Extended Data Fig. 1: Characterizing the calcium sensor, AAV injection time, and anesthetic used during in vivo NTS imaging.
a, Mice were injected in the NTS with AAVs encoding H2B-jRGECO1a and GCaMP6m, and NTS imaging was later performed to measure gastric distension-evoked calcium transients. A heat map depicting time-resolved responses of all 74 NTS neurons (2 mice) that displayed both H2B-jRGECO1a (left) and GCaMP6m (right) responses to a series of gastric distensions (green bars of increasing thickness: 150, 300, 600, and 900 μl), white bar: 10 s. b, Representative traces depicting normalized ΔF/F over time for individual neurons (same neurons on left and right), scale bar: 10 s. c, Maximal ΔF/F of NTS neurons responsive to 900 μl gastric distension. d, Mice were injected in the NTS with an AAV encoding H2B-jRGECO1a at 2 (left) and 5 (right) weeks of age, and NTS imaging was later performed. Representative two-photon images of H2B-jRGECO1a fluorescence in the NTS (transverse view). Neurons are color-coded based on their relative peak response amplitudes above threshold to laryngeal water (blue, 100 μl), stomach stretch (green, peak response to 150, 300, 600, or 900 μl), or duodenal stretch (red, peak response to 90, 115, or 140 μl), scale bar: 100 μm. e, Positions of neurons selectively responsive to stimuli indicated are charted with axis origin corresponding to the centroid for stomach-stretch responsive neurons, top left: 695 neurons, 4 mice, bottom left: 1106 neurons, 4 mice, top right: 2331 neurons, 8 mice, bottom right: 2215 neurons, 6 mice, color scale depicts neuron density (see methods), axis length: 150 μm, R: rostral, M: medial. f, Quantifying the percentage of neurons singly tuned to either gastric distension, duodenum distension, or laryngeal water, dots: individual mice, n: 4 (left), 8 (right), mean ± sem. g, Representative two-photon images of H2B-jRGECO1a fluorescence in the NTS (transverse view) of mice anesthetized with urethane (2.2–2.6 mg/mg) or isoflurane (1.5%–2.5%). Neurons are color-coded based on their relative peak response amplitudes above threshold to laryngeal water (blue, 100 μl), stomach stretch (green, peak response to 150, 300, 600, or 900 μl), or duodenal stretch (red, peak response to 90, 115, or 140 μl), scale bar: 100 μm. h, Positions of neurons selectively responsive to stimuli indicated are charted with axis origin corresponding to the centroid for stomach-stretch responsive neurons, top left: 1136 neurons in 8 mice randomly selected from 49 mice, bottom left: 2525 neurons in 9 mice randomly selected from 98 mice, top right: 1010 neurons, 8 mice, bottom right: 1707 neurons, 9 mice, color scale depicts neuron density (see methods), axis length: 150 μm, R: rostral, M: medial. i, Quantifying the percentage of neurons singly tuned to either gastric distension, duodenum distension, or laryngeal water, dots: individual mice, n: 63 (left), 10 (right), mean ± sem.
