(A– D) Time course of paw withdrawal threshold following i.t. injection of GSK101 in vehicle-treated and GSK219-treated WT mice (A), WT control littermates and Trpv4^–/– mice (B), Trpv4^fl/fl control littermates and Cx3cr1^CreER/+:Trpv4^fl/fl mice (C); Trpv4^fl/fl control littermates and Cdh5^Cre/+:Trpv4^fl/fl mice (D). GSK219 (25 μg) was i.t. injected 1.5 hours after GSK101 (1 μM) administration. n = 7–10 mice per group. ^#P < 0.05 versus vehicle group; *P < 0.05, **P < 0.01, ***P < 0.001 versus GSK101 group by 2-way repeated ANOVA with Bonferroni’s post hoc test. (E–H) Schematic illustration of in vivo 2-photon Ca²⁺ imaging (E), representative 2-photon images (F), ΔF/F hot map (G), and ΔF/F peak amplitude (H) of spinal Vglut2⁺ neurons responses to GSK101 (1 μM) and GSK219 (25 μg) i.t. injection. n = 105–120 neurons from 4 mice. ***P < 0.001 by unpaired 2-tailed Student’s t test. Scale bar: 50 μm. (I–K) Representative recorded Vglut2-tdTomato⁺ neuron in the lamina IIo of spinal cord slice (I), ramp protocol of depolarizing current was applied to assess the rheobase, RMP, and threshold (J), and step protocol of depolarizing currents was applied to assess the number of AP firings (K). (L–S) Representative traces and quantification of AP firings from Vglut2^Cre/+:tdTomato mice with GSK101 (300 nM) and GSK219 (300 nM) perfusion. n = 10–15 neurons from 3–4 mice. (T–W) Representative traces and quantification of AP firings from Cx3cr1^CreER/+:Trpv4^fl/fl mice with GSK101 perfusion. n = 10 neurons from 3 mice. ***P < 0.001 by paired 2-tailed Student’s t test (M, Q, and U), *P < 0.05, **P < 0.01, ***P < 0.001 by 2-way repeated ANOVA with Bonferroni’s post hoc test (O, S, and W). Data are presented as mean ± SEM.