorded neurons categorized as low-firing. Bath application of CVN527 reduced the firing of low-firing DRNVGAT neurons (n = 11; Fig. 3F, G), and these effects were partially reversed during the washout phase. Interestingly, we found that a small proportion of vGat neurons (n = 4) with a high-firing baseline rate showed no response to CVN527 and exhibited a trend towards increased firing frequency (Supplementary Fig. 4). We speculate that this subpopulation of vGat neurons may have been disinhibited by low-firing vGat neurons that were directly suppressed by CVN527. Altogether, the reduced excitability in the low-firing group of neurons after CVN527 application is consistent with the pharmacologic finding that CVN527 is an inverse agonist for GPR6. Together, the pharmacologic and electrophysiologic studies demonstrate that CVN527 is a brain penetrant, highly selective, and potent inverse agonist of GPR6.

Intra-DRN and oral CVN527 administration reduce body weight by enhancing thermogenesis
We next tested whether CVN527 treatment could replicate the effect of chemogenetic inhibition of DRN/vlPAGVGAT neurons using two different approaches: (1) a direct pump infusion of 300 ng (0.1 µl/min) of CVN527 in the DRN through an intraparenchymal brain cannula and (2) by feeding mice 10 mg/kg of CVN527 mixed in a 0.2 g peanut butter pellet. In both cases, the treatment started when mice on a chow diet were switched to an HFD (Figs. 4A, I). The intra-DRN infusion of CVN527 for 14 days reduced body weight when lean mice transitioned from a chow diet to an HFD (Fig. 4B). This effect was significantly diminished when the drug infusion was changed to vehicle (washout period).

Proper maintenance of energy balance depends on the brain’s ability to integrate circulating hormones and fuels into major neural networks that regulate feeding and thermogenesis1,31. Since DRN neurons have been linked to both feeding behavior and thermoregulation17,18, we next assessed whether the reduction in DRNVGAT neuron activity induced by intra-DRN infusion of CVN527 reduced food intake, induced thermogenesis, or both. Surprisingly, no effects on food intake were observed (Fig. 4C). However, when metabolic phenotyping was performed, we found increased oxygen consumption (VO2) (Fig. 4D) and energy expenditure (Fig. 4G), with no effect on carbon dioxide production (VCO2) (Fig. 4E) or locomotor activity (Fig. 4F). To further assess if BAT might have contributed to the overall increase in metabolic rate, we implanted wireless temperature transponders (IPTT-300) into the BAT and found acute increases in the BAT temperature 20 min after intra-DRN infusion of CVN527 (Fig. 4H). We next repeated these studies in animals receiving CVN527 orally simultaneously as animals were switched from chow to HFD (Fig. 4I). After 14 days of treatment, we observed a significant reduction in body weight, with no change in food consumption, compared with mice given peanut butter alone (Fig. 4J, K). Consistent with the effects we observed in the intra-DRN trial, we also observed increased oxygen consumption (VO2) (Fig. 4L) and energy expenditure (Fig. 4O) in mice given CVN527. No changes in carbon dioxide production (VCO2) (Fig. 4M), locomotor activity (Fig. 4N) and RER (Supplementary Fig. 5) were observed. To further confirm that the effect on the prevention of weight gain was associated with the activation of BAT, we also assessed the acute effects of oral consumption of CVN527 on BAT temperature and, as with the DRN infusions, we observed a significant and persistent temperature increase after 20 min (Fig. 4P). Possibly consistent with this, patients from clinical trials with lead compound CVN424 (a different GPR6 inverse agonist) for Parkinson’s disease reported a heat sensation, and bodily temperature increases between 0.5 and 1 °C were observed32. Importantly, metabolic phenotyping experiments were conducted in male mice to minimize variability; however, given the potential sex differences in energy homeostasis and thermogenesis, future studies for the translational potential of GPR6 will be needed to determine whether similar effects are observed in females.

The absence of GPR6 expression in BAT and the persistent thermogenic effect prompted us to further study metabolism and transcriptional changes in this tissue. Using the Clark electrode, we observed increased O2 consumption in BAT 60 minutes after oral CVN527 (Fig. 4Q). Next, we sought to measure the expression level of canonical genes involved in the thermogenic response 1 and 3 days after CVN527 consumption. While we did not observe any significant difference in a panel of thermogenic genes 1 day after CVN527 consumption (Fig. 4R), we observed significantly increased expression of Ucp1, Elovl3, and Dio2 in BAT of mice given oral CVN527 for 3 consecutive days (Fig. 4S). A putative explanation for the differences we found in this time-course experiment is that the BAT gene expression is highly variable depending on the pro-thermogenic stimuli33. For example, mice treated with CL316,243 (a selective Adrb3 agonist) exhibit increased expression of Ucp1 and Pgc1a in the inguinal adipose depot as early as one day34. Conversely, mice exposed to cold (6.5 °C) exhibit more pronounced expression of thermogenic genes after 3 days34. Importantly, our findings suggest that GPR6-positive DRN/vlPAGVGAT neurons primarily regulate energy expenditure, and that broad inhibition of these neurons also reduces food intake. Furthermore, the pro-thermogenic effect of CVN527 may also be linked to GPR6 activation in non-VGAT neurons. Future studies could explore intersectional or dual-inhibitor approaches to target a broader population of DRN/vlPAGVGAT neurons, aiming to activate BAT thermogenesis and suppress food consumption.

We previously showed that activating GABAergic input from DRN onto the raphe pallidus (RPa) decreases BAT temperature18. Based on this, one possibility is that the increased thermogenic response we observed after CVN527 administration was driven, at least in part, by a similar mechanism involving stimulation of BAT sympathetic preganglionic neurons, resulting in increased BAT sympathetic nerve activity. Within the DRN, DRNVGAT neurons locally inhibit DRN serotonergic neurons35. Thus, an alternative neural mechanism to explain the increase in thermogenic response is that the disinhibition of DRN serotonergic neurons increases BAT temperature and energy expenditure upon activation36. Further studies will be necessary to distinguish between these and other possibilities.

Finally, to confirm the safety of CVN527, we conducted an array of behavioral tests to identify potential side effects. We first evaluated anxiety responses, which often align with overt side effects. The behavior and performance of mice treated with CVN527 were evaluated in an open-field test, an elevated plus maze, and a marble burying test. Mice receiving CVN527 showed no increase in anxiety in any of these tests (Supplementary Fig. 6A–D). Consistent with our chemogenetic studies, oral administration of CVN527 was associated with increased locomotor activity, as indicated by greater distance traveled and velocity.

Overall, our findings indicate that oral CVN527 dosing significantly enhances thermogenic pathways in BAT, which appears to account for the decrease in weight gain of mice transitioning to HFD. This significant reduction in weight gain underscore the potential therapeutic value of CVN527 in the treatment of obesity.

Methods
Mice
All animal experiments were approved by the Ethical Review Committee at the local institutes and in accordance with the National guidelines on the use of animals in research. For experiments conducted at Rockefeller University, mice were group housed in a 12–12 h dark/light cycle at 22 °C with ad libitum access to a regular chow diet (PicoLab Rodent Diet 205053) and water. Diet-induced obese (DIO) wild-type, vGat-IRES-Cre male mice (JAX#016962) and vGat-IRES-Cre mated to EGFP-L10a (JAX#024750) were fed on 45% high-fat diet (HFD, Research Diets, Cat# D12451). Experiments for metabolic phenotyping were performed in adult male mice. For all Cre-mouse experiments, only heterozygous animals were used, and littermates of the same sex were randomly assigned to either experimental or control groups. For cFos stainings and iDISCO+/ClearMap analysis, all mice were fasted for 2 h prior to perfusion and sacrificed at the same circadian time (ZT2-ZT4). Environmental variables such as stress, noise and odor were controlled to improve reliability.

Viral vectors
All viral vectors used in these studies have been extensively used in neuroscience. For chemogenetic studies, AAV5-EF1a-DIO-hM4Di-mCherry (Addgene, 44632) or AAV5-EF1a-DIO-mCherry (Addgene, generated from plasmid 50462) were used. For viral-mediated translating ribosomal affinity purification (TRAP) to molecularly profile DRN/vlPAGVGAT neurons, AAV5-IV-GFPL10 (Addgene 98704, UNC Vector Core) was used.

Monosynaptic rabies tracing
Mice were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) in oxygen and placed in a stereotax apparatus (Kopf Instruments). For monosynaptic retrograde tracing, we injected 40 nL (20 nl/min) of helper AAV2-hSyn-FLEX-TVA-P2P-GFP-oG in the DRN of vGat-IRES-Cre male mice. After 21 days, we injected 200 nL (50 nl/min) of G-deleted rabies virus (EnvA-dG-RV-mCherry) in the same location. For both injections, we used a glass capillary attached to a Drummond Scientific Nanoject III. Mice were anesthetized under 5% isofluorane and perfused 7 days after the injection of the rabies virus. To count the number of input cells, 50 μm coronal sections across the whole brain were mounted and imaged with a SlideView microscope (VS200, Olympus). Input cells were manually counted using a Fiji plugin, and the percentage of input cells expressing mCherry was calculated from each of the brain regions. The following abbreviations are used in Supplementary Fig. 2: LC locus coeruleus, VTA ventral tegmental area, LH lateral hypothalamus, CeA central amygdala, SNc substantia nigra pars compacta, BNST bed nucleus of the stria terminalis, MPA medial preoptic area, LHb lateral habenula, PSTh posterior subthalamic nucleus.

Stereotaxic surgery
Mice were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) in oxygen and placed in a stereotax apparatus (Kopf Instruments). Viruses were delivered into the DRN/vlPAG through a glass capillary using a Drummond Scientific Nanoject III. The following coordinates relative to the lambda were used: −2.8 mm DV, 0 mm ML, 0 mm AP. For pharmacology studies, wild-type mice had a cannula (Plastics One) placed in the DRN (coordinates, relative to lambda: +0.8 mm ML, 0 mm AP, −3.0 mm DV: 15°).

Chemogenetic studies
Mice were injected with DREADD (hM4D(Gi)) or control virus (mCherry) in the DRN/vlPAG, followed by a recovery period of at least 3 weeks. Mice were habituated with sham injections at least 5 days prior to the assay. Mice were fed a high-fat diet for 16 weeks before starting the clozapine-N-oxide (CNO)-mediated activation of DRN/vlPAGVGAT neurons (curative) or given a high-fat diet at the same time as the CNO-mediated activation of DRN/vlPAGVGAT neurons (preventive). In the curative approach, food intake and body weight were measured daily after CNO injection intraperitoneally at a dose of 1 mg/kg/day. In the preventive approach, body weight and food intake was measured on a daily basis for 11 weeks and mice received 1 mg/kg/day of CNO twice a day (7:00 a.m. and 7 p.m.). Adiposity measurements were performed using a magnetic resonance equipment (EchoMRI) system at week 1 and week 11 in the preventive setting and at day 7 and 21 in the curative setting. Thermogenesis assays were performed in the home cage during the animal’s light phase at week 1 and week 11 in the preventive setting and at day 7 and 21 in the curative setting. Mice were given ad libitum access to a chow diet or a high-fat diet during the entire experiment. Control studies were performed by injecting a vehicle (saline) instead of CNO. All CNO injections were at a concentration of 1 mg/kg.

Histology
Mice were anesthetized under 5% isofluorane and transcardially perfused with PBS (Fisher, Cat# BP39920), followed by 4% PFA (EMS Cat# 15714-S). Brains were dissected and post-fixed in 4% PFA at 4 °C for 12 h. Brains were sectioned into 50-μm coronal slices using a Cryostat (Leica). For the immunohistochemistry reactions, brains were blocked in blocking buffer (0.2% Triton X-100, Thermo, Cat# 85111 in PBS, 3% bovine serum albumin, Sigma, Cat# A9647-500G, 2% normal donkey serum, Jackson ImmunoResearch, Cat# 017-000-121) for 1–2 h. For cFOS staining, sections were incubated with primary antibody (1:1000 rabbit anti-cFOS, Synaptic Systems 226008) overnight at 4 °C. Sections were then washed three times in PBS and incubated with secondary antibody (1:500 donkey anti-rabbit IgG Alexa 546, Thermo Fisher A10040) for 2 h at room temperature, washed three times in PBS and mounted with DAPI Fluoromount-G (Southern Biotech Cat# 0100-20). Images were taken using an LSM780 confocal microscope (Zeiss), and ImageJ software (NIH) was used for minimal processing, including brightness and contrast adjustments. cFOS counts were conducted manually using ImageJ in 4–5 DRN sections of five animals from anterior posterior regions spanning −4.1 to −4.8 mm relative to bregma.

