Author response: A zebrafish and mouse model for selective pruritus via direct activation of TRPA1

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Little is known about the capacity of lower vertebrates to experience itch. A screen of itch-inducing compounds (pruritogens) in zebrafish larvae yielded a single pruritogen, the TLR7 agonist imiquimod, that elicited a somatosensory neuron response. Imiquimod induced itch-like behaviors in zebrafish distinct from those induced by the noxious TRPA1 agonist, allyl isothiocyanate. In the zebrafish, imiquimod-evoked somatosensory neuronal responses and behaviors were entirely dependent upon TRPA1, while in the mouse TRPA1 was required for the direct activation of somatosensory neurons and partially responsible for behaviors elicited by this pruritogen. Imiquimod was found to be a direct but weak TRPA1 agonist that activated a subset of TRPA1 expressing neurons. Imiquimod-responsive TRPA1 expressing neurons were significantly more sensitive to noxious stimuli than other TRPA1 expressing neurons. Together, these results suggest a model for selective itch via activation of a specialized subpopulation of somatosensory neurons with a heightened sensitivity to noxious stimuli. https://doi.org/10.7554/eLife.32036.001 eLife digest Itch is a common and uncomfortable sensation that creates a strong desire to scratch. This mechanism may have evolved so animals can remove harmful parasites or substances from themselves. Feelings like touch, pain, and itch arise when stimuli such as mechanical pressure, temperature, or chemicals activate groups of specialized neurons in the skin. This response takes place when certain proteins – or receptors – at the surface of the neurons are stimulated. For instance, TRP ion channels such as TRPA1 play an important role in both the itch and pain responses. In mammals, directly activating these channels elicits pain. Itch is felt when itch responsive receptors are activated on a distinct set of neurons, which in turn activate TRP receptors. Although these processes have been well-studied in mammals, little is known about the existence of itch sensation in other animals. To explore this, Esancy, Condon, Feng et al. exposed zebrafish to chemicals that induce itch in mammals, and found that imiquimod, a medicine used to treat certain skin conditions, can elicit itch in fish. When this chemical was injected into the lips of a fish, the animal rubbed them against the walls of its tank, akin to scratching an itch. Further experiments showed that imiquimod directly activated the pain-sensing ion channel TRPA1. In fact, this receptor was essential to the ‘scratching’ behavior: fish genetically engineered to lack TRPA1 did not react to the drug. Fluorescent proteins were then used to track when the neurons that carry TRPA1 were activated.This revealed that, in the skin of zebrafish, there are at least two functionally distinct populations neurons that express TRPA1. One population, whose activation is associated with the animal ‘scratching’, responds even when TRPA1 receives a low level of stimulation. The other population is less sensitive: it responds only to high-intensity stimuli and is associated with a pain response such as freezing and slower movements. Further experiments in the mouse suggest that this mechanism is present in mammals as well. This coding strategy explains how pain and itch can be experienced when the same receptors are being activated. Studying how animals like fish experience itch gives an insight into how detecting these sensations could have evolved. In turn, understanding this mechanism at the molecular and cellular levels may help find new ways to design better treatments for itch and pain disorders. https://doi.org/10.7554/eLife.32036.002 Introduction Itch is an unpleasant sensation that elicits a scratch behavior in terrestrial vertebrates. In mammals, chemically-induced itch is thought to be mediated by pruritic receptors on somatosensory neurons (Bautista et al., 2014; Hoon, 2015). These receptors are typically G-protein coupled receptors (GPCRs) that, upon activation, prompt the opening of downstream transient receptor potential (TRP) channels, facilitating activation of the neuron (Ross, 2011; Zhang, 2015). This coupling of pruritic receptors to TRPA1 or TRPV1 is especially intriguing in that these TRP channels