Fig. 5. Most cold-sensitive DPANs use TRPM8, but TRPA1 and TRPC5 are also functional cold transducers.

(A) Characterization and quantification of cold-sensitive cultured DPANs (C57BL/6J: n = 23 of 136) based on Ca²⁺ transients measured with Fura-2 AM and sensitivity to TRP channel modulators menthol (me, n = 7), carvacrol (crv, n = 3), both (n = 10, descending diagonal stripes), neither (n = 4, ascending diagonal stripes), and riluzole (rlz, n = 1). Cold-sensitive DPANs insensitive to menthol and carvacrol (ascending diagonal stripes) are unchanged in TRPC5^−/− (n = 1 of 14 in 93) versus C57BL/6J (3 of 23 in 136; n.s. P = 0.86), but increased in TRPM8A1-DKO (n = 23 in 457; ^‡‡P = 0.002 and ^‡‡‡P = 0.00003). In TRPC5A1-DKO, cold-sensitive cells (n = 20 of 100) were mostly menthol sensitive (n = 15; crv+ n = 10; both, n = 7 and none, n = 2). TRPC5M8-DKO cold-sensitive cells (n = 5 of 103) were mostly sensitive to carvacrol (n = 3; me+ n = 2; both, n = 1 and none, n = 1). Inset: Photomicrograph of cultured mouse TG neurons with red DiI retro-labeled DPANs. (B and C) Ca²⁺ transient traces from (B) C57BL/6J representative of four types of cold-sensitive neurons and (C) TRPM8A1-DKO (red), TRPC5A1-DKO (green), and TRPC5M8-DKO (blue). Bottom: Temperature stimulator command. (D) TRPM8/A1-DKO control (cntrl) cold responses (n = 23) were smaller than in C57BL/6J (n = 23; ^###P = 0.0007) but not in TRPC5^−/− neurons (n = 13; P = 0.2). ML204 (circles = treated neurons) blocked most cold responses in TRPM8/A1-DKO (≥50% block = filled circles, n = 12; *P = 0.01) and some in C57BL/6J neurons (n = 15; *P = 0.02), but not in TRPC5^−/− neurons (n = 13; n.s. P = 0.2). n.s., not significant. Lines are medians, squares mean, boxes IQR, whiskers 2.2-fold IQR after exclusion of >2.2 IQR outliers identified by crosses.