Human tissue analysis
The expression of GPR6 mRNA in raphe nuclei from human subjects was evaluated in frozen sections of midbrain from two non-diseased male donors. Fully consented, postmortem human tissues were obtained from Tissues for Research (UK). Duplex RNAscope assays (Advanced Cell Diagnostics, Biotechne) were completed using probes to SLC32A1 (c1) and GPR6 (c2). On completion of the assays, sections were scanned in bright-field at 40x (Hamamatsu Nanozoomer) and analyzed for the presence and distribution of single or double-labeled raphe neurons.

Intra-DRN administration of compound CVN527
Drug infusions were achieved employing an infusion pump (Harvard Apparatus) attached to glass syringes (Hamilton), with an infusion rate of 0.1 µl/min. Total drug injection never exceeded 0.5 µl in local DRN studies for a total of 300 ng. Mice were habituated for 1 week to be plugged into the infusion system and minimize stress. After habituation, mice were measured (body weight and food intake) during 7 days in a vehicle phase. Next, mice were divided into age-matched and weight-matched groups. The first group of mice was treated with CVN527, and the second group of mice received only the vehicle without the CVN527 compound. After the 14 treatment days, mice returned for 7 days on vehicle only to assess whether there were irreversible consequences in weight due to the chronic treatment. The correct cannula placement was confirmed through post hoc analysis of brain slices containing the DRN region, and data from mice with misplaced or mistargeted cannulae were excluded.

Oral administration of compound CVN527
Cerevance Therapeutics proprietary preclinical specific oral GPR6 inverse agonist CVN527 has been administered at a dose of 10 mg/kg daily mixed in a pellet of 0.1 g of peanut butter in mice fed for 16 weeks in HFD until they reach DIO. Mice were habituated 1 week prior to the study to receive one pellet of peanut butter. All mice successfully ate the pellet on the first day of habituation in less than 2 min. After habituation, mice were measured (body weight and food intake) during 7 days in a vehicle phase while only receiving peanut butter. Next, mice were divided into age-matched and weight-matched groups. The first group of mice was treated with CVN527 and the second group of mice received only the peanut butter pellet without the CVN527 compound. Treatment lasted 14 days to mimic DRN treatment. A lean control group was also evaluated in parallel following the same paradigm. After the 14 treatment days, mice returned for 7 days on peanut butter only to assess whether there were irreversible consequences in weight due to the chronic treatment.

iDISCO+ and ClearMap analysis
Whole-brain staining was performed following the iDISCO+ protocol previously described16. Briefly, perfused brains were dehydrated in methanol 20, 40, 60, 80 and 100% (Sigma-Aldrich) and incubated overnight in a 66% dichloromethane solution (Sigma-Aldrich) in methanol. Samples were bleached overnight at 4 °C in methanol containing 5% hydrogen peroxide (Sigma-Aldrich) and then rehydrated after incubation in methanol 60, 40, and 20%. After permeabilization, samples were incubated with rabbit cFOS antibody, 1:4000 (Synaptic Systems 226 008) at 37 °C for 10 days. Secondary antibodies conjugated to Alexa 546 were used (Life Technologies). After a final incubation in methanol for dehydration, brain samples were washed in dichloromethane 100% for clearing and stored in dibenzyl ether (Sigma-Aldrich). The imaging acquisition was performed in a light sheet microscope (LaVision Ultramicroscope II) equipped with a sCMOS camera and LVMI-Fluor x4 objective lens equipped with a 6 mm working distance dipping cap. Images were taken every 6 μm (z steps), and the numerical aperture was set to 0.03 NA.

TRAP
Mice were sacrificed by cervical dislocation and a ventral piece including the DRN was rapidly dissected in ice-cold buffer containing 10 mM HEPES [pH 7.4], 150 mM KCl, 5 mM MgCl with 0.5 mM DTT (Sigma), 80 U/ml RNasin Plus (Promega), 40 U/ml SUPERase In RNase Inhibitor (Life Technologies), 100 mg/ml cycloheximide, protease inhibitor cocktail (Roche) and then cleared by centrifugation to isolate polysome-containing cytoplasmic supernatant. Polysomes were immunoprecipitated using monoclonal anti-EGFP antibodies (clones 19C8 and 19F7 bound to biotinylated-protein L-coated streptavidin-conjugated magnetic beads (Pierce, Thermo Fisher; Life Technologies). A small amount of RNA was taken prior to immunoprecipitation (Input) and both the Input and immunoprecipitated (IP) RNA were purified using the Absolutely RNA Nanoprep Kit (Agilent). For RNA-seq, cDNA libraries were prepared with the SMARTer Ultra low Input RNA for Illumina Sequencing Kit (Clontech) and sequenced on an Illumina HiSeq 2500 platform.

RNA-seq analysis
Sequence and transcript coordinates for the mouse mm10 UCSC genome and gene models were retrieved from the Bioconductor Bsgenome.Mmusculus.UCSC.mm10 (version 1.4.0) and TxDb.Mmusculus.UCSC.mm10.Known Gene (version 3.4.0) Bioconductor libraries, respectively. Details on the FASTQ data can be found in “Data availability”. Transcript expressions were calculated using the Salmon quantification software37 (version 0.8.2), and gene expression levels as TPMs and counts were retrieved using Tximport (version 1.8.0). Normalization and rlog transformation of raw read counts in genes were performed using DESeq238 (version 1.20.0). For visualization in genome browsers, RNA-seq reads are aligned to the genome using Rsubread’s subjunct method (version 1.30.6) and exported as bigWigs normalized to reads per million using the rtracklayer package39 (version 1.40.6). GSVA analysis of selected genes was performed using the GSVA (version 1.34.0) R package. Visualization of genes and gene-sets as heat maps was performed using the Pheatmap R package (version 1.0.10). The GENSAT samples was also visualized using the Complex Heatmap Bioconductor R package40.

Homecage indirect calorimetry assessment
Energy expenditure was assessed through measurement by indirect calorimetry of oxygen consumption (VO2), carbon dioxide production (VCO2), heat production, 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-h light/12-h 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. 1), intra-DRN infusion of CVN527 and peanut butter (0.1 g) with (6 days) or without CVN527 (4 days) (Fig. 4). Data were collected and analyzed as recommended by the manufacturers. Averaged measurements of control periods or treatment periods (CNO/CVN527) are represented. Locomotor activity was recorded as beam breaks converted into distance/velocity, measuring activity in 3D. 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 were conducted using CalR software41 and ANCOVA regressions.

RNA extraction and BAT gene expression analysis
Intrascapular BAT was extracted 1-h post CVN527 treatment and snap frozen. Brown fat pads were pulverized in liquid nitrogen, and tissue powder was collected for RNA. Afterwards, the RNA was obtained using a combined TRizol (Life Technologies, Cat. 15596018) and RNeasy spin column (Qiagen, Cat. 74104) protocol. A total of 2 ug RNA was used for cDNA synthesis (Thermo Scientific, Cat. 4368813) per sample. Expression of Ucp1, Elovl3, Cidea, Cox8b, Ppargc1a, Esr1, Dio2, Cpt1b, Cox4l1, Fabp4 and Prdm16-ex9 (exon 9) was assessed with Sybr Green (Applied Biosystems, Cat. 4367659) in a 384-well plate. Cyclophylin was used as a reference gene for delta analysis. Forward and reverse primer sequences are listed below (Table 1).

Brown adipose respiration in the Clark electrode
Oxygen consumption was assessed using a Clark electrode (Si Strathkelvin Instruments, Mitocell MT200) in freshly dissected intrascapular BAT from mice 1-h-post CVN527 oral treatment. Polypropylene electrodes were first washed with acetone, then electrode jackets were filled with KCl and bubbles were removed to ensure the electrode’s membrane was watertight. Electrodes were connected to respirometry chamber and left in H2O overnight to stabilize to 0 pA. Oxygen solubility at 37 °C for salinity of 0 and a pressure of 760 mmHg was set to 6.73 mg/L. Electrodes were then calibrated using prewarmed (37 °C) O2-low (0.25% Na2SO3; pA 0–8) and O2-high (oxygen-saturated H2O; pA 600). One sample was tested at a time in 15-min intervals. BAT was dissected, placed in saline on ice and immediately divided into three pieces (50–100 mg each). Each tissue piece was weighed using analytical scale, minced with spring scissors (40 snips) and buffered in 0.5 mL of respiration buffer (DPBS, 20% BSA, 4.5% glucose, 1 mM Na pyruvate). Resuspended tissue pieces were immediately placed in the respiration chamber with a stirring bar, and O2 consumption rates were recorded by the electrode for 2 min each. The respiration chamber was rinsed with water and fresh respiration buffer between samples. Consumption rates were normalized to tissue weights and auto-calculated by the Si Strathkelvin software. iBAT temperature was measured using wireless implantable temperature probes IPTT-300 (BioMedic Data Systems), and core body temperature was assessed using an anal probe (Braintree Scientific).

Patch-clamp electrophysiology
Animals (vGat-Cre::EGFP) were anesthetized with 4% isoflurane for 5 min until reflexes were not present and then decapitated. About 250 µm thick coronal brain slices containing the dorsal raphe nucleus (DRN) were obtained using a Leica VT1000S vibratome (Leica Microsystems, Wetzlar, Germany). DRN was identified by the presence of an aqueduct (Aq). Slicing was done in a high sucrose solution containing (in mM): 87 NaCl, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 10 D-glucose, 50 Sucrose, 3 MgCl2, 0.5 CaCl2 (pH 7.3). Slices were maintained in a holding chamber containing extracellular solution for 45 min at 35 °C. Whole-cell patch-clamp recordings were performed using an extracellular solution containing (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 0.1 MgCl2, 3 CaCl2 (pH 7.4, continuously bubbled with 95% O2/5% CO2). Recording pipettes were made from borosilicate glass (Sutter Instrument) and had resistances of 5–6 MOhm when filled with a pipette solution containing (in mM): 135 potassium methylsulfate (KCH3SO4), 4 Mg-ATP, 0.3 Na-GTP, 8 NaCl, 10 HEPES, 5 Na2-P-creatine; pH 7.31, adjusted with KOH. Whole-cell current-clamp recordings were obtained under visual control using a 40x objective using an EPC-10 patch-clamp amplifier under the control of Patch-Master software (HEKA Elektronik, Reutlingen, Germany). The recordings were done at room temperature. Brain slices were imaged using a CMOS camera (C11440-36U; Hamamatsu Photonics, Hamamatsu City, Japan) under control of HOKAWO 3.0 software. To visualize the EGFP fluorescence, the fluorophores were excited using LEDs (465 nm, Green; CoolLED pT-100). One current injection step round consisted of 1 s steps ranging from −100 to +180 pA in 20 pA increments relative to the holding current. CVN527 was dissolved in DMSO with a final bath perfusion concentration of DMSO 0.01% and CVN527 100 nM. Baseline of 5 min of current injection steps was recorded. Subsequently, 0.01% DMSO, alone followed by a drug or direct drug application was performed. The washout was performed after 10–20 min of the drug application. Electrophysiological recordings were analyzed in Igor Pro 7 (WaveMetrics) software using the NeuroMatic plug-in42. Cells with a leak current of more than 300 pA were rejected. APs were detected using automated selection, where the threshold of −10 mV was manually selected. In Fig. 3G, firing rates of two current injection step rounds were averaged for baseline; two current injection step rounds, 3 min after the drug application and 2 to 1 current injection step rounds 2–4 min into washout were averaged.

