Figure 6.

H₂O₂/ROS as a signal for TRPA1 activation during noxious heat responses. a) Heterologously expressed Smed-TRPA1 is activated by H₂O₂. b) Dose-response of H₂O₂ activation for Drosophila TRPA1-C (yellow trace and points), and Smed-TRPA1 (brown trace and points, AV±STD, n=5 cells/condition). c–e) The ROS dye Carboxy-H₂DCFDA demonstrates in vivo ROS production in response to heat in living planarians. c and d) Representative frames of tissues and cells undergoing rapid fluorescent changes in response to heat (scale bar=200µm). e) Traces are an ROI around the cell in d (arrow) and the square, light green box. f) Exposure of planarian worms to 35°C for 30” results in a significant increase in fluorescence (unpaired t-test, *P= 0.003, t(8)=4.1258; n=5/condition). g) Carboxy-H₂DCFDA fluorescence in response to heating in Drosophila salivary gland tissue (AV±SD). h) Exposure of salivary glands to 40°C for 1” results in a significant fluorescence increase (unpaired t-test, *P= 3.26 × e⁻⁵, t(8)=8.3283; n=5/condition). i) Acute feeding with pro-oxidants sensitizes adult Drosophila to heat. Feeding Paraquat (orange bars) or H₂O₂ (yellow bars), results in increased heat voidance in a 2-choice behavioral assay in both wild-type and heterozygous TRPA1/+ controls (unpaired t-tests; from left: *P= 0.003, t(26)=3.2093, n=16,12; *P= 1.26 × e⁻⁴, t(27)=4.472, n=16,13; *P= 0.005, t(26)=3.058, n=16,12; *P= 0.036, t(27)=2.201, n=16,13; *P= 0.027, t(12)=−2.51, n=7,7; *P= 0.003, t(14)=−3.53, n=7,9; *P= 0.021, t(12)=−2.64, n=7,7; *P= 0.033, t(14)=−2.35, n=7,9). In contrast, heat avoidance does not increase in TRPA1 mutants (n.s. =not significant in unpaired t-tests; from left: *P= 0.16, t(34)=−1.43, n=21,15; *P= 0.075, t(39)= −1.82, n=21,20; *P= 0.11, t(34)=−1.62, n=21,15; *P= 0.93, t(39)=0.088, n=21,20;. In f, h and i: Line=mean; outer boxes = +- STD; inner boxes = 95% Confidence Interval).