Genetic Identification of Vagal Sensory Neurons That Control Feeding

Abstract

Energy homeostasis requires precise measurement of the quantity and quality of ingested food. The vagus nerve innervates the gut and can detect diverse interoceptive cues, but the identity of the key sensory neurons and corresponding signals that regulate food intake remains unknown. Here, we use an approach for target-specific, single-cell RNA sequencing to generate a map of the vagal cell types that innervate the gastrointestinal tract. We show that unique molecular markers identify vagal neurons with distinct innervation patterns, sensory endings, and function. Surprisingly, we find that food intake is most sensitive to stimulation of mechanoreceptors in the intestine, whereas nutrient-activated mucosal afferents have no effect. Peripheral manipulations combined with central recordings reveal that intestinal mechanoreceptors, but not other cell types, potently and durably inhibit hunger-promoting AgRP neurons in the hypothalamus. These findings identify a key role for intestinal mechanoreceptors in the regulation of feeding.

Keywords: AgRP Neurons; RNA sequencing; chemogenetics; fiber photometry; hypothalamus; optogenetics; satiation; stretch; vagal afferents; vagus nerve.

Figure 1.. Anatomical characterization of vagal sensory neurons that innervate the GI tract

(A) Anterograde tracing strategy. (B-C) Schematic for the distribution (B) or morphology (C) of vagal sensory terminals. (D-H) Whole-mount (D-F and H, top-down view) and cross sections (G) showing the innervation of tdTomato⁺ vagal sensory terminals (magenta) in different tissues. Autofluorescence in the UV channel (gray) shows villi or crypts. FluoroGold (green) labels enteric neurons. (I-L) Distribution of tdTomato⁺ vagal sensory terminals (mean ± SEM). (M) Schematic of the two ways vagal-GI innervation could be organized. (N-O) Whole-mount nodose ganglion showing segregation of vagal sensory neurons that are retrogradely labeled from different GI targets (N) and quantification (O). Scale bar: 100 μm. See also Figure S1.

Figure 2.. Target-scSeq identifies vagal cell types innervating distinct visceral organs

(A) Target-scSeq strategy. (B-C) Spectral tSNE colored by gene-based clusters (B) or retrograde-tracing targets (C). (D-E) Percentage of cells retrogradely labeled from different targets within individual clusters (D), or vice versa (E). (F) The relative expression of subtype-enriched genes (rows) across cells sorted by cluster (column). (G) Dendrogram showing relatedness of clusters, followed by violin plots showing the expression of cluster-specific marker genes. (H) Immunostaining reveals the overlap between vagal cell-type markers (magenta) and retrograde tracer (green). (I) RNAscope reveals that Gpr65⁺ cells are partially labeled by Dbh (t10-t12) and Edn3 (t09). (J) Quantification of (H), showing the percentage of stomach- or intestine-projecting cells that express each marker gene (mean ± SEM). (K) Quantification of (I), n = 4 mice. Scale bar: 100 μm. See also Figure S2.

Figure 3.. Comparison of whole-nodose and target-scSeq reveals the global organization of vagal sensory subtypes

(A) Spectral tSNE plot shows gene-based clusters, combining GI target-scSeq and unbiased whole-nodose scSeq. (B-C) tSNE plots (left) or stacked bar graphs (right) showing neurons in each cluster that originated from individual target-scSeq clusters (B), or were labeled from individual GI targets (C). (D-H) Dendrogram showing relatedness of clusters (D), followed by violin plots showing gene expression across clusters of combined nodose scSeq (top, n1-n27) and GI target-scSeq (bottom, t01-t12) (E-H). Genes included are cluster markers of combined scSeq (E), markers enriched or excluded in subdiaphragmatic clusters (F), markers used for later anatomical characterizations (G), and genes listed in prior literature (H). Blue shadow marks putative subdiaphragmatic clusters from the combined whole-nodose scSeq. See also Figure S3.