also serve as nociceptors, mediating responses to algogenic (painful) stimuli (Laing and Dhaka, 2016; Ikoma et al., 2006). Zebrafish (Danio rerio) have proven to be a valuable tool in the study of nociception (Gau et al., 2013). The zebrafish ortholog of Trpa1, trpa1b, is required for nociceptive responses to aversive pungent chemicals (Prober et al., 2008). Orthologs of genes involved in mammalian itch transduction are also present in the zebrafish (Kaslin and Panula, 2001; Pei et al., 2016; Xu et al., 2011). Studying how these itch genes operate in the somatosensory system of zebrafish could reveal conserved itch transduction mechanisms, providing insight into the evolution of itch. Results Imiquimod evokes itch in zebrafish In an effort to determine if pruritic stimuli are capable of eliciting somatosensory activity in zebrafish, we screened five compounds known to both induce acute pruritus in mammals and act on receptors expressed by zebrafish (Schön and Schön, 2007; Bell et al., 2004; Lieu et al., 2014; Yamaguchi et al., 1999; Tsujii et al., 2009), excluding pruritogens that act on receptors that do not have a zebrafish ortholog, such as MRGPR agonists. Allyl isothiocyanate (AITC), a known algogen and TRPA1 agonist (Prober et al., 2008; Jordt et al., 2004), was used as the positive control. We evaluated somatosensory neuronal responses to pruritic compounds using transgenic larvae that pan-neuronally express the neuronal activity indicator CaMPARI (elavl3:CaMPARI), a fluorescent protein that permanently photoconverts from green to red in the presence of calcium and 405 nm light (Fosque et al., 2015). Using this approach, we were able to view trigeminal neuronal activity in 3 day post fertilization (dpf) larval zebrafish following the application of each pruritogen (Figure 1A–H). Of the pruritogens screened, only imiquimod (IMQ) significantly (p<0.05) activated zebrafish trigeminal ganglia (TG) neurons (Figure 1I). Figure 1 with 1 supplement see all Download asset Open asset Imiquimod produces behavioral and neuronal responses in the zebrafish. (A–H) 3dpf live elavl3:CaMPARI TG imaging of larvae exposed to 405 nm light and control (A), 50 μM allyl isothiocyanate (AITC) (B), 100 μM imiquimod (IMQ) (C), 100 μM histamine (HIST) (D), 500 μM serotonin (5-HT) (E), 100 μM SLIGRL-NH2 (F), 100 μM deoxycholic acid (DCA) (G), and 100 μM loxoribine (LOX) solutions (H). (I), Counts of photoconverted cells from experiments (A-H). (J), 5dpf WT larval locomotor behavior screen. (K) Representative traces of adult behavior. (L) Adult lip-rubbing behavioral assay. (I, J, L) ***p<0.001, **p<0.01, one-way ANOVA. Bars represent mean ± s.e.m. https://doi.org/10.7554/eLife.32036.003 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Normal adult zebrafish swimming behavior. This video is an example of typical swimming behavior following a cutaneous injection of 1% DMSO into the upper lip of an adult zebrafish. Note that the zebrafish exhibits minimal interaction with the walls of the tank, and its swimming capacity is not compromised. https://doi.org/10.7554/eLife.32036.005 Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Adult zebrafish injected with AITC demonstrate nocifensive behaviors. This video is an example of typical swimming behavior following a cutaneous injection of 10 µM AITC into the upper lip of an adult zebrafish. The zebrafish exhibits previously described nocifensive behaviors, such as dramatically reduced locomotion, bouts of freezing, and heightened respiration. https://doi.org/10.7554/eLife.32036.006 Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Adult zebrafish injected with IMQ demonstrate pruritic behaviors. This video provides a typical example of swimming behavior observed in adult zebrafish following a cutaneous upper lip injection of 100 µM IMQ. The fish not only exhibits increased swimming velocity in response to this stimulus, but also frequently rubs its lips against the walls of its tank, engaging in a behavior that is potentially analogous to mammalian scratching. https://doi.org/10.7554/eLife.32036.007 We have previously reported that noxious stimuli evoke locomotion in larval zebrafish (Gau et al., 2013). When 5dpf larval zebrafish were exposed to individual pruritogens, only IMQ elevated baseline locomotion (p<0.001), producing a dose dependent increase (Figure 1—figure supplement 