Figure 3

Calcium imaging of trigeminal neurons after stimulation with allyl isothiocyanate (AITC). (A and B) Both Cdk5 and its activator p35 are co-expressed with TRPA1 in cultured trigeminal ganglia neurons. Ca2⁺ imaging was performed using cultured trigeminal neurons from mice genetically engineered to have either increased (Tgp35) or decreased (p35KO) Cdk5 activity. Phalloidin, which binds to F-actin, was used as a control to stain individual cells. Cultured neurons were treated with either a low (1–3 μM) or high (300 μM) concentration of the TRPA1 agonist AITC. The percent of responders was normalized to KCl. (C) Cultured neurons from the p35KO mice (n = 7). Averaged Ca²⁺ imaging traces ± SEM from 7 experiments using p35KO mice and littermate controls. (D,E) The p35KO mice show fewer % responders at a low dose of the TRPA1 agonist AITC as compared to the wild type mice (unpaired t-test, *P < 0.05), but no significant differences were seen at high doses of AITC. (F) Averaged Ca²⁺ imaging traces ± SEM from 6 Tgp35 mice and wild type controls. (G,H) In contrast to the p35KO mice, cultured neurons from Tgp35 mice (n = 6) tend to be more responsive to AITC at both low and high concentrations.