Asymmetric control of food intake by left and right vagal sensory neurons

Figures

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  Figure 1.. LNGCart and RNGCart Asymmetrically Innervate the Duodenum (A) subpopulation of NG neurons that express the Cartpt gene within the Phox2b cluster. (B) Dot plot showing expression of selected genes involved in mechanosensing and nutrient sensing in NGCart neurons. (C) Unilateral DIO tdTomato injection (n=3–4). (D) tdTomato detected in LNG and RNG. (E) Quantification of (C). (F) Whole mount imaging of the duodenum showing vagal terminals from LNGCart or RNGCart fibers. (G-J) Quantification of IGLEs and mucosal endings in the duodenum (n=3, unpaired Student’s t test and two-way ANOVA). Data are expressed as mean ± SEM; ns, p > 0.05; t tests and post hoc comparisons, *p < 0.05. Scale bars 100 μm.

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  Figure 2.. Activity of vagal sensory neurons in response to intraduodenal nutrient infusion or distension. (A) In vivo calcium imaging of NGCart neurons in response to intestinal distension. (B-C) Heat maps depicting time-resolved responses (ΔF/F) of 250 LNGCart and 85 RNGCart neurons identified as distension responders (green bar, 20 seconds). (D) Percentage of NGCart neurons identified as distension responders in LNG and RNG (n=6/group, unpaired t-test, *p=0.041). (E) Average GCaMP6s signal in LNGCart and RNGCart neurons that were responsive to duodenal distension. Grey shaded area represents duration of stimulus. Dark lines represent means and lighter shaded areas represent SEM. Two-way ANOVA, *p<0.0001. (F) In vivo calcium imaging of NGCart neurons in response to intraduodenal fat infusion. (G-H) Heat maps depicting time-resolved responses (ΔF/F) of 210 LNGCart and 219 RNGCart neurons identified as fat responders (red bar, 5 minutes). (I) Percentage of NGCart neurons identified as fat responders in LNG and RNG (n=7/group, Unpaired t-test, *p=0.049). (J) Average GCaMP6s signal in LNGCart and RNGCart neurons that were responsive to intraduodenal fat infusion. Grey shaded area represents…

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  Figure 3.. Stimulation of RNGCart neurons promotes flavor-conditioning. (A) Flavor nutrient-conditioning test with mice that had RNGCart neurons ablated with flex-taCasp3-TEVp. (B) Post-conditioning preferences for controls, and RNGCart-Casp (n=6/group, paired t test). (C) Flavor CNO-conditioning test with LNGCart-hM3Dq and RNGCart-hM3Dq mice. (D) Post-conditioning preferences for LNGCart-hM3Dq mice, and RNGCart-hM3Dq mice (n=5–7/group, paired Student’s t test). (E) Cart-Cre mice unilaterally injected in the LNG or RNG with DIO-ChR2-YFP. (F) Mice implanted with optogenetic posts above vagal terminals in the NTS were trained to perform an operant task using a two-hole nose poke apparatus. One of the nose holes was designated as the active hole and was associated with a blue light laser pulse triggered by nose pokes, while the other hole remained inactive. (G) Preferences for the laser-paired nose poke over 3 consecutive days of stimulation for LNGCart-ChR2 and RNGCart-ChR2 mice (n=3–4/group, paired Student’s t test). Data are expressed as mean ± SEM; ns, p > 0.05; t tests and post hoc comparisons, *p < 0.05.

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  Figure 4.. Stimulation of LNGCart neurons is sufficient and necessary to reduces food intake. (A) Cart-Cre mice unilaterally injected in the LNG or RNG with DIO-hM3D(Gq)-mCherry. (B) Quantification of hM3D-mCherry expression in LNG and RNG. (C) hM3D-mCherry detected in LNG and RNG. (D) Paradigm for temporal analysis of chow intake in LNGCart- and RNGCart-hM3Dq mice. (E-H) 4-h food intake of (E) LNGCart-hM3Dq mice, and (G) RNGCart-hM3Dq mice (n=9/group, two-way ANOVA with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli post hoc) following CNO or saline injections; Average 4-h meal size of (F) LNGCart-hM3Dq, and (H) RNGCart-hM3Dq mice (n = 9/group, paired t test, ns) following CNO or saline injections. (I) Blood collection for gastric emptying assay from LNGCart- and RNGCart-hM3Dq mice following CNO injection and intragastric delivery of acetaminophen solution. (J) Gastric output measured as blood acetaminophen levels over time in LNGCart- and RNGCart-hM3Dq mice (n=4–8/group, two-way ANOVA with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli post hoc; Control vs. LNG, *p = 0.018; RNG vs. LNG, #p = 0.033). (K) Cart-tdTomato mice unilaterally…

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  Figure 5.. LNGCart and RNGCart recruit distinct downstream circuits in the brain. (A) Schematic of of the FosTRAP model for comparing neural response to intraduodenal fat and distension in the same animal. (B) Quantification of tdTomato labeled FatTRAP neurons and neurons labeled with Fos immunoreactivity after duodenal distension in the DVC (unpaired Student’s t test) (C) Venn diagram representing the separation in neuronal activity between the two stimuli. (D) Representative images of the NTS showing active populations in response to fat (magenta) and distension (cyan). (E) Unilateral injection of DIO-ChR2-YFP. (F) Optogenetic stimulation of the duodenum. (G) Representative images of the NTS after optogenetic stimulation of LNGCart or RNGCart fibers that innervate the duodenum. (H) Quantification of cFos expression on the DVC 90 min after stimulation (unpaired t test). (I) Brain-wide screen of FosTRAP mice that received intestinal distension and fat infusion. (J) Brain-wide screen of LNGCart-ChR2 or RNGCart-ChR2 mice that received intraduodenal optogenetic stimulation. HPC=hippocampus; LHA=lateral hypothalamus; CeA=Central amygdala; ACC=anterior cincular cortex; PVH=paraventricular…