1A). When coupled with our findings in CaMPARI transgenics, these results indicate that IMQ likely acts through somatosensory neurons to evoke behavioral responses. While 5-HT did produce a reduction in locomotion, it did not activate somatosensory neurons (Figure 1I,J). Given the limited sensitivity of the locomotor assay we were unable to differentiate nocifensive behavior elicited by AITC from potentially pruritic behavior elicited by IMQ. To address this issue, we employed an adult zebrafish behavioral assay (Correia et al., 2011; Maximino, 2011). Injection of IMQ into the lip elicited a lip-rubbing behavior that may constitute a form of itch-scratch response in zebrafish, a behavior that was absent in sham-injected control fish (p<0.001) (Figure 1K,L; Videos 1—3) and distinct from previously described zebrafish nocifensive, escape, or exploratory behaviors (Colwill and Creton, 2011; Egan et al., 2009; Levin et al., 2007). Consistent with studies of nocifensive behaviors, injection of AITC produced freezing behavior (p<0.01) (Figure 1—figure supplement 1C) as well as a significant decrease (p<0.05) in velocity not observed in control or IMQ injected fish (Figure 1—figure supplement 1B). Such distinct behavioral responses imply that zebrafish are capable of experiencing, and responding differentially to, discrete stimuli analogous to itch and pain in mammals. Trpa1b, but not tlr7, is required for both neuronal and behavioral responses to imiquimod The mechanism by which IMQ elicits itch in mammals is unclear. IMQ, a treatment for various skin disorders, acts through TLR7 to stimulate an immune response, with intense itching and painful burning commonly reported as side effects (Chang et al., 2005; Lebwohl et al., 2004). In mice, however, IMQ is reported to be itch selective, and only elicits scratching, but not nociceptive, behaviors (Liu et al., 2010; Kim et al., 2011). Furthermore, there is dispute surrounding TLR7’s role in IMQ-induced itch. One study found that Tlr7-/- mice showed deficits in IMQ evoked itch and proposed that Tlr7 expressed in dorsal root ganglion (DRG) neurons was mediating IMQ transduction (Liu et al., 2010). TLR7 has also been reported to couple with TRPA1 in DRG neurons to evoke nociception in response to other TLR7 agonists (Park et al., 2014). However, conflicting studies found that Tlr7-/- mice exhibited no deficits in IMQ-evoked itch, and RNAseq analysis of DRG neurons found no evidence for Tlr7 expression (Kim et al., 2011; Usoskin et al., 2015; Li et al., 2016). We therefore investigated whether TLR7 and/or TRPA1 were involved in transducing the neural and behavioral responses to IMQ in zebrafish. In situ hybridization studies in zebrafish larvae revealed that tlr7 mRNA expression is restricted to known hematopoietic regions in larval zebrafish (Bresciani, 2014; Du et al., 2011), and was notably absent in both TG and Rohon-Beard (RB) neurons (Figure 2A; Figure 2—figure supplement 1I). As expected, trpa1b expression was observed in both TG and RB neurons (Figure 2B; Figure 2—figure supplement 1C). Therefore, any role TLR7 could play in IMQ-evoked behavior would be via indirect mechanisms. Figure 2 with 1 supplement see all Download asset Open asset Trpa1b, but not Tlr7, is required for both neuronal and behavioral responses to IMQ. (A, B) In situ hybridization in 3dpf WT larvae probing for tlr7 and trpa1b mRNA respectively. (C, D) Larval locomotor assay of 5dpf tlr7+/+ and tlr7-/- (C) or trpa1b+/+ and trpa1b-/- (D) larvae. (E, F) Adult lip-rubbing behavioral assay of tlr7+/+/tlr7-/- (E) and trpa1b+/+/trpa1b-/-(F) fish. (C), (D), (E), (F), 100 μM IMQ used. ***p<0.001, **p<0.01, Student’s t-test. Bars represent mean ± s.e.m. (G, H) Representative calcium imaging traces of 3dpf trpa1b+/+ (G) and trpa1b-/- (H) larvae in a transgenic elavl3HuC:GCaMP5 background exposed to 100 μM IMQ, 50 μM AITC, and 1 mM 2-APB. B = blood, TG = trigeminal ganglion. https://doi.org/10.7554/eLife.32036.008 To determine if trpa1b and/or tlr7 are required for IMQ induced behaviors, we introduced early nonsense mutations in the coding sequences of both genes (Kimura et al., 2014). Tlr7-/- zebrafish larvae exhibited a significant (p<0.001) increase in total locomotion when exposed to IMQ (100 µM) that was indistinguishable from controls (Figure 2C). However, trpa1b-/- larvae demonstrated no response