In vitro pharmacology
Functional activity assessment of inverse agonists against GPR6 has been previously described43 Briefly, CHO-K1 cells (ATCC cat# CCL-61) stably transfected with mouse GPR6 cDNA (Origene) were incubated in 96-well plates for 24 h at 37 °C before being assessed via functional LANCE HTRF cAMP assay system (PerkinElmer). CVN527 was serially diluted in DMSO and added to the plates in Krebs’ Ringer’s buffer at 1x concentration (20 µl/well) and incubated for 45 min at 37 °C. Final concentrations ranged from 30 µM to 0.03 nM. In a final step, Eu-labeled cAMP tracer (10 µl) diluted in cAMP detection/lysis buffer with 10 µl ULight-anti-cAMP diluted in the same buffer was added to the cells. After a 60-min incubation at room temperature, time-resolved energy transfer was measured on an EnVision plate reader (Perkin Elmer). CVN527 showed full inverse agonism (Emax 100%), and efficacy values were calculated by fitting data to the four-parameter variable slope model without constraints using nonlinear regression in Prism (Graphpad).

Receptor occupancy, Ki determination, and pharmacokinetics
Ex vivo, dose-dependent receptor occupancy was determined in male C57/Bl6 mice (Charles River, UK). Since receptor occupancy is expected to be related to exposure and this ratio not to be sex dependent, we chose to use one sex in these studies, furthermore this relates to the HFD and behavior studies. The mice were dosed with vehicle (10% DMSO, 0.5% Tween 80, 89.5% PBS) or CVN527 at 1, 3, 10, or 30 mg/kg for p.o (n = 5/group) and then euthanized after 1 h. Terminal blood samples were taken by cardiac puncture and placed into lithium/heparin tubes (Sarsedt). Blood samples were centrifuged (1900 × g for 5 min) to extract the plasma, which was transferred to a microtube and frozen at −80 °C until bioanalysis. The brains were excised, and a coronal section was taken for receptor occupancy (rapidly frozen in isopentane cooled to −20 °C), and the remaining brain tissue was stored at −80 °C until bioanalysis. A GPR6-rich brain region previously pharmacologically validated (striatum) was cut into 20 µm sections and placed in humidified chambers for 120 min in 200 µl buffer (50 mM Tris buffer, pH 7.4 containing 50 mM NaCl, 6 mM MgCl2 and 0.1% BSA) containing either 2.5 nM [3H]-RL338 (total binding) and CVN527 at four different doses (1, 3, 10, 30 mg/kg) or 2.5 nM [3H]-RL338 and 10 µM CVN527 (non-specific binding). After incubation, the slides were washed 3x in ice-cold assay buffer and 1x distilled water. Slides were analyzed on a β-imager. occupancy (O) data analysis was determined according to Mugnaini M. et al.44. Brain samples for bioanalysis were homogenized in 0.1 M PBS before further processing. Both brain and plasma samples were analyzed via LC-MS/MS. The Ki of CVN527 for mouse striatal expressing GPR6 was determined using a similar method to the receptor occupancy, except the mice were not dosed with CVN527 prior to euthanasia, and the sections (8/animal, n = 9 mice) were incubated with one of nine concentrations of CVN527 (10–11 to 10–4 M). The binding (CPM/mm2) was used to determine the Ki using the Cheng–Prusoff equation45.

Behavioral studies
The OFT was used to evaluate locomotion activity after treatment with CVN527 (10 mg/kg) in peanut butter or peanut butter alone as a control (vehicle). Standard OFT arenas were used. Of note, mice had never been exposed to the OFT arena prior to studies but had been thoroughly habituated to the room where behavioral experiments occurred. Experiments were performed in 5-min sessions (pre- and post-drug). Total distance traveled, velocity and time spent in the center versus time spent in the borders of the OFT arena (anxiety-related behaviors) were calculated by using an automated tracking system (Ethovision XT).

The EPM was used to evaluate anxiety after treatment with CVN527 (10 mg/kg) in the same experimental groups aforementioned. Standard EPM arenas were used. Of note, mice had never been exposed to the EPM arena prior to studies but had been thoroughly habituated to the room where behavioral experiments occurred. Experiments were performed in 5-min sessions. Total distance traveled, frequency and cumulative duration in the open arms versus closed arms, velocity and mean distance to each arm were evaluated.

The marble burying test was used to assess anxiety, 12 glass marbles were evenly spaced in three rows, on an ~5 cm layer of sawdust bedding lightly pressed down to make a flat, even surface, in a plastic cage. Each mouse was placed in the cage and left for 30 min, after which the number of marbles buried with sawdust was counted as a measure of anxiety.

