Figure 3.

Sensory neuron activation/inhibition using calcium imaging. (a) Quantification of calcium imaging responses in primary culture DRG sensory neurons to the vehicle (1% ethanol in 1X PBS [v/v]); 9-HODE; PGE₂; 9,13EHL; 9,10,13THL; stable analogs of 9,13EHL and 9,10,13THL and their small molecule pharmacophores. All compounds were tested at a concentration of 1 μM and normalized to 100 mM KCl responses unless noted. (b) Dose‒response curve of the endogenous mediator 9,10,13-THL; each dot represents a single coverslip. n = 2. (c) DRG neurons preincubated with vehicle or gallein (100 μM) exposed to 9,10,13-THL and capsaicin (∗P = 0.0339) as determined by a two-tailed paired Student’s t-test. (d) DRG neurons preincubated with vehicle or PTX (1 ng/ml) and exposed to 9,10,13-THL and capsaicin; ns based on a two-tailed paired Student’s t-test. (e) Neurons responding to 9,10,13-THL, AITC (100 μM), and capsaicin in WT, Trpa1-KO, and Trpv1-KO mice by measuring calcium transients. Significance was determined using two-way ANOVA with a Holm-Šídák correction for multiple comparisons. For 9,10,13-THL, ∗∗P = 0.0311 (WT vs. Trpa1 KO) and ∗∗P = 0.002 (WT vs. Trpv1 KO). For AITC, ∗∗∗P = 0.001 (WT vs. TRPA1 KO). For capsaicin, ∗∗∗∗P < 0.0001 (WT vs. Trpv1 KO). All data are presented as mean ± SD. Each data point represents an average of coverslips/mouse; n ≥ 3. 9,10,13-THL, 9,10,13-trihydroxy-octadecenoate; 9,13-EHL, 13-hydroxy-9,10-epoxy octadecenoate; AITC, allyl isothiocyanate; DRG, dorsal root ganglia; HODE, hydroxyoctadecenoate; KCl, potassium chloride; KO, knockout; ns, not significant; PGE₂, prostaglandin E₂; PTX, pertussis toxin; WT, wild type.