to IMQ (100 µM), while their WT siblings displayed normal IMQ induced behaviors (p<0.01, Figure 2D). These data support a mechanism where trpa1b, but not tlr7, is necessary for mediating behavioral responses to IMQ in larval zebrafish. As expected based on previous reports, behavioral responses to AITC were absent in trpa1b-/- fish (Figure 2—figure supplement 1F) (Prober et al., 2008). Notably, trpa1b-/- fish demonstrated an equivalent increase in locomotor behavior as their WT siblings when exposed to increased temperatures (Figure 2—figure supplement 1E). This indicates that the trpa1b mutation specifically affects Trpa1b-mediated sensations, rather than causing generalized sensory impairment. To test whether the presence of Trpa1b was necessary to mediate neuronal responses in larval zebrafish TG, we conducted in vivo calcium imaging using elavl3:GCaMP5g larvae (Akerboom et al., 2012) (Figure 2G). In WT larvae, IMQ activation was seen exclusively in a subset of AITC responsive neurons (4/36, n = 5 larvae). Trpa1b-/- fish, however, exhibited no response to either IMQ or AITC (0/89 total neurons, n = 5 larvae) (Figure 2H). Notably, the highly specific TLR7 agonist loxoribine did not evoke TG neuron activation, larval locomotion, or adult lip-rubbing behavior, further strengthening the finding that Tlr7 does not play a role in IMQ evoked behaviors in zebrafish (Figure 1I,J; Figure 1—figure supplement 1D). This finding is similar to reports in the mouse demonstrating that loxoribine does not elicit pruritic behavioral responses (Kim et al., 2011). Imiquimod directly activates TRPA1 To determine if TRPA1 directly interacts with IMQ to produce itch, we utilized the TLR7-deficient cell line HEK293T (Hornung et al., 2005). HEK cells transfected with zebrafish, mouse, and human Trpa1 showed a dose-dependent increase in intracellular calcium following application of IMQ and AITC that was not observed in HEK cells alone (Figure 3A–F; Figure 3—figure supplement 1A,C). Importantly, we found that loxoribine did not activate HEK cells transfected with zebrafish or mouse Trpa1 (Figure 3—figure supplement 2A–D), indicating that TRPA1 is responsive to IMQ, and not to TLR7 agonists in general. Figure 3 with 2 supplements see all Download asset Open asset Imiquimod directly activates TRPA1. (A–C) Calcium imaging of HEK cells transfected with zebrafish (A), mouse (B), and human (C) Trpa1 or Trpa1 + Tlr7. (D, E, F) Calcium imaging dose response curves of HEK cells transfected with zebrafish (D), mouse (E), and human (F) Trpa1, exposed to IMQ or AITC. (A–F) Numbers represent total cell counts per condition. (G) Patch clamp of HEK cell transfected with zebrafish trpa1b exposed to 100 μM IMQ. (H) Patch clamp dose response curve for HEK cells transfected with zebrafish trpa1b, mouse Trpa1, or mouse Trpa1 + mouse Tlr7. (I) Current density values of HEK cells exposed transfected with zebrafish trpa1b or mouse Trpa1 and exposed to 100 μM AITC or 100 μM IMQ. (G–I) n = 5 cells per condition. **p<0.01, Student’s t-test. Bars represent mean ± s.e.m. https://doi.org/10.7554/eLife.32036.010 While we observed no expression of tlr7 in the zebrafish TG (Figure 2A), given the lack of consensus over its functional role we sought to determine whether TLR7 might serve as a pruritic co-receptor that potentiates the TRPA1 response. We co-transfected HEK cells with Trpa1 and Tlr7 and examined the calcium responses following treatment with IMQ and observed no discernible differences in the average peak responses to IMQ between Trpa1 and Trpa1 + Tlr7 conditions (Figure 3A–C, Figure 3—figure supplement 1C). Whole cell electrophysiological experiments corroborated these findings. When stimulated with IMQ, voltage-clamped HEK cells transfected with mouse or zebrafish Trpa1 demonstrated a significant increase in current (Figure 3G; Figure 3—figure supplement 1G). Co-transfecting mouse Tlr7 with mouse Trpa1 in HEK cells had no demonstrable effect on current influx (Figure 3—figure supplement 1H). Additionally, no difference was found in the IMQ current density dose-response curves for cells transfected with zebrafish trpa1b, mouse Trpa1, or mouse Trpa1 + mouse Tlr7 (Figure 3H). In contrast to previous reports, we found no evidence that TLR7 coupled with TRPA1 in the presence of loxoribine as measured by ratiometric calcium imaging and whole cell