Quantification and statistical analysis
Statistical parameters reported in the Figures and Figure Legends are displayed as mean ± SEM. Significance was defined as P < 0.05. Significance annotations are annotated with the actual value. Mice were randomized into control and 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.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "All my earlier comments have been resolved. The manuscript is original, contributes meaningful data, and is supported by a robust multidisciplinary methodology. Overall, it is an excellent study.", "reviewer_2": "The revised manuscript has addressed the majority of my concerns. However, my enthusiasm is tempered by the lack of a synergistic effect on body weight loss when CVN527 is co-administered with semaglutide. It appears that CVN527 itself has only a very subtle effect in the DIO mouse model (Rebuttal Figure 3). Furthermore, if the original study tested CVN527 in a preventive setting by switching mice from a chow to a high-fat diet at the time of compound administration, then these mice should not be referred to as \"diet-induced obese (DIO)\". Consequently, it is inaccurate to state that the compound \"significantly reduced weight gain in DIO mice.", "reviewer_3": "The authors were responsive, and the revised manuscript by Moura-Assis et al. is significantly improved. However, there are a few remaining comments/clarifications/concerns.\n1) Given the vast expression of VGAT in and around the DRN, the authors should either include representative histology showing hM4Di expression in Figure 1 or a more extensive histological assessment as an extended figure, as the authors propose.\n2) My original comment #7 about only 50% of Gpr6-expressing neurons being VGAT positive was partially misinterpreted. I apologize if I was not clear. The authors comment on the phenotype observed with local pharmacology and provide the example of GLP1R agonists targeting a small population of AP/NTS neurons. I agree with their response, however; my intent was to raise the issue that the effects the authors see may not be mediated completely by VGAT neurons since half of the Gpr6-positive neurons do not express VGAT. CNV527 obviously cannot target only the VGAT neurons that express Gpr6, so how do the authors know that it’s the VGAT neurons that are responsible for the effects they are seeing? Perhaps CVN527 causes its effects on energy balance through both VGAT neurons and some other population. This is further supported by the fact that CVN527 treatment does not fully recapitulate the different phenotypes observed with chemogenetic inhibition of DRN VGAT neurons (e.g., chemogenetic inhibition reduces feeding). Thus, the authors should revise the text after line 444 (“Importantly, our findings suggest…”) to include the possibility that CVN527 may produce some of its effects via non-VGAT neurons.\n3) The electrophysiology study is still problematic.\nFirst, it is not required to generate a Gpr6-Cre mouse in order to know which neuron subtypes in the region express the receptor. I did not mean for this to be my takeaway message as I agree with the authors that generating a new mouse line requires considerable time. Instead, there are likely alternative Cre lines available that could be used as a proxy for Gpr6-positive neuron types, if a genetic marker were deciphered by analyzing single-cell RNA-seq data. This is one of the reasons I suggested such a study in a different comment. Further, instead of running a new study, the authors could utilize publicly available scRNA-seq data from the Allen Brain Institute or other publications (PMID: 31411560, biorxiv.org/content/10.1101/2022.06.27.497769v2) to find such a marker.\nSecond, even in the absence of an improved method of recording from Gpr6-expressing neurons, the issue of CVN527 increasing the firing rate of high-frequency firing DRN VGAT neurons remains unaddressed. There were 11 recordings from low-firing DRN VGAT neurons, and CVN527 reduced firing by approximately 33% at current injections of 0 pA and above. Of note, the legend in this figure says the statistical test performed was a paired t-test, which is inappropriate given there were repeated measures and would require multiple t-tests. Are the p-values reported adjusted for multiple comparisons? Meanwhile, CVN527 increased firing at current injections at or above 100 pA, reaching a nearly two-fold increase in action potential firing at currents of 140 pA and above. However, there is no mention of the increased firing rate in these recordings in the text, probably because there was no statistical significance – but this was likely a result of only having 4 recordings from high-firing DRN VGAT neurons. Together, there seems to be justification for more recordings, revision of the text related to the electrophysiology studies, or both."}, "author_response": "We sincerely thank Reviewer #1 for the thoughtful and encouraging comments on our manuscript. Below, we provide detailed responses to each of the reviewer’s insightful points:\nSome minor comments need to be addressed:\n1. In the ANCOVA analysis, representation of regression lines would be useful to better visualize the data.\nWe agree with the reviewer that visualizing group-specific\nregression lines in the ANCOVA plot would significantly enhance\nthe interpretability of the data. We have revised the metabolic 8.0\ncage data in the manuscript (Original Figure 1H-K and Figure\n7.5\n7.0\nanalysis to illustrate individual regression lines for each\n6.5 experimental group (Extended Data Figure 1C, Extended Data\n6.0 Figure 5A and C and Rebuttal Figure 1).\n5.5\n22 24 26 28 30 32\nIn the figure legend, we now describe the covariance analysis and\nBody Weight (g)\nexplicitly state that the parallel slope assumption was met (see\nExtended Data Figure 1C, Extended Data Figure 5A and C),\nsupporting the significance observed. Additionally, we revised\nthe Results section to clarify how these regression lines support\nthe data. We added a sentence about how we calculated it in the\nMethods section, representing indirect calorimetry studies.\n)gk/h/lack(\nEE\nmCherry\nhM4Di\n7.5\n7.0\n6.5\n6.0\n5.5\n5.0\n4.5\n20 25 30 35 40\nBody Weight (g)\n)gk/h/lack(\nEE\nCT\nGPR6\n9\n8\n7\n6\n5\n25 30 35 40\nBody Weight (g)\n)gk/h/lack(\nEE\nA\nB\nFigure 1 Energy expenditure ANCOVA plot for the 3 major\nstudies conducted in our manuscript. A) ANCOVA plot of mice\nCT treated with CNO at the same time as they transitioned to a high\nC\nGPR6 caloric diet exposed to CNO at the end point of the studies\n(mCherry refers to the control group and hM4Di the animals in\nwhich vlPAG/DRN Vgat neurons are silenced. B-C) Metabolic\ncage ANCOVA assessments of mice treated with GPR6 inverse\nagonist CVN527 in the DRN (B) and after oral delivery (C).\n2. Data on RER would add value to the data and could be showed.\nWe appreciate this suggestion. Respiratory exchange ratio (RER) provides 1.5 mCherry\nessential insight into substrate utilization and energy metabolism. We have hM4Di\nincluded RER data collected during indirect calorimetry studies in the\n1.0\nrevised manuscript (new Extended Data Figure 1D, Extended Data\nFigure 5B and D Rebuttal Figure 2).\n0.5\n3. Experiments were performed in male mice. This should be stated in the\n0.0\ntitle and the abstract.\nThank you for pointing out this necessary clarification. Experiments\ninvolved in metabolic phenotyping (Figure 1 and 4) in this study were\nconducted in adult male mice to avoid potential variability due to estrous\ncycle–related hormonal fluctuations and to ensure consistency across\nmetabolic and pharmacological readouts. Moreover, our previous studies\nhave already demonstrated that, in terms of feeding behavior and weight\nregulation, DRNVgat neurons exhibit similar behavior in males and females\n(PMID: 31257028). However, to comply with transparency and rigor, we\nnoted in the abstract, and discussion section that metabolic phenotyping\nwas done in male mice.\nIn response to the reviewer’s suggestion:\n• The abstract now also states clearly \"in male mice\".\n• The Methods section now includes this information, but we have\nemphasized it more clearly by moving it to the opening lines of the\n“Animals” subsection.\n• Additionally, in the Main section, we have included a brief\nparagraph acknowledging the limitation of using only male mice,\nstating:\n“Importantly, metabolic phenotyping experiments were conducted\nin male mice to minimize variability; however, given potential sex\ndifferences in energy homeostasis and thermogenesis, future studies for\ntranslational potential of GPR6 will be needed to determine whether similar\neffects are observed in females. ”\nRER\nLight Dark\nCT\nGPR6\n1.0\n0.8\n0.6\n0.4\n0.2\n0.0\nRER\nLight Dark\nCT\n1.0 GPR6\n0.8\n0.6\n0.4\n0.2\n0.0\nRER\nA\nB\nC\nLight Dark\nFigure 2 Respiratory Exchange\nRate (RER) plot for the 3 major\nstudies conducted in our\nmanuscript. A) RER plot of mice\ntreated with CNO at the same time\nas they transitioned to a high caloric\ndiet exposed to CNO at the end\npoint of the studies (Mcherry refers\nto the control group and hM4Di the\nanimals in whicvh vlPAG/DRN Vgat\nneurons are silenced. B-C)\nMetabolic cage RER assessments\nof mice treated with GPR6 inverse\nagonist CVN527 in the DRN (B) and\nafter oral delivery (C).\nReviewer #2 (Remarks to the Author):\nThis article by Alexandre et al. demonstrated that chronic activation of VGAT-positive neurons in the dorsal\nraphe nucleus (DR) reduces the body weight of obese mice. The authors also focused on a G-protein-coupled\nreceptor, GPR6, which is specifically enriched in VGAT neurons within the DR, and identified an effective\ninverse agonist for GPR6, CVN527. CVN527 was shown to reduce the excitability of VGAT neurons in the DR\nand stimulate brown adipose tissue thermogenesis. The authors concluded that CVN527 could be a promising\ndrug to treat obesity. While the data are exciting, aspects of the study seem preliminary, and as such make\ndrawing conclusions from this data premature at this point.\nMajor Concerns:\n1. One major concern of this review is that whether the CVN527 could have a cumulative effect in term of\nreducing body weight when applied together with semaglutide (sema). This is an important question since\nsema has been widely used for obesity treatment. New drugs for obesity treatment need to be shown the\ncombination medication potential with sema.\nWe thank the reviewer for raising this important and timely point. We fully agree that the potential for\ncombinatorial efficacy with Semaglutide would be critical for weight management in obesity.\nFirst of all, we would like to note that all studies involving GPR6 in this project were conducted preventively,\ngiven its ability to trigger a thermogenic effect. We hypothesized that stimulating energy expenditure before\nWestern Diet exposure could reduce weight gain. Regarding Semaglutide, which mainly targets feeding and has\nan anorexigenic effect, most studies are performed in a curative manner. Therefore, to address the reviewers'\nconcerns, we believe that an additive effect should be evaluated in this curative setting.\nIn brief, we co-administered CVN527 with a clinically relevant dose of Semaglutide in diet-induced obese (DIO)\nmale mice (100nmol/kg). Notably, the combination treatment was well-tolerated, with no obvious signs of\ndistress. Unfortunately, we did not observe a reduction in body weight beyond the effect of Semaglutide alone.\nThese findings suggest that, under the conditions tested, CVN527 does not exert a synergistic or additive effect\nwhen administered concomitantly with Semaglutide (Rebuttal Figure 3)\n30\n20\n10\n0\n1 2 3 4 5 6 7 8 9\nDays\nFigure 3 Semaglutide combination treatment with CVN527 does not show synergistic effects. Cumulative\nfood intake and body weight assessment when combining CVN527 and Semaglutide (GLP-1 agonist) does not\nlead to synergistic effects. Notably, both drugs function as expected in monotherapies. Data are represented as\nmean ± s.e.m. P values were calculated using a two-way ANOVA with a multiple-comparisons test using a Tukey\npost-hoc approach (n = 5-7).\nWe hypothesize that this may reflect a ceiling effect on food intake suppression, in which CVN527 via GPR6\nantagonism cannot drive further weight loss, particularly given Semaglutide’s robust anorectic action. It is also\npossible that overlapping downstream pathways limit combinatorial efficacy. Nonetheless, we are actively\nexploring additional dosing paradigms (e.g., sequential treatment, dose titration, or in different metabolic\ncontexts) that may unmask a synergistic interaction.\nWe appreciate the reviewer’s insightful suggestion.\n)g(\nekatni\ndoof\nevitalumuC\n110\n100\n* 90\n80\n70\n0 1 2 3 4 5 6 7 8 9\nDays\n)%(\nthgieW\nydoB\nA B\nCT\nCVN527\nSema\n* CVN527 + Sema\nRebuttal Figure 2: (A) Cumulative food intake, (B) Body weight at days 0 vs 9 (n=5-7), two-way ANOVA\nwith Tukey’s multiple comparison tests. All error bars represents mean ± SEM. *p<0.05.\n2. The figures and figure legends in the article contain numerous errors and unclear descriptions (especially\nregarding the labeling of \"n\" and p-values), reducing the credibility of the results.\nFor example:\n1. Figure 1B lacks the value of \"n\" and what \"n\" represents for.\n2. Figure 2E is unclear whether “n” refers to the number of brain slices or the number of mice.\n3. The meaning of x-axis in Figure 3C is unclear. Also, why these is a negative point?\n4. Starting from panel K in Figure 4, the figure legends contain errors. For instance, Figure 4K is not about\nCVN527 levels, and legends for Fig4 T is not included.\n5. Many panels in Extended Figure 4 lack annotations on p-values.\nWe thank the reviewer for this detailed and constructive feedback regarding figure clarity and legend accuracy.\nWe take these issues seriously and have undertaken a thorough revision of all figures, legends, and statistical\nannotations across the primary and extended data to ensure consistency, accuracy, and transparency. Below,\nwe address each specific point raised:\n1. Figure 1B:\nWe have now added the value of “n” to the legend and a clear statement that “n” refers to the number of\nbrain slices of DRN using 3 per animal (n= 24 slices (8 mice)). This change ensures transparency in\nexperimental design and sample size. Need to do that\n2. Figure 2E:\nThe legend has been revised to explicitly state that “n” refers to the number of brain slices used, with 3\nbrain slices analyzed per animal. We also included this detail in the Methods section for clarity and\nreproducibility. Need to do that\n3. Figure 3C:\nWe thank the reviewer for pointing out the ambiguity in the x-axis labeling. The axis now clearly denotes\nthe parameter measured (dose mg/kg).