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


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.

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Fig. 1: Smed-TRPA1 is required for noxious heat avoidance in the planarian worm S. mediterranea.
Fig. 2: Smed-TRPA1 is required for behavioral avoidance of the irritant chemical AITC.
Fig. 3: Smed-TRPA1 expressed in Drosophila cells is activated by AITC but not by heat.
Fig. 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).
Fig. 5: Across-phylum rescue of Drosophila TRPA1 mutant phenotypes by planarian and human TRPA1.
Fig. 6: H2O2 and/or ROS as a signal for TRPA1 activation during noxious heat responses.


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We thank D. Tracey and R. Carthew for reagents; A. Kuang and L. Vinson for technical assistance; L. Macpherson, D. Yarmolinsky, and members of the Gallio Lab for comments on the manuscript; I. Raman for technical advice, and M. Stensmyr for the kind gift of the fly drawing in Fig. 1. Work in the Gallio lab is supported by NIH grant R01NS086859 (to M.G.), the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust (to E.E.Z.), and by training grant 2T32MH067564 (to O.M.A.). Work in the Petersen Lab is supported by an NIH Director’s New Innovator award (1DP2DE024365-01).

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Authors and Affiliations



M.G. designed the study, analyzed the data, and wrote the paper with critical input from all authors; M.G. and O.M.A. designed and built the behavioral assays. O.M.A. performed all planarian behavioral experiments and electrophysiology and analyzed the corresponding data. E.E.Z. performed all fly rescue experiments and ROS assays and analyzed the corresponding data. A.P. cloned Smed-TRPA1, produced rescue constructs and transgenics, and analyzed sequences with help from C.P.P; A.P., O.M.A., and C.V.D. performed q-PCR and ISH experiments. E.E.Z. and O.M.A. generated human-TRPA1-expressing flies.

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Correspondence to Marco Gallio.

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Integrated supplementary information.

Supplementary Figure 1 Design of the arena used to measure heat avoidance behavior in planarians (the Planariometer).

a) Planariometer design. In each trial two opposing tiles are set to 24°C and two to 32°C (noxious heat). Animal movement is then recorded for 4 minutes. The spatial configuration of hot and cool tiles is then reversed for 4 additional minutes (and a second movie acquired) to control for potential spatial biases. The hot and cold tiles are covered by thin anodized aluminum foil; on it, a hydrophobic ink pen is used to create a circular barrier. A thin film of water (1-2 mm) forms a central pool in which the worms can move freely. The experiment is conducted in the dark, under IR-LED illumination; movies are acquired using an IR-sensitive CCD camera. b) Images acquired using a thermal imaging camera, showing sharp thermal boundaries between hot and cool quadrants in the two spatial configurations used for each experiment. c) Snapshots from video recordings of planarian movement, corresponding to the thermal configurations shown in b.

Supplementary Figure 2 Smed-TRPA1 RNAi does not impair negative phototaxis.

a) A simple assay to measure negative phototaxis in planarians. Worms are released in chamber 1 either in the dark or in bright room light, while chamber 2 is kept in constant darkness. b) Both controls and experimental animals avoid the light, and efficiently escape into the dark chamber (chamber 2). Outer boxes = +- STD; Inner boxes = 95% Confidence Interval.

Supplementary Figure 3 Dye, imaging, and feeding controls.

a) The fluorogenic ROS dye, carboxy-H2DCFDA does not increase in fluorescence as a result of heating. Drosophila tissue (larval salivary glands) that was heat-killed prior to dye loading did not display the prominent heat-dependent increase in fluorescent observed in live tissue (see Figure 6g). b) Carboxy-H2DCFDA fluorescence increases are not due to imaging conditions. Imaging dye-loaded, live tissue (larval salivary glands) at constant temperature, produces a modest fluorescent changes (compare to Figure 6g). In all panels: thick line=average, grey shading= +-STD, imaging conditions are as in Figure 6g. c) Feeding controls, related to the experiments in Figure 6i. A green food dye demonstrates that, prior to their performance in the arena, flies indeed ingest H2O2 or Paraquat laced food.

Supplementary Figure 4 The genetically encoded ratiometric H2O2 indicator roGFP2-Orp1 reports increase in H2O2 levels in fly larvae upon brief (~5 s) exposure to noxious heat.

A, B and C above are schematics of the experiment and correspond to the x-axis legend of the boxplot. A is the negative control (no treatment), while C is the positive control (H2O2 treatment). Fly genotype: tub-cyto-roGFP2-Orp1, (a transgenic line expressing the H2O2 indicator roGFP2-Orp1 in all cells, under a tubulin promoter). Red line = mean; Outer boxes = +- STD; Inner boxes = 95% Confidence Interval; from left: *P=0.006 and *P=1.12 x e-4 in unpaired t-tests; t(15)=-3.2 n=9,8; t(13)=-5.44 n=9,6. Control fluorescence (i.e. of untreated tissue) was set to 1.

Supplementary Figure 5 Knockdown of Smed-TRPA1 reduces scrunching following a physical injury (i.e., a tail snip).

a) Selected frames from a video including and immediately following a tail snip (shown at 1, time=0). “Scrunching” is described as the unusual gate planarians engage in following injury, and is characterized by strong contraction/expansion cycles – quite different from the planarian’s normal smooth gliding movement. b) Normalized body area throughout representative movies for control (blue, UNC22 RNAi) and Smed-TRPA1 RNAi (red; for each animal 1=average length across the movie, movie=15 seconds at 3 frames/sec). c) Amplitude of contractions calculated throughout the movie. Line=mean. Outer boxes = +- STD; Inner boxes = 95% Confidence Interval; *P=0.0022 t(13)=3.79, unpaired t-test; n=7,8

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Supplementary Figures 1–5

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Supplementary Video 1 A behavioural assay designed to measure noxious heat avoidance in Planarian worms.

The videos show heat avoidance behaviour of control worms as well as Smed-TRPA1 RNAi worms (S. mediterranea; videos are accelerated 24X). In this arena, worms are given a choice between 24°C (top right and bottom left tiles) and 32°C (top left and bottom right tiles). Control worms are essentially confined to the cool quadrants; in contrast Smed-TRPA1 RNAi planarians glide around the chamber but do not appear confined to the 24°C regions (i.e. they are defective in heat avoidance)

Supplementary Video 2 A behavioural assay designed to measure AITC avoidance in Planarian worms.

This set up consists of shallow circular chambers filled with water and interconnected by narrow corridors that worms do not readily traverse (note that connected chambers form an inverted U shape, i.e. chambers 1 at the bottom right and 4 at the bottom left do not communicate). At the beginning of each experiment, worms are introduced in chamber 1 in the presence of either a mock Agar pellet or of a pellet laced with AITC. AITC avoidance is apparent as the worms perform quick withdrawal manoeuvres in the vicinity of the AITC pellet and eventually escape to distant chambers (videos are accelerated 24X; Note that the setup used to acquire the data presented in Figure 2 was a simplified version, with two chambers rather than four)

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Arenas, O.M., Zaharieva, E.E., Para, A. et al. Activation of planarian TRPA1 by reactive oxygen species reveals a conserved mechanism for animal nociception. Nat Neurosci 20, 1686–1693 (2017).

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