Trpm3 mRNA levels in dorsal root ganglion neurons innervating the inflamed paw, and augmented TRP channel-mediated calcium responses, both in the cell bodies and the intact peripheral endings of nociceptors. In particular, inflammation provoked a pronounced increase in nociceptors with functional co-expression of TRPM3, TRPV1 and TRPA1. Finally, pharmacological inhibition of TRPM3 dampened TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw, but not in those innervating healthy tissue. These insights into the mechanisms underlying inflammatory heat hypersensitivity provide a rationale for developing TRPM3 antagonists to treat pathological pain.Genetic ablation or pharmacological inhibition of the heat-activated cation channel TRPM3 alleviates inflammatory heat hyperalgesia, but the underlying mechanisms are unknown. We induced unilateral inflammation of the hind paw in mice, and directly compared expression and function of TRPM3 and two other heat-activated TRP channels (TRPV1 and TRPA1) in sensory neurons innervating the ipsilateral and contralateral paw. We detected increased  Painful stimuli are detected by nociceptors in peripheral tissues and are transmitted as action potentials towards the central nervous system to elicit pain sensation . Under p-/-/Trpv1-/-/Trpa1-/-Trpm3 triple knockout mice  superfamily of cation channels play key roles as primary molecular sensors in nociceptor neurons, directly involved in translating external stimuli into neuronal activity and pain . For insout mice . These rout mice  or only out mice  is suffiTo address this problem, we induced experimental inflammation in one hindpaw of mice and evaluated changes in expression and function of TRPM3, TRPA1, and TRPV1 in sensory neurons. By combining retrograde labeling and quantitative in situ hybridization using RNAscope, we obtained evidence suggesting an increase in mRNA encoding TRPM3 in dorsal root ganglion (DRG) neurons innervating the inflamed paw. Furthermore, we developed GCaMP3-based confocal imaging of DRG neurons in situ, which allowed us to measure inflammation-induced changes in the activity of TRPM3, TRPA1, and TRPV1, both in the neuronal cell bodies and in the intact nerve endings in the skin. These experiments indicate that tissue inflammation provokes a pronounced increase in the activity of nociceptors co-expressing TRPM3 with TRPV1 and TRPA1 channels. Notably, pharmacological inhibition of TRPM3 not only eliminated TRPM3-mediated responses but also reduced TRPV1- and TRPA1-mediated responses in DRG neurons innervating the inflamed paw. These findings elucidate a prominent role of TRPM3 in inflammatory hyperalgesia, and provide a rationale for the development of TRPM3 antagonists to treat inflammatory pain.To investigate the consequences of tissue inflammation on the expression and function of TRPM3, TRPA1, and TRPV1, we used a mouse model of complete Freund’s adjuvant-induced (CFA-induced) peripheral inflammation. In this established model, unilateral hind paw injection of CFA produces a strong inflammatory response associated with pronounced hyperalgesia, and the contralateral hind paw can be used as an internal control.+) and unlabeled (WGA-AF647-) neurons on the ipsilateral side, and between WGA-AF647+ neurons on the ipsi- versus contralateral side. Importantly, this analysis revealed a significant increase in the TRPM3 mRNA levels in the ipsilateral, WGA-AF647+ DRG neurons, both when compared to WGA-AF647+ neurons on the contralateral side, and to WGA-AF647- neurons on the ipsilateral side  of the levels in the WGA-AF647+ neurons on the contralateral side, and to 133%  of the levels in WGA-AF647- neurons on the ipsilateral side. Notably, almost all (98 ± 2%) of the ipsilateral, WGA-AF647+ DRG neurons in the six tested animals showed a positive RNAscope signal for TRPM3, compared to between 70% and 80% TRPM3-positive neurons in the ipsilateral WGA-AF647- and contralateral DRG neurons  of the level in the contralateral WGA-AF647+ DRG neurons and to 83%  of the level in ipsilateral WGA-AF647- neurons  of the level in the contralateral WGA-AF647+ DRG neurons and to 126%  of the level in ipsilateral WGA-AF647- neurons , TRPA1  and TRPV1 (capsaicin), and of a depolarizing high K+ solution resulting in TRP channel-independent Ca2+-influx via voltage-gated Ca2+ channels  when compared to the ipsilateral WGA-AF647- neurons