\n• Regarding the negative value, receptor occupancy is calculated by measuring the displacement of a\nradio-labelled ligand for GPR6. The principle of this can be found in PMID: 22968817, amongst\nothers. Essentially, the % receptor occupancy is a calculation of how much drug is displaced at a given\ndose or concentration. The calculation is\nIn which SB is the SB in the animal treated with the test item, and the mean SB of the animals treated\nT\nwith the vehicle. (SB: Specific binding)\nBecause this calculation is essentially a ratio, at very low doses where there is no displacement, that\ncalculation for an individual animal may appear negative. This should be regarded as a mathematical\nartefact; in reality, the receptor occupancy is 0.\n4. Figure 4, panels K–T:\nWe carefully reviewed all figure panels and legends from Figure 4K onward and have corrected multiple\nlabeling inconsistencies:\no Figure 4K now correctly states that it shows food intake (g) during the treatment.\no We thank the reviewer for noting that there was an editing issue, and the legend from 4J to 4S\nhad shifted one position, showing 4T. We have now revised it and it correctly represents each\nfigure panel.\no All subpanel identifiers and descriptions were reviewed for consistency and accuracy.\n5. Extended Figure 5 (now 6):\nWe acknowledge that several p-values were missing in the original figure. These have now been added\ndirectly to the graphs or in the corresponding legends, as appropriate. Just note that given Reviewer 3’s\nconcern on sample size, we have now redone the experiment in this figure. The new experiment includes\n10 mice in each group. As a result, we now have better statistical power. Of note, the same conclusions\nremain in which the only visible effect is an increase in locomotion activity, consistent with our\nchemogenetic, pharmacologic, and published studies (PMID: 31257028).\nThese revisions are now reflected in the updated figures and legends in the revised manuscript. We believe\nthese changes substantially improve the clarity, rigor, and credibility of our data presentation. We again thank\nthe reviewer for helping us enhance the quality of the manuscript.\n3. Figures 1A-B: The c-fos statistical analysis is more suited for representing short-term neuronal activity changes\nrelated to specific behaviors due to its strong dependence on the expression window, and is not appropriate for\nrepresenting long-term neuronal excitability changes. Also, whether these c-fos positive cells are GABAergic or\nglutamatergic is not demonstrated.\nWe thank the reviewer for this thoughtful and essential comment regarding interpreting c-fos as a marker of\nneuronal activity. We agree that c-fos is classically used to capture transient or acute neuronal activation and is\ninfluenced by temporal proximity to behavioral or physiological stimuli. However, we would like to clarify the\nrationale and context of our approach.\nOur goal in Figures 1A–B was to perform an unbiased, whole-brain screening of neuronal populations whose\nactivity is persistently altered in response to diet-induced obesity. While c-fos is an immediate early gene best\nsuited to detect short-term changes, when applied under tightly controlled and consistent conditions across\ngroups, it can also provide comparative insights into chronic alterations in basal neuronal activity states. We\nminimized variability by analyzing mice at the same circadian time point and controlling for confounding factors,\nincluding stress, satiety state, and locomotion. This information has been added to the Methods section We\nimproved the interpretability of c-fos expression as a proxy for relative activity differences across experimental\nconditions.\nFurthermore, we did not rely on a single time point. We conducted c-fos analysis at multiple stages of high-fat\ndiet (HFD) exposure—including 3 days, 2 weeks, and 16 weeks—to capture both early and sustained\nadaptations in neuronal activation. These data, together, indicate that activity changes in the dorsal raphe\nnucleus (DRN) are present not only acutely but also persist during long-term exposure to obesogenic conditions,\nstrengthening the relevance of our findings to chronic metabolic states.\nTo address the second point, we agree it is essential to identify the neurochemical identity of the c-fos–positive\ncells. To this end, in a separate set of experiments, we performed co-labeling of c-fos with:\n• TPH2 immunofluorescence to identify serotonergic neurons, and (Rebuttal Figure 4-B)\n• VGAT using Vgat:GFP-L10 reporter mice, which selectively label GABAergic neurons in the DRN\n(Rebuttal Figure 4C-D).\nFigure 4 Immunofluorescence\nfor TPH2 and cfos to\nA\nTPH2 cFOS Merge demonstrate that neurons\nactivated by high calorie\ncontent are not serotonin\nexpressing neurons. A) IHC for\ncontrol (top panels) and HFD fed\nanimals for 2 weeks (bottom\npanels) in which TPH2 is in the\n488 channel (green) and cFOS in\nthe 594 channel (red). Arrows\nindicate colocalization. B)\nQuantification of cFOS positive\ncells per animal in either chow\ndiet (RCD) or high fat diet (HFD).\nC) Overlap cFOS/TPH2\nquantification. Ns = non\nsignificant. Data presented as\nmean +/- SEM. Scale bar =\n200um. N = 12-14 mice\nThese studies revealed that a substantial proportion of c-fos–positive cells in the DRN overlapped with VGAT-\npositive neurons, confirming that the activity changes we observed occur predominantly within the GABAergic\npopulation (Rebuttal Figure 4). These data support the hypothesis that DRNVGAT neurons are specifically\nrecruited or persistently active in the context of HFD-induced obesity.\nFigure 5 Immunofluorescence for cfos in Vgat::GFPL10 mice in HFD exposed mice. A) Example of IHC for\ncontrol (top panels) and HFD fed animals for 2 weeks (bottom panels) in which endogenous Vgat encoded GFP\nis in the 488 channel (green) and cFOS in the 594 channel (red). Arrows indicate colocalization. B) Quantification\nof number of cfos positive cells in either chow diet (RCD) or high fat diet (HFD). C) Percentage of colocalization\nwith Vgat. *p<0.05 ***p<0.001. Data presented as mean +/- SEM. Scale bar = 200um. N = 3-4 mice\nDCR\nDFH\nB\n80\n60\n40\n20\nC\necils/sllec\nevitisop\nSOFc\nns\n30\n20\n10\n0\n-10\nRCD HFD\nsllec\ngnippalrevo\nfo\nrebmuN\nns\nWe have clarified these points in the revised Main section and added the co-labeling data as part of Extended\nFigure 1B.\nWe appreciate the reviewer’s comment, which allowed us to clarify the rationale, limitations, and interpretive\nvalue of our use of c-fos in this context.\n4. In Fig3, the expression pattern change of GPR6 in the DR of DIO mice is unclear. Electrophysiological\nrecordings of DR VGAT-positive neurons are needed to explore the effects of CVN527 on DR VGAT-positive\ncells in high-fat diet induced obesity mice.\nWe appreciate the reviewer’s important suggestion regarding the utility of electrophysiological recordings to\ncharacterize further the effects of CVN527 in the dorsal raphe (DR) of diet-induced obese (DIO) mice. We agree\nthat such data could provide additional mechanistic insight into the modulation of DRNVGAT neurons by GPR6 in\nthe context of obesity.\nHowever, there are several technical and conceptual considerations that we hope will clarify why these\nrecordings were not included in the current study:\nFirst, the whole-cell patch-clamp recordings we performed were already technically demanding. Each\nexperiment required a prolonged 40–45 minute recording window to encompass the baseline, CVN527\napplication, and washout phases, and was conducted in acute slices from adult Vgat:GFP-L10 mice. Maintaining\nstable recordings from DR neurons for this duration necessitated high-quality tissue viability, which is significantly\ncompromised in older, DIO mice due to age-related and obesity-associated deterioration in brain slice quality\nand reduced neuronal integrity. These factors dramatically decrease the success rate and reliability of such\nrecordings in obese animals.\nSecond, logistical constraints must be considered. To obtain DIO mice for electrophysiological recordings,\nanimals must be subjected to a high-fat diet for at least 16 weeks. Including such a cohort would not only extend\nthe experimental timeline by several months but also require replicating the full electrophysiological workflow in\nmore fragile tissue—a technically intensive process with a low yield.\nThird, we respectfully note that the core objective of this study is to evaluate the effect of GPR6 modulation in\npreventing the development of obesity, rather than characterizing its reversal in established obesity. In alignment\nwith this aim, our molecular profiling (Figure 2) and pharmacological intervention studies (Figure 4) were initiated\nin lean mice before diet-induced obesity. Electrophysiological characterization was therefore performed under\nthe same baseline conditions to match the experimental context and support the interpretation of CVN527’s\neffects in a preventive paradigm.\nFinally, while we did not repeat recordings in DIO mice, our transcriptomic analyses (Figure 2) demonstrated\nGPR6 enrichment in DRNVGAT neurons, and our chemogenetic and pharmacological manipulations (Figures 3–\n4) functionally confirmed that modulation of this population significantly alters energy expenditure and body\nweight. Thus, the combined molecular, functional, and behavioral data offer a coherent and comprehensive\nframework that supports our conclusions.\nWe have now clarified this rationale in the revised Main section. We hope the reviewer agrees that, while\nelectrophysiological recordings in DIO mice would be of interest for future studies focused on therapeutic reversal\nof obesity, they are not essential to support the central claims of this prevention-focused work.\n5. Chronic inhibiting DR VGAT neurons through chemogenetic reduces food intake while increasing BAT\nthermogenesis in mice (Fig1). However, CVN527 treatment, inhibiting DR VGAT neurons, only increases BAT\nthermogenesis without affecting food intake. The data show that approximately 50% of DRVgat neurons express\nthe GPR6 receptor that is targeted by CVN527, raising a question of whether different subtypes of DR VGAT\nneurons are involved. Does this mean that DRVGAT neurons expressing GPR6 are unrelated to feeding\nbehavior?\nWe thank the reviewer for this important observation and thoughtful question, which touches on the functional\nheterogeneity of DRNVGAT neurons and the specificity of GPR6 modulation.\nIt is correct that chemogenetic inhibition of the entire DRNVGAT population results in reduced food intake and\nincreased thermogenesis, while CVN527, a selective GPR6 inverse agonist, increases thermogenesis without\naffecting feeding. Despite targeting overlapping neuronal populations, this behavioral outcome divergence\nsuggests that distinct DRNVGAT neuron subpopulations may differentially regulate feeding and energy\nexpenditure.\nAs the reviewer noted, our GPR6 staining revealed that about 50% of DRNVGAT neurons express GPR6,\nindicating molecular and likely functional diversity within this inhibitory population. Based on our findings, we\npropose the following working model:\n• GPR6-expressing DRNVGAT neurons are preferentially involved in regulating energy expenditure and\nthermogenesis, likely via downstream projections to sympathetic regulatory centers (e.g., the rRPa or\nspinal intermediolateral nucleus).\n• In contrast, the GPR6-negative subset of DRNVGAT neurons may be more directly involved in\ncontrolling feeding behavior, potentially via connections to hypothalamic appetite-regulating regions such\nas the arcuate nucleus or lateral hypothalamus.\nThis interpretation is further supported by our behavioral pharmacology results showing that CVN527 did not\nalter acute or cumulative food intake, even at doses that robustly increased BAT thermogenic activity and energy\nexpenditure. The global chemogenetic approach, which likely silences in a bulk effect most DRNVgat neurons\n(both GPR6-positive and -negative subpopulations), producing a broader phenotype that includes feeding\nsuppression.\nWhile we cannot definitively exclude subtle or context-dependent effects of GPR6 modulation on feeding, our\ndata (as the reviewer notes) suggest that the GPR6-expressing subset of DRNVGAT neurons is primarily involved\nin energy expenditure control, and dissociated mainly from feeding regulation. We now discuss this model more\nexplicitly in the revised Main section and highlight the potential for future studies to dissect the projection targets\nand behavioral roles of DRNVGAT subpopulations using intersectional or circuit-tracing approaches.\nAgain, we thank the reviewer for raising this key point, which allowed us to refine our interpretation and clarify\nthe functional specificity of GPR6 signaling in the DRN.\nMinor Concerns:\n1. Figure 1E: Does this refer to the 24-hour food intake of mice? The food intake testing method needs to be\ndescribed.\nYes, Figure 1E reflects 24-hour cumulative food intake following chemogenetic inhibition of DRNVGAT neurons.\nWe have now clarified this in the Figure legend, where we describe the food intake measurement protocol.\n2. In Figures 3C-D, why was the effect of CVN527 tested in the striatum rather than in the DR?\nWe appreciate the opportunity to clarify. CVN527 was tested in the striatum in these panels to confirm target\nengagement and downstream cAMP modulation in a GPR6-rich region previously validated pharmacologically.\nThese experiments served as a pharmacodynamic control for compound activity before DRN-specific testing.\nWe now state this rationale more clearly in the Methods section.\n3. The cannula placement traces in Figure 4A indicate that the cannula tip is in the fourth ventricle rather than\nthe DR.\nThank you for pointing this out. The schematic in the revised Figure 4A (Rebuttal Figure 6) has been corrected\nto reflect the cannula tip position within the DRN accurately by showing a higher magnification, in which it can\nbe appreciated that the tip is actually in the DRN. We also verified all placements histologically and confirmed\naccurate targeting, as noted in the updated Methods section.\nFigure 6 Cannulation representative image. Updated image to illustrate cannula placements in the DRN for\nGPR6 inverse agonist CVN527 infusions.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Moura-Assis et al. shows that inhibitory neurons of the DRN, that they previously showed are\ninvolved in energy homeostasis, are activated by high fat diet feeding. Neurons within and around the dorsal\nraphe are activated after high fat feeding, of which approximately 25% express VGAT. The authers go on to build\non prior work to show that chronic inhibition of DRN VGAT neurons attenuates body weight gain from HFD and\nreduced body weight in 16-week HFD fed mice. Next, the authors profile VGAT neurons in the region to identify\ndruggable targets of these neurons to be used to reduce their activity. Through RNA profiling and drug screens\nthe authors identify GPR6 and an inverse agonist for the receptor, CVN527. CVN527 treatment reduces body\nweight gain from HFD feeding when administered directly to the DRN or when given orally. In summary, this\nstudy confirms prior work by the authors on DRN VGAT neurons, but the new findings lack specificity due to\nsome of the approaches used, which decreases enthusiasm.