biostudies-arrayexpressBrian GuralMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17566Glucagon-like peptide-1 receptor (GLP1R) agonists such as semaglutide reduce food intake and body weight, and Glp1r-expressing neurons in the hypothalamus and hindbrain mediate these effects. To profile these cells directly, Glp1r-Cre mice were crossed to the Gt(ROSA)26Sor-CAG-Sun1-sfGFP reporter, which labels the nuclear envelope of Glp1r-expressing cells with GFP. The hypothalamus, dorsal vagal complex, and nodose ganglion were dissected and flash-frozen; tissue was pooled from two or more mice of both sexes per library. Nuclei were isolated, stained with propidium iodide, and GFP+/PI+ nuclei collected by fluorescence-activated sorting. Libraries were prepared using the 10x Genomics Chromium Single Cell 3' v3 platform, targeting approximately 10,000 recovered nuclei per library, and sequenced on an Illumina NovaSeq 6000 as 151 bp paired-end reads. Reads were mapped with Cell Ranger 6.0.0 to the mm10-2020-A reference. Each library contains nuclei from all three regions; region of origin was assigned informatically rather than by separate library preparation. Animals received semaglutide or vehicle, indicated per sample, though treatment was not a variable in the reported analysis, which used the data to identify cell-type marker genes.biostudies-arrayexpressGrowth Protocol - Glp1r-Cre mice (Jackson 029283) were crossed to the Gt(ROSA)26Sor-tm5.1(CAG-Sun1-sfGFP)Nat reporter (Jackson 021039). Mice were housed in specific-pathogen-free, temperature-controlled (22 C) rooms on a 12 hour light/dark cycle with Purina Lab Diet 5LOD and water available ad libitum. Animals were on a segregating C57BL/6;SJL;129 background. Both male and female mice were used.Sample Collection - Mice were euthanized with isoflurane and decapitated. Brains were removed and sectioned into 1 mm coronal slices using a brain matrix; the hypothalamus and dorsal vagal complex were dissected, and nodose ganglia were collected. Tissue was flash-frozen in liquid nitrogen and pooled from two or more mice per library.Nucleic Acid Extraction - Frozen tissue was homogenized in lysis buffer (EZ Prep Nuclei Kit, Sigma) with Protector RNase Inhibitor and filtered through a 30 µm MACS strainer. Nuclei were pelleted at 500 rcf for 5 min, resuspended in wash buffer (10 mM Tris pH 8.0, 5 mM KCl, 12.5 mM MgCl2, 1% BSA, RNase inhibitor), strained and re-pelleted. Nuclei were stained with propidium iodide and sorted on a MoFlo Astrios cell sorter; GFP+/PI+ double-labelled nuclei were collected, pelleted at 100 rcf for 6 min, and resuspended at 750-1200 nuclei/uL.Library Construction - Reverse transcription mix was added targeting approximately 10,000 recovered nuclei and loaded onto the 10x Chromium Controller. Reverse transcription, cDNA amplification and library preparation used the Chromium Single Cell 3' Library and Gel Bead Kit v3, Chromium Chip B Single Cell kit, and Chromium i7 Multiplex Kit according to manufacturer instructions. Each library was generated from nuclei pooled across multiple male and female mice; libraries do not represent individual animals (n >= 2 per library).Sequencing - Libraries were sequenced on an Illumina NovaSeq 6000, paired-end with 151 bp reads. bcl2fastq2 Conversion Software (Illumina) was used to generate demultiplexed FASTQ files.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - FASTQ files were processed with Cell Ranger 6.0.0 against the refdata-gex-mm10-2020-A mouse reference with intron inclusion enabled.Data Transformation - Cell Ranger output was analysed in R. Genes expressed in at least 5 cells were retained, as were cells with at least 600 detected genes. Doublets were scored using Scrublet; clusters with a median doublet score above 0.3 and individual cells scoring above 0.3 were removed. Data were normalized using scran, then centred and scaled for each dataset independently. Genes called variable by Seurat FindVariableFeatures were input to principal component analysis, and the top principal components were retained at the elbow of the scree plot (typically 15-30 depending on the dataset). These were used for dimension reduction by UMAP and