Figure 1.

Menthol-sensitivity is restricted to Vglut3^lineage DRG neurons. A, Representative confocal images of DRG sections (25 μm) from adult Slc17a8^iCre; Rosa26^Ai14 mice immunostained with anti-DsRed (TdTomato; red), anti-neurofilament heavy (NFH; blue), and anti-β3-Tubulin (green). Images were acquired using a 20× 0.8 NA air objective. B, C, Baseline normalized, representative traces depicting Fura2-AM ratio (F340/F380) versus time traces of averaged responses from Vglut3^lineage (B) and non-Vglut3^lineage (C) DRG neurons to various chemosensory stimuli [menthol (M) 100 μm, blue trace; chloroquine (CQ) 1 mm; capsaicin (Cap) 1 μm, red trace; and high K⁺ Ringer's (K⁺) 140 mM, black trace]. Colored bar indicates time of agonist application. D, Images of calcium transients in live, dissociated Slc17a8^iCre;Rosa26^Ai14 DRG neurons quantified in B and C. Top, Fura2-AM calcium microfluorimetry following menthol application. Green circles indicate menthol-sensitive DRG neurons. Bottom, Fluorescent image showing TdTomato-expressing (Vglut3^lineage) DRG neurons. E, Quantification of percentage of Vglut3^lineage (n = 331) and non-Vglut3^lineage (n = 453) neurons responding to individual agonists. F, Quantification of baseline calcium signals between Vglut3^lineage menthol-sensitive neurons and menthol-insensitive neurons (both Vglut3^lineage and non-Vglut3^lineage). Significance was determined using an unpaired Student's t test. *p < 0.05. Data represented as mean ± SD. Scale bars, 100 μm.