\nWe thank the reviewer for the thoughtful summary and for recognizing the value of our prior and current work\nlinking DRNVGAT neurons to energy homeostasis. We appreciate the acknowledgment of the comprehensive\napproach used in this study, including chronic circuit manipulation, transcriptomic profiling, and pharmacological\ntargeting of a novel GPCR. We hope that after addressing the concerns raised by the reviewers, we will increase\nthe reviewers’ enthusiasm for the updated manuscript.\n1) The rationale for running Fos studies after 3 days, 7 days, 2 weeks and 16 weeks of high fat diet feeding is\nunclear. Additionally, there is no explanation of how the experiments were run. Were the animals sacrificed\nduring the inactive or active phase? Had the animals recently ingested high fat diet? Also, there is a discrepancy\nwith the “Clearmap” experiment which uses 7 days of 60% HFD, while the other Fos experiments at 3 days, 2\nweeks, and 16 weeks use 45% HFD.\nWe thank the reviewer for this excellent observation and welcome the opportunity to clarify the rationale,\nexperimental design, and apparent discrepancies across the Fos datasets. We aimed to map the temporal\ndynamics of neuronal activity changes in the DRN in response to high-fat diet (HFD) exposure across distinct\nphysiological stages of obesity progression.\nSpecifically:\n• 3 days: Represents the acute response to HFD, following ~2 days of taste adjustment. This time point\ncaptures early sensory and homeostatic responses before major changes in weight or metabolism occur.\n• 7 days: Reflects a transition phase, where the diet has changed but no significant weight gain is typically\nobserved. This is also the time point used in the CLEARMAP iDISCO-based whole-brain analysis, which\noffers an unbiased view of DRN activation during early diet exposure.\n• 2 weeks: Corresponds to the period when initial weight gain and early metabolic alterations become\ndetectable in most animals.\n• 16 weeks: Represents the chronic phase of diet-induced obesity, at which point there is a consensus in\nthe field that mice develop leptin and insulin resistance, consistent with established metabolic\ndysfunction.\nThese time points were selected to provide a longitudinal view of DRN engagement from early exposure to full\nmetabolic disease.\nExperimental Conditions:\nTo ensure consistency and minimize variability across time points:\n• All animals were sacrificed during the inactive (light) phase, between ZT2 – ZT4., a time window chosen\nto control for circadian variability in neuronal activity.\n• Food was removed at 7:00 a.m. (2 hours prior to perfusion) to standardize the nutritional state across\nanimals and eliminate acute ingestion effects, while maintaining a close link to the habitual feeding\ncontext.\n• Mice were perfused under baseline, unstimulated conditions—i.e., not acutely exposed to novel HFD\nprior to sacrifice, allowing us to assess the basal Fos signal reflective of their diet-adapted homeostatic\nstate.\nDietary Composition Differences:\nWe acknowledge the reviewer’s observation regarding the use of different HFD formulations across datasets:\n• The 3-day, 2-week, and 16-week time points were part of a longitudinal Fos study conducted\nat Rockefeller University, using a 45% kcal-from-fat HFD. This diet was selected to match the conditions\nused in our GPR6 inverse agonist treatment experiments, ensuring consistency in our chemogenetic and\npharmacological studies.\n• In contrast, the 7-day CLEARMAP experiment was performed independently at Yale University, where\nour group routinely uses a 60% kcal-from-fat HFD for iDISCO-based mapping studies focused on\nobesity-induced brain dysfunction, including links to neurodegeneration.\nWhile this difference in fat content is noted, we find it striking—and scientifically reassuring—that DRN\nactivation is robustly observed across both paradigms and all time points, from as early as 3 days to as late as 16 weeks\nof HFD exposure. This temporal persistence of DRN engagement aligns with recent work from the Garret Stuber\nlab, which showed sustained inactivation of lateral hypothalamic Vglut2 neurons from 2 to 12 weeks of HFD\n(PMID: 31249056), reinforcing the concept that discrete neuronal populations remain tonically active during\nchronic overnutrition.\nWe have now revised the Methods sections to include this information, clarify experimental timelines and\nconditions, and highlight the cross-institutional consistency of DRN activity as a meaningful and reproducible\nbiological signal.\nAgain, we thank the reviewer for raising this critical point, which helped us improve the clarity and contextual\nrelevance of our study.\n2) In the “Clearmap” Fos experiment is Figure 1 the arrow points to a strong increase along the midline within\nthe DRN at Bregma -4.1, where serotonin/VGLUT3 neurons are concentrated. Why did the authors only assess\nFos in VGAT neurons? Also, at Bregma -4.36 arrows point to an activated area which seems ventral and lateral,\nperhaps not DRN. Nonetheless, given that only 25% of Fos labeling was accounted for by VGAT neurons\naccording to Supplemental Figure 1, the majority of activated neurons are not VGAT positive – suggesting they\nare either serotonergic/VGLUT3 or VGLUT2. Further, the data in Supplemental Figure 1 is not convincing. There\nis no quantitative comparison to a control group and only 2 animals were used for Fos and VGAT labeling after\nHFD. Thus, it is possible that the increase in Fos expression is occurring in other cell types after HFD, not in\nVGAT neurons.\nWe thank the reviewer for this thoughtful and detailed critique, which highlights important considerations\nregarding the cellular specificity of HFD-induced neuronal activation in the DRN. We appreciate the opportunity\nto strengthen this aspect of the study and clarify the interpretation of our CLEARMAP and c-Fos results.\n1. Regarding the apparent midline Fos activation at Bregma -4.1:\nWe agree that this region contains a high density of serotonergic/VGLUT3-expressing neurons. However,\nthe Clearmap iDISCO+ analysis is a whole-brain, unbiased tool designed to identify brain regions that show\nconsistent activation in response to a physiological stimulus—in this case, 7 days of HFD. This analysis broadly\npointed us to the DRN, prompting follow-up studies to dissect the specific cell types involved.\n2. Why we focused on VGAT neurons:\nWe selected VGAT-expressing neurons for deeper analysis because our prior work demonstrated that DRNVGAT\nneurons are critical regulators of energy homeostasis. Based on this established link, we prioritized investigating\nwhether these neurons are part of the HFD-responsive population. Nonetheless, we fully agree that serotonergic\nand glutamatergic populations are abundant in the DRN and may also contribute to the Fos signal.\n3. New data to address cell-type specificity of Fos expression:\nTo directly address this concern, we performed two additional experiments following 2 weeks of HFD exposure:\n• (i) Double immunofluorescence for c-Fos and TPH2, the rate-limiting enzyme for serotonin synthesis,\nto assess Fos expression in serotonergic neurons.\n• (ii) c-Fos labeling in Vgat::GFP-L10 mice, which enables visualization of VGAT-expressing neurons in\nthe DRN.\nThese new data demonstrate that at this time point, the majority of cFos–positive neurons within the DRN co-\nlocalize with VGAT-expressing cells, and only a minority overlap with TPH2+ serotonergic neurons. This supports\nour interpretation that VGAT neurons represent the primary activated subpopulation in response to HFD, at least\nduring the early and intermediate phases of exposure (Rebuttal Figure 5)\n4. Interpretation of lateral/ventral activation at Bregma -4.36:\nWe appreciate the reviewer’s observation. While the region at Bregma -4.36 is anatomically adjacent to the DRN,\nthe Fos signal is still within the extended periaqueductal gray/raphe complex, which includes functionally\nconnected neurons involved in energy regulation. Given the resolution limitations of light-sheet imaging and\nregistration, we now acknowledge this uncertainty and have updated the manuscript to refer to the vlPAG/DRN.\nIn summary, we now provide additional cell-type–specific evidence confirming that DRNVGAT neurons are indeed\nFos-activated in response to HFD. These new data strengthen the link between diet-induced DRN activation and\nthe inhibitory subpopulation previously shown to modulate energy expenditure.\nWe thank the reviewer for prompting this necessary clarification and believe these additions substantially\nimprove the mechanistic rigor of our study.\n3) If the VGAT neurons are being activated after high fat diet, why? Since it is unclear when the animals were\nsacrificed, it is also unclear what may be causing the activation of VGAT neurons. Is it in response to eating the\nhigh fat diet? Are these VGAT neurons leptin-sensitive and elevated leptin from HFD feeding is driving their\nactivity? Is the increased activity in response to “overnutrition” sensing by upstream neurons? This is all\nunknown and not addressed or discussed in the manuscript.\nWe thank the reviewer for this insightful question. We agree that understanding the upstream drivers of DRNVGAT\nneuron activation is crucial, and we appreciate the opportunity to clarify both our experimental design and our\ncurrent interpretation of the underlying mechanisms.\n1. Sacrifice timing and fasting status: As previously noted, all animals were sacrificed during the inactive/light\nphase, specifically between ZT2-ZT4., and food was removed at 7:00 a.m. to standardize nutritional status and\nminimize acute feeding effects. Thus, the c-Fos signal we observe reflects a baseline state of neuronal activity,\nnot an immediate response to food consumption.\n2. Leptin sensitivity of DRNVGAT neurons: We have addressed the possibility of leptin signaling and can\nconfidently state that, based on our transcriptomic profiling, DRNVGAT neurons do not express leptin receptor.\nTherefore, the observed activation is not due to direct leptin signaling, despite elevated leptin levels typically\nassociated with HFD feeding. We now include this clarification in the revised Main section. Moreover, Fos\nmapping in OB/OB mice also turns on the DRN, further confirming that DRNVGAT neurons are activated in states\nof hunger (Rebuttal figure 7). This data is part of another publication and will not be included since the focus of\nthis manuscript is DIO.\nCoronal View\nControl Ob/ob Atlas pvlas\nDorsomedial nucleus of the hypothalamus\nField CA3\nHypothalamus\nVentro Lateral Periaqueducal gray\nDorsal Raphe\nFigure 7 Whole brain activity mapping for FOS reveals that the DRN/vlPAG region is activated in ob/ob\nfurther suggesting an important role for this region in obesity management. IDISCO+ based volumetric\nimaging for the marker FOS in a cohort of control C57Bl6 mice and ob/ob mice followed by quantification using\nCLEARMAP software analysis indicates that several midline sturctures including the DRN/vlPAG are activated\nin adlibitum fed obese mice. Experiments conducted at ZT2-ZT4 and in a sample size of 8 mice per group. First\ntwo columns are the averged heatmaps of activity of control (first column) and ob/ob (second column) of FOS\nbased activity. Third column is the coronal slice of the Allen Brain Atlas and the last coumn is the p-value map\nin which in green we can see regions with a p<0.05 increased activity and in red regions with decreased activity\nat a p<0.05\n3. Prior work on DRNVGAT function and energy state sensitivity: Our previous studies (PMID: 28753423)\nhave shown that DRNVGAT neurons are activated by energy deficit and promote feeding, suggesting they are part\nof a hunger-sensitive circuit. Paradoxically, their persistent activation in HFD-fed mice, who are not calorically\ndeprived, raises important questions about how hedonic or metabolic cues may override normal satiety signaling.\n4. A possible model: hedonic override of satiety circuits: We propose that palatable, energy-dense diets\nmay induce a persistent “hunger-like” state, in which DRNVGAT neurons remain chronically active despite energy\nsurplus. This could be due to altered input from upstream metabolic or limbic regions that normally regulate\nfeeding behavior, such as the lateral hypothalamus and ventral tegmental area. We have now performed a\nRabies-based, monosynaptic tracing experiment to determine the overall distribution of presynaptic partners of\nDRNVGAT and found input from upstream metabolic or limbic regions that normally regulate feeding behavior,\nsuch as the lateral hypothalamus (LHA), ventral tegmental area (VTA) and Substantia nigra compacta (SNc)\n(PMID: 40185087, PMID:38242133) (Extended Fig 2 and Rebuttal figure 8). We speculate\nthat overconsumption of rewarding food may decouple homeostatic feedback, leading to the chronic recruitment\nof hunger circuits and reinforcing further intake, a mechanism analogous to those described in addiction\nneuroscience (PMID: 32747789).\nWhile we do not directly test the afferent drivers of DRNVGAT neuron activation in this study, we agree that this is\nan important area for future investigation. We now incorporate this mechanistic discussion into the revised\nmanuscript, including possible upstream sources and behavioral implications.\nAgain, we thank the reviewer for raising this foundational question, which has helped us improve the mechanistic\nframing of our findings.\n4) Line 113 begins, “Since activating these neurons increases body weight,…”, but there is no citation. What is\nthe evidence that activating DRN VGAT neurons increases body weight? The authors showed in previous\npublications that their activation increases food intake and decreases energy expenditure acutely, which likely\nwould improve body weight under chronic conditions, but no body weight data were reported. On the other\nhand, inhibiting DRN VGAT neurons chronically had been shown to decrease body weight previously in ob/ob\nmice.\nWe thank the reviewer for this critical clarification and sincerely apologize for the overstatement. The reviewer is\ncorrect—our previous studies (PMID: 28753423; PMID: 31257028) demonstrated that acute activation of\nDRNVGAT neurons increases food intake and reduces energy expenditure, physiological changes that would be\nexpected to promote weight gain if sustained chronically. However, as the reviewer points out, we did not directly\nmeasure body weight outcomes following chronic activation in those studies, and therefore should not have\nimplied causality without supporting data. In contrast, we demonstrated that chronic inhibition of DRNVGAT\nneurons results in significant body weight reduction in ob/ob mice, supporting their relevance in long-term energy\nbalance regulation.