clustering with the Seurat FindNeighbors and FindClusters functions. FindClusters was optimized for cluster consistency by varying the resolution parameter from 0.2 upward in steps of 0.2 until a maximal mean silhouette score was found. Clusters were hierarchically ordered by Euclidean distance in principal component space. Cell types were identified by projecting labels from published single-cell RNA-seq datasets using the Seurat CCA method. Neuron cluster names were chosen from genes found in an unbiased marker gene search (Seurat FindMarkers); clusters without unique marker genes were labelled by neurochemical identity (GABA or GLU) plus a number.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000GLP-1 receptor agonists (GLP1RAs) effectively reduce feeding to treat obesity, although nausea and other aversive side effects of these drugs can limit their use. Brainstem circuits that promote satiation and that mediate the physiologic control of body weight can be distinguished from those that cause aversion. It remains unclear whether brainstem <i>Glp1r</i> neurons contribute to the normal regulation of energy balance and whether GLP1RAs control appetite via circuits distinct from those that mediate aversive responses, however. Hence, we defined roles for AP and NTS <i>Glp1r</i>-expressing neurons (AP^Glp1r and NTS^Glp1r neurons, respectively) in the physiologic control of body weight, the GLP1RA-dependent suppression of food intake, and the GLP1RA-mediated stimulation of aversive responses. While silencing non-aversive NTS^Glp1r neurons interfered with the physiologic restraint of feeding and body weight, restoring NTS^Glp1r neuron <i>Glp1r</i> expression on an otherwise <i>Glp1r</i>-null background failed to enable long-term body weight suppression by GLP1RAs. In contrast, selective <i>Glp1r</i> expression in AP^Glp1r neurons restored both aversive responses and long-term body weight suppression by GLP1RAs. Thus, while non-aversive NTS^Glp1r neurons control physiologic feeding, aversive AP^Glp1r neurons mediate both the anorectic and weight loss effects of GLP1RAs, dictating the functional inseparability of these pharmacologic GLP1RA responses at a circuit level.RNA-seq of coding RNA from single cellsMus musculusA single dorsal vagal complex circuit mediates the aversive and anorectic responses to GLP1R agonistsBrian GuralMartin MyersTune PersWarren T. Yacawych, Yi Wang, Guoxiang Zhou, Shad Hassan, Stace Kernodle, Frederike Sass, Martin DeVaux, Iris Wu, Alan Rupp, Abigail J. Tomlinson, Zitian Lin, Anna Secher, Kirsten Raun, Tune Pers, Randy J. Seeley, Martin Myers, Weiwei QiuWeiwei QiuShad HassanfalseSingle-nucleus RNA-seq of FACS-enriched Glp1r-Sun1-sfGFP nuclei from mouse hypothalamus, dorsal vagal complex, and nodose ganglionGlucagon-like peptide-1 receptor (GLP1R) agonists such as semaglutide reduce food intake and body weight, and Glp1r-expressing neurons in the hypothalamus and hindbrain mediate these effects. To profile these cells directly, Glp1r-Cre mice were crossed to the Gt(ROSA)26Sor-CAG-Sun1-sfGFP reporter, which labels the nuclear envelope of Glp1r-expressing cells with GFP. The hypothalamus, dorsal vagal complex, and nodose ganglion were dissected and flash-frozen; tissue was pooled from two or more mice of both sexes per library. Nuclei were isolated, stained with propidium iodide, and GFP+/PI+ nuclei collected by fluorescence-activated sorting. Libraries were prepared using the 10x Genomics Chromium Single Cell 3' v3 platform, targeting approximately 10,000 recovered nuclei per library, and sequenced on an Illumina NovaSeq 6000 as 151 bp paired-end reads. Reads were mapped with Cell Ranger 6.0.0 to the mm10-2020-A reference. Each library contains nuclei from all three regions; region of origin was assigned informatically rather than by separate library preparation. Animals received semaglutide or vehicle, indicated per sample, though treatment was not a variable in the reported analysis, which used the data to identify cell-type marker genes.2026-09-14T00:00:00Z2026-09-17T10:39:18.137Z2026-09-04T11:06:23.202ZE-MTAB-17566ERP204940EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_000418410.1101/2025.01.21.634167