electrophysiology in mouse and zebrafish (Figure 3—figure supplement 2A–D) (Liu et al., 2010). Together, our data suggest that TLR7 plays no role in the direct activation of somatosensory neurons, and does not appear to potentiate the response of TRPA1 to IMQ. Following these results, we confirmed that mouse and human TLR7 were present and functional in our assays (Figure 3—figure supplement 1I–K) (Mitchell and Sugden, 1995). Intriguingly, zebrafish Tlr7 did not respond to either IMQ or loxoribine in a dual-luciferase assay, suggesting that zebrafish Tlr7 is not activated by mammalian TLR7 agonists (Figure 3—figure supplement 2E). However, due to a lack of a Tlr7 positive control, we were unable to confirm that Tlr7 was functional in our heterologous expression system. With this caveat in mind, the lack of zebrafish Tlr7 response to these TLR7 agonists lends further credence to the conclusion that Tlr7 is not involved in somatosensory neuronal activation or behavior in this species. If TRPA1 does not couple with TLR7, but is instead directly activated by both IMQ and AITC, how could IMQ be itch-selective in the mouse? To address this question we assessed the EC50 and peak responses to IMQ and AITC in HEK cells transfected with Trpa1 from different species. The EC50 of IMQ for zebrafish, mouse and human TRPA1 was consistently higher than the AITC EC50, demonstrating that IMQ is a weaker agonist than AITC (Figure 3—figure supplement 1K). Notably, while the IMQ EC50 of zebrafish Trpa1 and human TRPA1 were only 2–3 fold greater than that of the EC50 of AITC, mouse TRPA1 demonstrated a ~40 fold difference between the EC50 of AITC and IMQ. Furthermore, only mouse TRPA1 elicited significantly lower maximum responses to IMQ than AITC (Figure 3D–F). In similar electrophysiology experiments, HEK cells transfected with zebrafish trpa1b exhibited identical current density responses upon stimulation with the maximum dose of either AITC or IMQ, but cells transfected with mouse Trpa1 exhibited significantly higher current density responses following stimulation with AITC, relative to IMQ (Figure 3I). Such physiological differences in TRPA1 function between species could provide a potential mechanism for the itch selectivity of IMQ in mice, implying that IMQ is not a strong enough mouse TRPA1 agonist to elicit nociception in this species. Conversely, the ability of both zebrafish and human TRPA1 to respond equally to AITC and IMQ at maximal doses potentially explains how IMQ can elicit both itch and pain sensations in humans, and suggests that the same may be observable in fish. Imiquimod responsive neurons are a primed subpopulation of TRPA1-expressing neurons In the preceding in vivo calcium imaging experiments, we observed that only a small proportion of zebrafish Trpa1+ neurons, identified by their responsiveness to AITC, were also responsive to the IMQ stimulus. To further explore this result, we used elval3:H2BGCaMP6 (Chen et al., 2013) transgenic zebrafish to record the response properties of larval TG neurons to IMQ and AITC. No neurons were found larvae. neurons, were responsive to IMQ (Figure Figure with 2 supplements see all Download asset Open asset Imiquimod responsive cells are a primed subpopulation of TRPA1 positive cells and affects behavioral and neuronal responses. (A, B) Representative traces from calcium imaging experiments in 3dpf zebrafish exposed to 100 μM IMQ and 50 μM AITC. TG neuron is in (A), while an only neuron is in (C) of neuronal neurons. n = neurons, n = only neurons. (D) of the maximum the 50 μM AITC between neuronal (E) of photoconverted neurons in 3dpf elavl3:CaMPARI zebrafish TG following stimulation with TRPA1 agonists. (F) of lip-rubbing behavior in adult zebrafish following to TRPA1 agonists. Bars represent ± s.e.m. **p<0.01, ***p<0.001, Student’s (D), one-way (E, The data that neurons are a subset a population of Trpa1+ TG neurons, implying that a population coding strategy for pruritus might be at This does not the question of how such an itch-selective Trpa1+ subpopulation might be activated by a TRPA1 agonist other Trpa1+ neurons that may for nociceptive behaviors. on our finding that IMQ is a weaker TRPA1 agonist than AITC, potential is that such an itch-selective Trpa1+ population is more sensitive to TRPA1 and can be activated by weaker stimuli. In neurons, we that the average maximum