Figure 4.. Genetic markers identify vagal subtypes with unique morphologies and innervation patterns

(A-F) Distribution of gastric mucosal endings (A-D), intestinal mucosal endings (C-D), and gastrointestinal IGLEs (E-F) labeled by seven nodose-subtype Cre lines. (A, C, E) Schematic of distribution. (B, D, F) Top-down view of whole-mount GI tissue. Magenta: tdTomato⁺ vagal sensory terminals. Gray: villi visualized by the autofluorescence in UV channel. Green: FluoroGold-labeled enteric neurons. (G) Quantification of mucosal ending- and IGLE-distributions labeled by vGlut2^Cre, Nav1.8^Cre, and the seven nodose-subtype Cre lines (mean ± SEM). Scale bar: 100 μm. See also Figure S4.

Figure 5.. Activation of gastrointestinal mechanoreceptors potently inhibits food intake

(A) Expression of hormonal receptors and Trpv1 across target-scSeq clusters. (B) Summary of the four vagal Cre lines for functional analysis. (C) Optogenetic activation strategy. (D-E) ChR2-mCherry expression in the vagal sensory cell bodies (D) and their central terminals in the NTS and AP (E). (F-I) Cumulative food intake (F-H) or water intake (I) comparing trials with and without photostimulation across the four vagal-ChR2 lines and control (no ChR2). Blue indicates the period of photostimulation. (J) Chemogenetic activation strategy. (K) hM3D-mCherry expression in the vagal sensory cell bodies. (L-O) Cumulative food intake (L-N) or water intake (O) comparing trials with CNO or saline treatment across the four vagal-hM3D lines and control (no hM3D). (P) Place-preference assay. (Q-R) Percentage of time spent in photostimulation-paired chamber, comparing baseline and the third stimulation session, using fasted (Q) or fed (R) mice. (S-T) Cumulative food intake (S) or water intake (T) tested 30min after CNO or saline treatment. Error bars and shaded areas represent mean ± SEM. N mice is annotated within figures. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-way ANOVA, Sidak correction. Scale bar: 100 μm. See also Figure S5.

Figure 6.. Stimulation of gastrointestinal mechanoreceptors modulates feeding centers in the brain

(A-G) Immunostaining of Fos and Th in the NTS/AP (A-B), Fos and Calca in the PBN (C-D), and quantification (E-G), comparing Oxtr-hM3D and control mice after CNO treatment. (H) Concurrent Arc-photometry recording and vagal sensory neuron activation (left). The expression of GCaMP6m is restricted within the Arc (right). (I-J) Normalized AgRP neuron calcium signal in fasted mice presented with chow and caged chow (I) and quantification (J). (K-O) Normalized AgRP neuron calcium signal in fasted mice after CNO or saline treatment (top) and quantification (bottom), across the four vagal-hM3D lines or control. Values are reported as mean ± SEM. Comparisons were made between groups (*P < 0.05, **P < 0.01, ***P < 0.001) or from baseline (^#P < 0.05, ^##P < 0.01, ^###P < 0.001), two-way ANOVA, Sidak correction. Scale bar: 100 μm (A-D) or 1 mm (H). See also Figure S6.

Figure 7.. Gastrointestinal distension is sufficient to inhibit food intake and regulate AgRP neuron activity.

(A) Normalized weight of GI contents measured 5 min after sham treatment or oral gavage of 500 uL of various solutions. (B-C) Cumulative (B) and total (C) food intake of fasted mice after oral gavage of 500 uL of various solutions. (D-E) Average of normalized AgRP neuron calcium signal of fasted mice following oral gavage of 500 uL of various solutions (D) and quantification (E). Gray bars indicate the period of oral gavage. (F-G) Average of normalized AgRP neuron calcium signal in fasted mice, after gastric or intestinal infusion of 1mL various solutions (F) and quantification (G). Gray bars indicate the infusion period. Values are reported as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA, Sidak correction. See also Figure S7.