Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception

Abstract

All animals must detect noxious stimuli to initiate protective behavior, but the evolutionary origin of nociceptive systems is not well understood. Here we show that noxious heat and irritant chemicals elicit robust escape behaviors in the planarian Schmidtea mediterranea and that the conserved ion channel TRPA1 is required for these responses. TRPA1-mutant Drosophila flies are also defective in noxious-heat responses. We find that either planarian or human TRPA1 can restore noxious-heat avoidance to TRPA1-mutant Drosophila, although neither is directly activated by heat. Instead, our data suggest that TRPA1 activation is mediated by H₂O₂ and reactive oxygen species, early markers of tissue damage rapidly produced as a result of heat exposure. Together, our data reveal a core function for TRPA1 in noxious heat transduction, demonstrate its conservation from planarians to humans, and imply that animal nociceptive systems may share a common ancestry, tracing back to a progenitor that lived more than 500 million years ago.

Figure 1

Smed-TRPA1 is required for noxious heat avoidance in the planarian worm S. mediterranea. a) Phylogeny of Bilateria, showing the position of Schmidtea (C. elegans is circled). b) Phylogenetic tree constructed from an alignment of full-length TRPA1 protein sequences from a variety of species, Smed-TRPA1 is circled and a model of the channel’s structure is shown (circles=ankyrin repeats, cylinders=transmembrane domains). c) 2-choice assay for heat avoidance. In each trial two opposing floor tiles are set to 24°C and two to 32°C (noxious heat). Tracks of two worms during one such trial are shown in green and purple. Unlike wild-type, controls (unc22 RNAi), and ap2 RNAi, Smed-TRPA1 RNAi animals were not confined to the cool quadrants. d) Avoidance index for 32°C for RNAi animals. Smed-TRPA1 RNAi animals show a reduced avoidance index for heat (N= 5 groups of 10 animals, *P= 0.0054, Kruskal-Wallis; Chi-sq(3,16)=12.68). e) Smed-TRPA1 RNAi does not impact the animal’s speed of movement (N=10–13 animals; n.s. = not significantly different, P= 0.6, Kruskal-Wallis; Chi-sq(3,39)=1.48). f–i) In situ hybridization with a Smed-TRPA1 probe in (g) Control (unc22) RNAi, (h) Smed-TRPA1 RNAi and (i) ap2 RNAi animals (head region, see f), demonstrates overall reduction of mRNA by Smed-TRPA1 RNAi (independent quantification by Q-PCR is shown in j; N=4 replicates of 3 animals each, *P= 0.02, Kruskal-Wallis; Chi-sq(2,9)=7.65). k) In contrast, ap2 RNAi reduces the number of Smed-TRPA1-expressing cells in the brain region, but not in the periphery (N=9 animals, *P= 1.5 × e⁻⁵; unpaired t-test, t(16)=6.1048); in all plots, line=mean; outer boxes = +- STD; inner boxes = 95% Confidence Interval.

Figure 2

Smed-TRPA1 is required for behavioral avoidance of the irritant chemical AITC. a) Two-chamber arena designed to quantify behavioral avoidance of chemical agonists of TRPA1. Planarian worms are introduced in chamber 1 in the presence of a mock Agar pellet (empty squares) or Agar+AITC (50 mM; red squares); their movement is then recorded for 5 minutes. The panels are maximum-projections of 5’ movies, illustrating the extent of worm movement (white tracks). b) In the presence of agar alone, control (unc22), ap2 and Smed-TRPA1 RNAi worms do not readily cross the narrow channel connecting chambers 1 and 2. In the presence of AITC, both control (unc22) and ap2 RNAi worms exit chamber 1 and explore chamber 2. In contrast, Smed-TRPA1 RNAi animals overwhelmingly remain in chamber 1 (N= 5 groups of 10 animals, fraction was calculated on the last 1’ of video; *P=0.0078, Kruskal-Wallis comparing fraction of animals in chamber 1 or 2 across treatments; Chi-sq(2,12)=9.71; line=mean; outer boxes = +- STD; inner boxes = 95% Confidence Interval).