\nTo address this issue, we have revised the sentence in Line 113 to read:\n“Since activating these neurons increases food intake and reduces energy expenditure—physiological\nchanges that could lead to increased body weight if sustained over the long-term…”\nWe appreciate the reviewer’s close reading and thoughtful correction, which helped us improve our manuscript's\naccuracy and scientific integrity.\n5) Figure 1 shows that chronic inhibition of DRN VGAT neurons attenuates HFD-induced weight gain and\ndecreases body weight in mice that have been fed HFD for 16 weeks. This is consistent with prior studies by the\nsame authors showing that inhibiting DRN VGAT neurons chronically in ob/ob mice decreases body weight.\nHowever, no histology of injection sites demonstrating where hM4Di is expressed are shown leaving is unknown\nhow specific these effects are to the DRN VGAT neurons. For instance, how are you excluding VGAT neurons\nof the adjacent vlPAG which is abundant in VGAT?\nWe thank the reviewer for highlighting this important point. We fully agree that precise anatomical targeting is\nessential to interpreting circuit-level manipulations, particularly in densely packed brainstem regions such as the\nDRN and ventrolateral periaqueductal gray (vlPAG), which both contain abundant VGAT-expressing neurons. In\nour experimental design, we carefully targeted the DRN, guided by atlas coordinates and post-mortem\nconfirmation of needle tracks. However, we acknowledge that due to the anatomical proximity of the vlPAG and\nthe diffusion properties of viral vectors, we cannot definitively exclude some degree of hM4Di expression in\nadjacent vlPAGVGAT neurons. That said, it is worth noting that in our previous work (PMID: 28753423) and (PMID:\n31257028) have shown that vlPAGVGAT neurons and DRNVGAT neurons, are both activated during low energy\nstates, and are likely involved in overlapping or complementary roles in energy balance. Therefore, even if some\nviral spread occurred into the vlPAG, the observed phenotype, attenuation of weight gain and increased energy\nexpenditure, remains consistent with the functional role of these neurons in promoting feeding and conserving\nenergy. Moreover, Reis and co-authors (PMID: 38454000) critically identified that these neurons are necessary\nfor feeding control, ultimately highlighting an important role for these brainstem clusters in weight regulation.\nA\nTVA-eGFP-oG RV-mCherry\nAAV2-FLEX-TVA-eGFP-oG\nEnvA-dG-RV-mCherry\nDR\nTVA-eGFP-oG\nvGat-Cre\nB RV-mCherry\nLC VTA LH\nA\nCeA SNc BNST\nC 25\n20\n15\n10\n5\n)sllec\nretrats(\nstupni\n%\nAq Aq Aq\nDR\nvlPAG\nFigure 8. Input mapping from DRN Vgat cells using monosynaptic rabies. A) Scheme of experimental\nsetup: G-deleted EnvA-mCherry expressing Rabies virus are injected in the vLPAG/DRN of Vgat-Cre mice\ntogether with a TVA-eGFP Cre dependent helper virus. Images are representative of viral expression. B)\nSignificant inputs are observed in hedonic and metabolic centers such as the VTA/SNc and LHA. Other regions\nof significance such as the CeA, BNST and LC are also included with relevance to feeding regulation. C)\nQuantitative assessment of inputs to all regions illustrates which constitute the main projection sites. Scale bar\n= 200uM\nTo address this limitation transparently\n• We have now revised the manuscript\ntext to refer to “DRN/vlPAGVGAT\nAq\nneurons” when describing this\nmanipulation in Figure 1.\n• We have clarified this point in\nthe Methods and Main sections.\nDRN\n• While we did not include histology in\nthe original submission, we are\ncurrently compiling representative\nexpression maps and can include it as\na new Extended Data Figure upon\nrequest (Rebuttal Figure 9).\nAgain, we thank the reviewer for this\nvaluable comment and for helping us\nimprove our work's anatomical and\ninterpretive precision.\n50μm\nFigure 9. Representative targeting of vlPAG/DRN Vgat neurons.\nRepresentative image of Vgat-Cre mice injected with an hM4Di-\nmCherry adeno-associated virus to illustrate targeting region for our\nchemogenetic studies. Scale bar = 50um.\n6) Given that only 25% of DRN VGAT neurons are activated by HFD and that there are likely multiple types of\nDRN VGAT neurons, the rationale for using translating ribosomal affinity purification is unclear. A single cell\nRNA-Seq or spatial transcriptomic approach in HFD fed vs control mice could have shown cell type enrichment\nof VGAT and Fos (or other activity dependent markers). This type of approach would also allow for identification\nof the non-VGAT Fos-activated neurons in the region.\nWe thank the reviewer for this insightful comment. We fully agree that DRNVGAT neurons are a heterogeneous\npopulation. Only a subset (~25%) appears to be activated by HFD, as we observed in our Fos labeling studies.\nGiven the flaws of double-staining using FISH and immunohistochemistry, we have performed a new series of\nstainings using vGat-Cre::GFPL10a to increase the accuracy of Fos labeling in VGAT neurons and found that\napproximately 75% of VGAT cells co-localize with Fos after exposure to HFD. We also appreciate the\nsuggestion that single-cell RNA-seq or spatial transcriptomics could provide a more granular map of activity-related\ntranscriptional changes across cell types in the dorsal raphe region.\nWe selected translating ribosomal affinity purification (TRAP) for this study because it offers cell-type–specific\nmolecular profiling of mRNAs actively being translated in defined genetic populations—in this case, Vgat-\nexpressing neurons. Our goal was to identify druggable molecular targets enriched in DRNVGAT neurons,\nindependent of their real-time activation state, to enable the development of selective pharmacological tools. Of\nnote, this approach was successfully published in our previous work (PMID: 36411386). This approach aligned\nwell with our experimental pipeline, which targeted a stable molecular feature (GPR6) rather than transient\nactivity markers.\nWhile we acknowledge that TRAP does not resolve cellular heterogeneity or capture dynamic activation states\nat single-cell resolution, it was a practical and hypothesis-driven choice that successfully identified GPR6 as a\ncandidate for functional interrogation. Indeed, we believe that the strength of this study lies in integrating TRAP-\nderived target identification with in vivo functional validation via electrophysiology, pharmacology and\nchemogenetics (Figures 3 and 4).\nThat said, we fully agree with the reviewer that spatial transcriptomics or single-cell RNA-seq approaches—\nparticularly in HFD-fed animals—could uncover:\n• Distinct molecular subtypes of DRNVGAT neurons are involved in specific aspects of energy balance;\n• The identity of non-VGAT, Fos-activated populations (e.g., serotonergic, glutamatergic) within the DRN\nand surrounding regions.\nHowever, with regards to druggable targets a TRAP based pulldown reaches deeper depth in sequencing at the\nlevel of gene encoding plasma membrane GPCRs.\nWe are exploring spatial transcriptomic tools in ongoing studies and appreciate the reviewer’s encouragement\nto expand on this line of investigation.\nWe thank the reviewer again for this thoughtful comment, which helped us better clarify our approach's rationale.\n7) Related to above, if the goal is to find a druggable target that is enriched in DRN VGAT neurons, Gpr6 seems\nto fall short of this goal if only 50% of Gpr6-expressing neurons are VGAT positive. In addition, based on\nMERFISH data from the Allen Brain institute, Gpr6 is expressed in nearby vlPAG neurons that are also VGAT\npositive.\nWe thank the reviewer for this critical observation and agree that Gpr6 is not exclusively expressed in DRNVGAT\nneurons but also in VGAT-positive neurons within the adjacent vlPAG, as confirmed by the ABA MERFISH data.\nIn light of this, and as noted in our response to Comment 5, we have revised the manuscript to refer to\n“vlPAG/DRNVGAT neurons” when describing the population of interest. This terminology more accurately reflects\nthe anatomical overlap and ensures clarity regarding the likely cellular targets of CVN527.\nWhile Gpr6 may not be uniquely specific to DRNVGAT neurons, we believe its enrichment in this region, combined\nwith the potent and selective phenotype observed with local pharmacological inhibition, supports its relevance\nas a functionally meaningful and tractable target. Importantly, we demonstrate that modulating Gpr6-expressing\nneurons within the vlPAG/DRN axis is sufficient to enhance thermogenesis and attenuate weight gain, making\nthis pathway of interest for therapeutic development. Similarly, GLP1R in the AP/NTS, with a huge success in\nweight loss therapeutics, is only expressed in ~30% of the cells (PMID:38987598).\n8) Since approximately half of Gpr6-expressing neurons in the DRN express VGAT, the use of VGAT-IRES-\nCre::-L10-GFP mice for electrophysiological assessment of CVN527 in DRN slices is problematic. CVN527\ninhibited the low firing group, but looks to be potentiating the high firing group, though not significant with low n\nof 4. An approach where recordings can be done from known DRN Gpr6-positive and Gpr6-negative neurons is\nnecessary for testing this newly developed drug.\nWe appreciate the reviewer’s detailed critique of our electrophysiological approach and fully agree that identifying\nthe precise subpopulation of drug-responsive neurons—specifically, Gpr6-positive vs. Gpr6-negative—is an\nimportant goal for future work. However, we would like to respectfully push back on the implied requirement that\nrecordings be performed from Gpr6-identified neurons at this stage.\nAt present, there is no existing Cre or reporter mouse line for Gpr6, and generating a Gpr6-Cre or Gpr6-Flp\nknock-in line would be a technically and temporally intensive undertaking, requiring 1–2 years of development,\ncharacterization, and breeding before functional use. While such a tool would indeed provide ideal specificity,\nwe believe it is unrealistic to expect this level of genetic resolution for an initial validation of a newly identified\npharmacological target.\nOur use of Vgat::GFP-L10 mice was based on our established finding that Gpr6 is enriched within the VGAT+\npopulation of the DRN/vlPAG, and the goal of our recordings was to test whether CVN527 modulates the firing\nproperties of this physiologically relevant and genetically accessible population. The observed suppression of\nactivity in the low-firing group is consistent with an inverse agonist effect on neurons with higher baseline Gpr6\nsignaling, which aligns with the behavioral thermogenic phenotype.\nWe believe that the electrophysiology experiments included here provide meaningful, though preliminary, insight\ninto the cellular action of CVN527, and that the absence of a Gpr6-specific mouse line should not be viewed as\na limitation undermining the broader significance of the findings.\nWe thank the reviewer for their thoughtful suggestion and have revised the manuscript to clearly state the current\nlimitations and future directions for improving cell-type resolution.\n9) DRN infusion or orally ingested, peanut butter supplemented CVN527 attenuated body weight gain induced\nby a high-fat diet, but there was no effect on food intake. This is inconsistent with earlier DREADD inhibition\nstudies in Figure 1 and in prior publications by the authors. The effects on VO2, VCO2, and energy expenditure\nalso different compared to chronic DREADD inhibition. This raises the possibility that the CVN527 treatment\nis acting on non-VGAT neurons in the region to produce its effects.\nWe thank the reviewer for bringing this concern to our attention. Our interpretation, similar to Reviewer 2 Point\n5, is that in the global chemogenetic approach, which likely silences all DRNVgat neurons (both GPR6-positive\nand -negative subpopulations) through a bulk effect, produces a broader phenotype that includes feeding\nsuppression because it targets a larger group of Vgat cells. Conversely, the CVN527 compound only affects a\nGPR6-positive subcluster responsible for the thermogenic effect.\nThis is interesting, as it highlights the existence of different subpopulations in the vlPAG/DRN region with\ninhibitory Vgat identity, which mediate either feeding or thermogenic responses in a coordinated manner. Similar\nfindings are observed in other brain nuclei, such as the paraventricular hypothalamus, where TRH-expressing\nneurons regulate thermogenesis and MC4R neurons control feeding responses, both of which are\nglutamatergic.\nWe have expanded the Main section, mentioning the likely existence of different subclusters of DRNVgat\nneurons.\n10) Line 377 states, “We have previously shown that reducing tonic GABAergic input from DRN onto the raphe\npallidus (RPa) increases sympathetic tone to BAT”, and includes reference 18. In this reference, the authors\ninhibited all DRN VGAT neurons and showed increased core temperature, but not BAT temperature. Further,\nthey did not specifically inhibit DRN VGAT projections to the RPa in this study. They did, however, specifically\nactivate RPa-projecting DRN VGAT neurons and showed a decrease in BAT thermogenesis, which they also\nobserved when stimulating DRN VGAT projections to the forebrain (MPA, BNST, and DMH).\nWe thank the reviewer for this accurate and important clarification. The reviewer is correct that in reference 18,\nour prior study demonstrated that global inhibition of DRNVGAT neurons increased core temperature, but did not\ndirectly measure BAT temperature, nor did we selectively inhibit DRNVGAT projections to the RPa.\nAs the reviewer notes, we instead optogenetically activated RPa-projecting DRNVGAT neurons, which resulted in\na decrease in BAT thermogenesis, providing functional evidence that this pathway can suppress sympathetic\noutput to BAT. A similar suppressive effect was observed upon activating projections to the MPA, BNST, and\nDMH.\nIn light of this clarification, we have revised the statement in Line 472 to reflect the data and interpretation\nmore accurately. The sentence now reads:\n“We have previously shown that activating GABAergic input from DRN onto the raphe pallidus (RPa) decreases\nBAT temperature”\nWe also updated the relevant discussion paragraph to more clearly distinguish between global manipulation and\nprojection-specific effects.\nWe sincerely thank the reviewer for pointing out this discrepancy, which helped us improve the accuracy and\nscientific precision of our manuscript.