Extended Data Fig. 8: Vagal neurons neither express Tlr5 nor respond directly to flagellin.

(a) Quantification of the peak vagal response to flagellin in PyyCre_Tlr5^fl/fl and _Tlr5^fl/fl littermate controls (PyyCre_Tlr5^fl/fl: n = 5 mice; _Tlr5^fl/fl: n = 3 mice; *P < 0.05 by Kruskal Wallis test with non-parametric comparisons using the Wilcoxon method). (b) RNAseq of vagal nodose neurons showed no expression of Tlr5 (PyyGFP+ cells: n = 5 mice, PyyGFP- cells: n = 8 mice, nodose neurons: n = 8 mice; DESeq2 normalization). (c) qPCR of synaptic markers and Toll-like receptors in vagal nodose neurons (n = 3 mice). (d) In situ hybridization of Nissl (green) stained neurons of the nodose ganglion showed no expression of Tlr5 (red), representative of n = 3 mice. Scale bars=100μm. (e) Representative trace of calcium transient in acutely dissociated Neurod1Cre_Salsa6f nodose neurons showed responses in 57% of neurons to capsaicin, but not to flagellin (n = 227 neurons, n = 4 mice). (f) Secondary analysis of data from Bai et al., 2018 of the colon-projecting vagal neurons showed expression of the PYY receptor Npy2r (n = 44 cells). (g) Spatial overlay of vagal nodose neurons using tdTomato and GCaMP6s fluorescence. (h) Spatial overlay of vagal nodose neurons using compartment analysis of temporal activity by fluorescence in situ hybridization (CatFISH). (i) Representative image of CaTFISH mapping of in vivo calcium transients onto Npy2r positive neurons. Scale bars=10μm. (j) Calcium traces from vagal nodose neurons responsive to either only flagellin (2 µg/mL) (top), intralipid (7%) and flagellin (2 µg/mL) infused separately (middle), or only intralipid (7%) (bottom), representative of n = 520 neurons. Error bars and shades indicate S.E.M. f, adapted from ref. ⁵⁶, Cell Press.