Figure 3

Smed-TRPA1 expressed in Drosophila cells is activated by AITC but not by heat. a) S2R+ Drosophila cells voltage clamped at −60mV were stimulated by heat (red trace) and by bath application of AITC (500µM, grey bar). AITC application (but not heating) resulted in an inward current. b) Current/Voltage relationship from averages of three step protocols done at room temperature (blue trace), during the heat stimulation (red trace), and at the end of AITC application (orange trace; note that the inset is a IV curve from a mock transfected cell; the timing of each set of measurements is also labeled as A,B and C on the trace shown in panel A). c) Current density (Max/capacitance) at 32°C and in the presence of AITC recorded in mock-transfected, dTRPA1-A transfected, and Smed-TRPA1 transfected cells. line=mean; outer boxes = +- STD; inner boxes = 95% Confidence Interval. d). Dose-response for AITC activation of Smed TRPA1 (AV±STD; n=5 cells/condition; mustard flower and seed represent an iconic source of AITC).

Figure 4

Functional expression of Smed-TRPA1 in vivo in adult Drosophila further demonstrates that the channel is sensitive to AITC but not to heat (°C). a) Adult fruit flies expressing either Smed-TRPA1 or –as a control- the intrinsically heat sensitive Drosophila TRPA1-A splice variant, throughout the nervous system (under the control of elav-Gal4) were subjected to a brief step at 35°C (a temperature which does not normally impair fly activity). TRPA1-A expressing flies are readily and reversibly incapacitated by heat (presumably because of simultaneous depolarization of neurons, caused by channel opening) and fall to the bottom of the tube; Smed-TRPA1 flies appear instead unaffected. b) Quantification of the experiment in a. Blue trace = pooled controls (elav/+; UAS-Smed-TRPA1/+; UAS-TRPA1-A/+ ; N=4 groups of 10 animals for each, tested separately); purple trace = experimental animals (elav-Gal4>UAS-Smed-TRPA1; N=4 groups of 10 animals); orange trace = positive control (elav-Gal4>UAS-TRPA1-A; N=4 groups of 10 animals; for all traces shaded area ±SEM). c) Adult flies expressing either Smed-TRPA1 or TRPA1-A were reversibly incapacitated by brief exposure to AITC vapors (see methods for details; Groups and Ns as above; shaded area ±SEM).

Figure 5

Across-phylum rescue of Drosophila TRPA1 mutant phenotypes by planarian and human TRPA1. a) In a 2-choice assay, wild type Drosophila flies robustly avoid noxious heat (40°C). In contrast, TRPA1¹ mutants explore more readily the 40°C quadrants (the panels are maximum-projections of 3’ movies, illustrating the extent of fly movement, temperature in °C is indicated next to each quadrant). b) Schematic of the rescue experiments. c) Avoidance index of wild-type (black boxes), TRPA1¹ mutants (red), rescues (yellow, brown, green), and control genotypes (grey). TRPA1¹ mutants display a significantly lower avoidance index for heat (unpaired t-tests, 30°C: *P= 4.2 × e⁻⁴, t(19)=4.2578; 40°C: *P= 7.7 × e⁻⁷, t(19)=7.1991). Pan-neural expression (under the control of elav-Gal4) of mRNA encoding Drosophila TRPA1-C (a splice variant encoding a channel that is not heat-sensitive, yellow), Smed-TRPA1 (brown), or human TRPA1 (green, each under a UAS- promoter) significantly rescues noxious heat avoidance (40°C). Control genotypes: elav driver/+; TRPA1¹ and UAS-transgene/+; TRPA1¹ (see methods for full genotypes). For rescues, AI values for each test temperature were compared by two-way ANOVAs; asterisks denote a significant interaction between the Gal4 and UAS transgene (from left: *P=0.0001, F(1,32)=19.32; *P=0.0092, F(1,35)=7.6; *P=0.0011, F(1,34)=12.7). Thick line = mean; outer boxes = +- STD; inner boxes = 95% Confidence Interval.

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).