Primary mouse DRG cultures were prepared for Ca²⁺ imaging (A–C) or whole-cell current clamp recordings (D and E). (A) Representative original magnification ×40 images of cultured DRG neurons loaded with Fura 2 at baseline and upon treatment with 2AT (1 μM) and KCl (50 mM), a positive control for neuronal Ca²⁺ signaling. White arrows highlight an individual cell responsive to both 2AT and KCl (i.e., PAR2⁺ neurons). Scale bar: 20 μm. (B) Representative traces of 2 cultured DRG neurons showing changes in [Ca²⁺]_i (plotted as 340/380 nm ratiometric change) in response to 2AT and KCl. Baseline measures were recorded for 60 seconds in normal bath solution. Cells were then treated with 2AT (1 μM) for 120 seconds, washed in normal bath solution for 120 seconds, and treated with KCl (50 mM) for 10 seconds. Cells with at least 20% ratiometric change in response to KCl treatment were classified as neurons, and out of these, neurons with at least 40% ratiometric change in response to 2AT treatment were classified as PAR2⁺. (C) Pie chart illustrating the percentage of PAR2⁺ neurons in culture as characterized by response to 2AT (1 μM). About 3%–4% of primary cultured DRG neurons (KCl responsive) are PAR2⁺ (2AT responsive). (D) Whole-cell current clamp recordings reveal increased firing of TRPV1⁺CGRP^– cultured DRG neurons from CGRP^cre/+-ER Rosa26^{LSL-tDTomato/+} TRPV1-GFP reporter mice after activation with 2AT (1 μM). A linear ramp (0 to 0.1 nA for 1 second) was applied to the patched neuron to generate an action potential (AP) train before and after 2AT (1 μM) treatment. (E) Electrophysiological experiments demonstrate that 2AT (1 μM) induced hyperexcitability exclusively in TRPV1⁺CGRP^– neurons. Changes in neuronal excitability were based on the ratio of current ramp-generated AP frequencies after and before vehicle/2AT treatment. n = 7 for TRPV1⁺CGRP^– neurons treated with vehicle, n = 13 for TRPV1⁺CGRP^– neurons treated with 2AT (1 μM), and n = 8 for CGRP⁺ neurons treated with 2AT (1 μM). Data represent mean ± SEM. One-way ANOVA with Tukey’s multiple comparisons (E) **P < 0.01.