(A) Fura-2-based intracellular calcium measurements in mouse trigeminal neurons from TRPV1/TRPA1 double knockout mice showing increases in intracellular calcium in response to AITC (3 mM) and menthol (30 µM). The upper trace shows the time differential of the intracellular calcium concentration, which represents a measure of net calcium influx/extrusion mechanisms. The TRPM8-dependence of the responses was ensured based on full block by AMTB (as in Figure 1; not shown). (B) Same as (A), but in the presence of TTX (1 μM) to block neuronal action potentials. (C) Same as (A), but in a HEK293 cell expressing mouse TRPM8. Non-transfected cells did not show any detectable response to AITC or menthol. (C) Relative stimulatory effect of menthol and AITC in control trigeminal neurons (n = 81 from 9 different mice), trigeminal neurons treated with 1 μM TTX (n = 3 from 3 different mice) and HEK293 cells (n = 448). *, **, ***: p<0.05, 0.01 and 0.001, respectively, in paired t-test comparing the response to AITC and menthol within individual cells. ###: P<0.001 in unpaired t-tests comparing TG neurons and HEK293 cells.