Extended Data Figure 1. Sex-specific sensitization of mucosal afferents and visceromotor responses by isovalerate.
a, Graphical scheme of ex vivo mucosal afferent recordings (evMAR) from NaV1.8-ChR2 mice. b-c, Representative pelvic mucosal afferent fiber responses showing that isovalerate lowers the threshold to optogenetic activation (0.05 – 34 mW/mm2) in males whereas females display heightened baseline sensitivity. d, Fluorescent channelrhodopsin-eYFP signal in L6 DRG sections from NaV1.8-ChR2 mice, showing no difference between sexes. Scale bars=100μm. e, Heatmaps from two representative males showing in vivo NaV1.8-GCaMP6s responses of L6 DRG neurons to intracolonic application of an isovalerate bolus (200μM) after pretreatment with alosetron (10μM). f, Responses of dissociated DRG neurons from male (N=3) and female (N=3) NaV1.8-GCaMP6s mice showing no sex differences in responsiveness to ionomycin 5μM (females: 514 neurons from 15 coverslips, males: 521 neurons from 18 coverslips), potassium chloride (KCl 30mM; females: 478 neurons from 11 coverslips, males: 629 neurons from 18 coverslips) or capsaicin 20nM (females: 246 neurons from 11 coverslips, males: 363 neurons from 18 coverslips). g, Data from individual mice showing total area under the curve for all colonic distension pressures. VMRs are significantly increased in male but not female mice following intracolonic application of isovalerate (100μl bolus of 200μM) versus vehicle; male: N=12 vehicle, N=12 isovalerate, female: N=12 vehicle, N=12 isovalerate). h, total AUC compared across cohorts, similarly demonstrating increased VMRs over baseline (grey) following application of isovalerate (purple) in male but not female mice (male: N=15 vehicle, N=9 isovalerate, female: N=7 vehicle, N=7 isovalerate). i, Colonic compliance is unchanged with isovalerate in both male and female mice. Unpaired t-test, 2-tailed in panel d, Unpaired Mann Whitney U test, 2-tailed in panels f, h, Wilcoxon matched-pairs signed-rank test 2-tailed in panel g and two-way ANOVA in panel i. **p< 0.01, ***p< 0.001, error bars represent mean ± SEM. Graphical element in panel a is licensed from BioRender.