response to the IMQ was significantly lower than that of the AITC (p<0.001), implying that at the IMQ is a weaker TRPA1 than AITC in vivo (Figure 3—figure supplement Furthermore, we found that the maximum AITC response of neurons was significantly greater than that of only neurons (Figure neurons displayed a significantly greater average AITC response than only neurons Figure supplement 1B). These data suggest that TG neurons are primed to respond to TRPA1 agonists and support a model where weak TRPA1 such as IMQ at the could a potential subpopulation of Trpa1+ neurons. stimuli like AITC at the however, would activate the of Trpa1+ neurons to evoke nocifensive behaviors, with findings that nociception takes over itch sensation et al., et al., 2010; et al., 2011). To that IMQ activates a selective subset of Trpa1+ neurons as to activating Trpa1+ neurons we calcium imaging experiments in which 3dpf were exposed to of 100 μM IMQ. Of the neurons we identified five fish, to both of IMQ, only only to the of IMQ (Figure supplement Additionally, it is the single neuron that only to the of IMQ may also be a While the only our response the it is that the baseline may have a minimal response to the especially the low of the response. However, for of we to this in our Given the finding that only of AITC responsive neurons to of IMQ (100 if that this is the that any given AITC responsive neuron would respond to IMQ (100 and that responses to IMQ are in would only a small of neurons to be (Figure The finding that all neurons were that these neurons a distinct population of Trpa1 expressing neurons, primed to respond to low TRPA1 dependent stimuli. affects behavioral and neuronal responses Although the IMQ dose-response curve in zebrafish HEK cells was it was able to elicit the same of intracellular calcium that AITC This that at a IMQ might be able to neurons of the subpopulation observed in the larval calcium imaging experiments, eliciting neuronal and behavioral responses of AITC at nociceptive it is also that at low enough AITC is capable of eliciting the pruritic neuronal and behavioral responses we observed following application of IMQ. In CaMPARI fish we observed that the of AITC with a reduction in the of photoconverted neurons (Figure Additionally, higher IMQ an equivalent of neurons as of AITC (Figure suggesting that for TRPA1 eliciting pruritus or nociception is dependent more on than In vivo imaging our CaMPARI findings that affects which of Trpa1+ neurons are activated. In these experiments, we observed that the activated more neurons. a subset of neurons that to a of AITC to a lower of AITC and of those even neurons to 100 μM IMQ (Figure supplement 1C). Furthermore, the IMQ to µM in adult behavioral experiments evoked nocifensive behaviors such as elevated freezing and significantly reduced and the itch-like lip behavior seen at 100 µM was notably absent 2011; (Figure Figure 1—figure supplement Figure supplement 1D). Conversely, low of AITC elicited both itch-like lip behavior and increased velocity (Figure Figure supplement 1C). These data indicate that the subpopulation of Trpa1+ neurons that itch behavior in the zebrafish are distinct in their sensitivity to TRPA1 but can be activated by either AITC or IMQ at the to produce equivalent behaviors. TRPA1 itch behavior and neuronal responses in in the mouse We found that IMQ elicited responses in of DRG neurons (Figure from WT animals. To determine if TRPA1 IMQ responses in mice, we examined IMQ-evoked responses in DRG neurons from both WT and animals using ratiometric calcium We found that both IMQ and AITC responses were in DRG neurons from while neurons from WT siblings exhibited normal to both stimuli (Figure Furthermore, application of loxoribine did not elicit calcium responses in mouse DRG neurons, providing evidence that TLR7 stimulation does not in activation of somatosensory neurons (Figure supplement 1H). We whether IMQ-evoked scratching behavior was also dependent on TRPA1. IMQ did not evoke nocifensive behaviors in WT mice = with previous reports (Kim et al., 2011). However, scratching bouts at a low of IMQ were significantly in mice, demonstrating that TRPA1 is required for normal IMQ-induced scratching behavior (Figure a higher dose of IMQ evoked equivalent scratching behavior in both WT and mice (Figure supplement 1G). This result, with our