\n11) It is stated that CVN527 treatment was not associated with increased anxiety in behavioral tests; however,\nthere is a significant decrease in center time in the open field and a decreased open are entry in the elevated\nplus maze. The n is also low in these tests, thus there may actually be some anxiety related effects of CVN527.\nWe thank the reviewer for this careful observation and agree that the behavioral interpretation based on the initial\nsmall sample size warranted further investigation.\nTo address this, we have now expanded the behavioral study to include 10 mice per group, increasing statistical\npower across the open field test (OFT), elevated plus maze (EPM), and marble burying test. With this updated\ncohort:\n• No significant differences were observed in anxiety-related behaviors across any of the three paradigms.\n• In the OFT, while center time and entries were comparable between groups, we observed a significant\nincrease in total locomotion and average velocity in CVN527-treated mice, suggesting a mild hyper\nlocomotor effect rather than increased anxiety per se.\n• In the EPM and marble burying test, no differences in open arm entries, time spent in open arms, or\nnumber of buried marbles were observed between treated and control animals.\nWe have now updated the Main section and Extended Data Figure 5 (Rebuttal Figure 10) to reflect these\nexpanded data. We have revised the text to clarify that CVN527 does not induce anxiety-like behavior under the\nconditions tested but increases exploratory activity.\nWe thank the reviewer for prompting us to strengthen this part of the study and improve the interpretive rigor of\nour behavioral analysis.\n3\n80\n60\n40\n20\n)m(\ndevom\necnatsiD\nCenter\n200\n150\n100\n50\n)s(\nnoitarud\nevitalumuC\nPeriphery\nns 600\n400\n200\n)s(\nnoitarud\nevitalumuC\nns\n2500\n2000\n1500\n1000\n500\n)mc(\ndevom\necnatsiD\n8\n6\n4\n2\nns\n)s/mc(\nyticoleV\nClosed arms\n50\n40\n30\n20\n10\nycneuqerF\nClosed arms\nns\n250\n200\n150\n100\n50\n)s(\nnoitarud\nevitalumuC\nns\nOpen arms\n8\n6\n4\n2\nycneuqerF\nOpen arms\nns\n15\n10\n5\n0\n-5\nControlTreated\n)s(\nnoitarud\nevitalumuC\nns\nLatency Time\n40\n30\n20\n10\n)s(\nemiT\n25\n20\n15\n10\n5\ndeirrub\nselbraM\nrebmuN\nDuration Digging\n200\n150\n100\n50\n)s(\nnoitaruD\nControl\nA\nCVN527\nB\nControl\nCVN527\nC\nFigure 10. Behavioral phenotyping as side-effect testing. B6 mice were treated with either vehicle or\nCVN527 (10mg/kg) in peanut butter and behavioral assessment for Open-field-test (A), Elevated Plus\nMaze (B) and Marble Burying (C) was conducted. A) (Left to right) Behavioral response tracking, distance\ntraveled, cumulative duration in the center and periphery of control and CVN527 treated mice. B) (Left to\nright) Behavioral response tracking, velocity and distance moved, frequency in closed and open arms,\nand cumulative duration in closed and open arms of vehicle or CVN treated mice. C) Marble burying test\nscheme, number of marbles buried, latency to first burying and duration of digging in vehicle and CVN\ntreated mice. *p<0.05, **p<0.01 n = 10 mice per group.\nPoint by point response:\nReviewer #1 (Remarks to the Author):\nAll my earlier comments have been resolved. The manuscript is original, contributes meaningful\ndata, and is supported by a robust multidisciplinary methodology. Overall, it is an excellent study.\nWe sincerely thank the reviewer for their positive evaluation and supportive comments.\nReviewer #2 (Remarks to the Author):\nThe revised manuscript has addressed the majority of my concerns. However, my enthusiasm is\ntempered by the lack of a synergistic effect on body weight loss when CVN527 is co-administered\nwith semaglutide. It appears that CVN527 itself has only a very subtle effect in the DIO mouse\nmodel (Rebuttal Figure 3). Furthermore, if the original study tested CVN527 in a preventive\nsetting by switching mice from a chow to a high-fat diet at the time of compound administration,\nthen these mice should not be referred to as \"diet-induced obese (DIO)\". Consequently, it is\ninaccurate to state that the compound \"significantly reduced weight gain in DIO mice.\nWe thank the reviewer for the constructive comments and careful reading of our revision.\nRegarding the lack of a synergistic effect with Semaglutide, not always do dual agonists\noutperform GLP-1R mono-agonism in pre-clinical models, especially considering the strong\nappetite-suppressant effects of GLP-1R agonists (PMID: 31186439, PMID: 40399267).\nAdditionally, we performed this drug trial in mice fed a HFD for 16 weeks, and the pro-\nthermogenic effect of CVN527 might be impaired due to BAT dysfunction in these obese mice\n(PMID: 37318951, 26884382).\nWe also appreciate the reviewer’s point about terminology. In the preventive study, animals\nwere switched from chow to a high-fat diet at the time of compound administration. We have\ncorrected the text to avoid referring to these animals as “diet-induced obese (DIO)” and now our\nmanuscript states: “We next developed and administered a potent and highly selective GPR6\ninverse agonist, which significantly prevented weight gain in male mice exposed to a high-fat diet\n- line 58“\nThe revised language now clearly distinguishes between preventive and interventional settings.\nWe thank the reviewer again for this feedback, which has improved both the accuracy and\nprecision of our manuscript.\nReviewer #3 (Remarks to the Author):\nThe authors were responsive, and the revised manuscript by Moura-Assis et al. is significantly\nimproved. However, there are a few remaining comments/clarifications/concerns.\n1) Given the vast expression of VGAT in and around the DRN, the authors should either include\nrepresentative histology showing hM4Di expression in Figure 1 or a more extensive histological\nassessment as an extended figure, as the authors propose.\nWe thank the reviewer for this helpful suggestion.\nIn response, we have now included a representative histological image of hM4Di expression in\nthe DRN in Figure 1, as requested (Rebuttal Figure 1 and Figure 1C in the manuscript). This image\nbetter represents the specificity of hM4Di targeting within the DRN/vlPAG.\nRebuttal Figure 1. Representative targeting of vlPAG/DRN Vgat neurons. Representative\nimage of Vgat-Cre mice injected with an hM4Di-mCherry adeno-associated virus to illustrate\ntargeting region for our chemogenetic studies.\n2) My original comment #7 about only 50% of Gpr6-expressing neurons being VGAT positive was\npartially misinterpreted. I apologize if I was not clear. The authors comment on the phenotype\nobserved with local pharmacology and provide the example of GLP1R agonists targeting a small\npopulation of AP/NTS neurons. I agree with their response, however; my intent was to raise the\nissue that the effects the authors see may not be mediated completely by VGAT neurons since\nhalf of the Gpr6-positive neurons do not express VGAT. CNV527 obviously cannot target only the\nVGAT neurons that express Gpr6, so how do the authors know that it’s the VGAT neurons that\nare responsible for the effects they are seeing? Perhaps CVN527 causes its effects on energy\nbalance through both VGAT neurons and some other population. This is further supported by the\nfact that CVN527 treatment does not fully recapitulate the different phenotypes observed with\nchemogenetic inhibition of DRN VGAT neurons (e.g., chemogenetic inhibition reduces feeding).\nThus, the authors should revise the text after line 444 (“Importantly, our findings suggest…”) to\ninclude the possibility that CVN527 may produce some of its effects via non-VGAT neurons.\nWe thank the reviewer for clarifying this important point. We agree that, given only ~50% of\nGpr6-expressing neurons are VGAT-positive, CVN527 may exert its effects on energy balance\nthrough both VGAT and non-VGAT neuronal populations.\nWe have revised the text to acknowledge this possibility explicitly and to note that the\npharmacological effects of CVN527 may extend beyond VGAT neurons, thereby accounting for\ndifferences relative to the phenotypes observed with selective chemogenetic inhibition of DRN\nVGAT neurons.\nNow it reads: “Furthermore, the pro-thermogenic effect of CVN527 may also be related to the\nactivation of GPR6 in non-VGAT neurons.”\n3) The electrophysiology study is still problematic.\nFirst, it is not required to generate a Gpr6-Cre mouse to know which neuron subtypes in the\nregion express the receptor. I did not mean for this to be my takeaway message as I agree with\nthe authors that generating a new mouse line requires considerable time. Instead, there are likely\nalternative Cre lines available that could be used as a proxy for Gpr6-positive neuron types, if a\ngenetic marker were deciphered by analyzing single-cell RNA-seq data. This is one of the reasons\nI suggested such a study in a different comment. Further, instead of running a new study, the\nauthors could utilize publicly available scRNA-seq data from the Allen Brain Institute or other\npublications (PMID: 31411560, biorxiv.org/content/10.1101/2022.06.27.497769v2)\nto find such a marker.\nWe thank the reviewer for this clarification and for the thoughtful suggestions. Using scRNA-seq\nanalysis, we examined overlapping markers that could inform the use of alternative Cre lines as\nproxy markers for GPR6.\nIn our initial examination of available DRN single-cell datasets (PMID: 31411560), Gpr6\nexpression was observed in only a very small number of cells (27 out of approximately 2,000)\n(see Rebuttal Figure 2A and B). We believe this likely reflects the well-known limitations of older\nsingle-cell technologies, especially their high dropout rates for genes expressed at lower levels.\nWe then examined another study that sequenced serotonin neurons in the DRN (PMID:\n31647409). However, the GPR6 expression level in this dataset was also low and inconclusive\n(Figure 2 C and D). Therefore, while these datasets can reliably identify abundant markers, they\nare less dependable for receptors like Gpr6 that may be expressed near the detection threshold.\nFor this reason, we could not find a suitable proxy for Gpr6-positive neurons.\nRebuttal Figure 2. (A and C) UMAP representation of publicly available single-cell RNA-seq\ndatasets from the dorsal raphe nucleus (DRN); (B and D) Gpr6 expression across correspondent\ndataset, showing overall low transcript detection with enrichment in GABAergic and\ndopaminergic neuronal clusters.\nSecond, even in the absence of an improved method of recording from Gpr6-expressing neurons,\nthe issue of CVN527 increasing the firing rate of high-frequency firing DRN VGAT neurons remains\nunaddressed. There were 11 recordings from low-firing DRN VGAT neurons, and CVN527\nreduced firing by approximately 33% at current injections of 0 pA and above. Of note, the\nlegend in this figure says the statistical test performed was a paired t-test, which is\ninappropriate given there were repeated measures and would require multiple t-tests. Are the p-values\nreported adjusted for multiple comparisons? Meanwhile, CVN527 increased firing at current\ninjections at or above 100 pA, reaching a nearly two-fold increase in action potential firing at\ncurrents of 140 pA and above. However, there is no mention of the increased firing rate in these\nrecordings in the text, probably because there was no statistical significance – but this was likely\na result of only having 4 recordings from high-firing DRN VGAT neurons. Together, there seems to\nbe justification for more recordings, revision of the text related to the electrophysiology studies,\nor both.\nWe thank the reviewer for raising this important point. We agree that the small number of\nrecordings from high-frequency firing DRN VGAT neurons (n=4) limits the statistical power to\ndetect effects of CVN527 on this subpopulation. We have now revised the text to explicitly\nacknowledge the observed trend toward increased firing at current injections ≥100 pA:\n“Interestingly, we found that a small proportion of vGat neurons (n=4) with a high-firing baseline\nrate showed no response to CVN527 and exhibited a trend towards increased firing frequency”.\nWe would also like to note that previous studies have shown that the firing properties of DRN\nneurons are electrophysiologically heterogeneous (PMID: 11325353; PMID: 14573555;\nPMID: 23275149). For example, PMID: 23275149 reported that GAD67⁺ neurons in the DRN\nexhibit relatively low firing frequencies comparable to our low-firing VGAT population, whereas\nGAD67⁻ neurons display higher firing frequencies that are more comparable to our high-firing\nVGAT neurons. Future work will be needed to determine which subtypes of DRNVGAT neurons\nexpress the GPR6 receptor, to elucidate the local connectivity between low- and high-firing VGAT\nneurons, and to test whether the observed firing increase in high-firing VGAT neurons following\nCVN527 is an indirect consequence of decreased activity in the low-firing population and\ndisinhibition of high-firing VGAT neurons. We have also included this possibility in the\nmanuscript: “We speculate that this subpopulation of vGat neurons may have been disinhibited by\nlow-firing vGat neurons that CVN527 directly suppressed”. Therefore, we also highlight this as\nan area for future work, as additional recordings will be necessary to definitively assess CVN527’s\neffects across DRN VGAT neuronal subtypes, using distinct cell-type markers as proxies for Gpr6-\npositive neurons.\nConcerning statistical testing, we acknowledge the reviewer’s concern and appreciate the\nopportunity to clarify. The original analysis used paired t-tests at each current step; however, we\nagree that this approach does not fully account for repeated measures. We have now re-analyzed\nthe data using a repeated-measures ANOVA with post hoc corrections for multiple comparisons.\nThe revised p-values are reported in the updated figure legend (Figure 3G). Importantly, the\noverall conclusion—that CVN527 reduces firing in low-frequency DRN VGAT neurons within a\nmargin of significance —remains unchanged after this correction.\nWe thank the reviewer for this critical feedback, which has improved both the transparency and\nrigor of our analysis.\nLi YQ, Li H, Kaneko T, Mizuno N. Morphological features and electrophysiological properties of\nserotonergic and non-serotonergic projection neurons in the dorsal raphe nucleus. An\nintracellular recording and labeling study in rat brain slices. Brain Res 900:110–118, 2001.\nS. G. Beck, Y-Z. Pan, A. C. Akanwa, and L. G. Kirby. Median and dorsal raphe neurons are not\nelectrophysiologically identical. Journal of Neurophysiology, 91(2):994–1005, 2004.\nGocho, Y., Sakai, A., Yanagawa, Y., Suzuki, H., and Saitow, F. (2013). Electrophysiological and\npharmacological properties of GABAergic cells in the dorsal raphe nucleus. J. Physiol. Sci. 63, 147–\n154. doi: 10.1007/s12576-012-0250-7"}, {"round": 2, "reviewer_comments": {"reviewer_2": "I have no further comments.", "reviewer_3": "All my comments have been addressed."}, "author_response": ""}]

Decision letter