and to 448%  when compared to the WGA-AF647+ contralateral neurons  when compared to the WGA-AF647+ contralateral neurons  when compared to the ipsilateral WGA-AF647- neurons and to 205%  when compared to the WGA-AF647+ contralateral neurons  when compared to the ipsilateral paw skins measured in the absence of isosakuranetin. In these isosakuranetin-treated skin preparations, the surface of nerve endings responding to the TRPA1 and TRPV1 agonists were similar in the ipsilateral and contralateral skin .2+ responses to specific agonists; this increased functionality is detected both in the neuronal cell bodies and in the nerve endings in the inflamed skin; (3) increased TRP channel activity in inflammatory conditions is associated with an increase in the fraction of cell bodies and nerve endings that functionally co-express TRPM3 with TRPV1 and TRPA1, and (4) pharmacological inhibition of TRPM3 not only eliminates Ca2+ responses to TRPM3 agonists, but also reduces responses to TRPV1 and TRPA1 agonists in neurons innervating the inflamed paw. Taken together, these data demonstrate, for the first time, an increased molecular and functional expression of TRPM3 in nociceptors innervating inflamed tissue, and indicate that tissue inflammation is associated with a functional upregulation of all three molecular heat sensors implicated in the normal pain response to noxious heat.In recent work, we demonstrated that three TRP channels  act as redundant sensors of acute heat: neuronal heat responses and heat-induced pain are preserved in mice in which two of these three TRP channels were genetically ablated, but combined elimination of all three channels fully abolishes the withdrawal reflex from a noxious heat stimulus . Notably+, we consider the possibility that an overall increase in TRPA1 mRNA in the order of 30% or less would not be revealed in our statistical analysis.Whereas this is, to our knowledge, the first study pointing at possible inflammation-induced alterations in TRPM3 expression at the mRNA level, earlier studies had already investigated alterations in TRPA1 and TRPV1 mRNA using the rodent CFA model. In line with our current results, several earlier studies report that transcript levels for TRPV1 were found unchanged after CFA treatment when assessed using quantitative PCR or RNA protection assays . However+ (see methods), it is not unlikely that a subset of DRG neurons that innervate the inflamed paw did not take up sufficient dye to be considered as retrogradely labeled. This may explain why we also observed a mild increase in TRPM3 mRNA  and TRPV1-GCaMP3 mice on a C57BL/6J background were used. TRPV1-GCaMP3 mice were generated by crossing Rosa26-floxed-GCaMP3 mice  with TRPReagents were purchased from Sigma-Aldrich  unless otherwise indicated.To specifically label afferents from the hind paw skin, 10 μl Alexa Fluor 647-conjugated wheat germ agglutinin  was injected intraplantar into both hind paws. The injections were performed 7 days prior to imaging. Initial experiments showed no edematous or hyperalgesic response to the retrograde label.Local inflammation was induced by injection of 10 µl complete Freund’s adjuvant  into the plantar surface of the ipsilateral hindpaw of the studied mouse. The contralateral hind paw was injected with 10 μl vehicle . All ipsilateral mouse hind paws showed substantial edema 24 hr after the injection, which was not observed in the control paw.2 inhalation, and skin and DRG tissue were collected immediately.Animals were euthanized using COSensory nerve ending recordings were obtained from isolated dorsal hind paw skin preparations. The fur was removed with tape and the skin was gently dissected from the underlying tissue.Bilateral L3-L6 DRGs were isolated. In brief, the spinal column was isolated, cleaned, and split sagittally. The spinal cord, meninges covering the DRG, and the distal axon bundles were removed. Finally, a small segment of the spinal column containing the DRG of interest was extracted.3, 1.67 NaH2PO4, 3.48 KCl, 0.69 MgSO4, 9.64 D-gluconic acid, 5.55 D-glucose, 7.6 Sucrose and 2 CaCl2. The pH was buffered to 7.4 using carbogen gas (95% O2 and 5% CO2). In some experiments, the latter 30 min incubation solution as well as the proceeding perfusion SIF solution contained TRPM3 blocker isosakuranetin . During image acquisition, skin tissue was fixed with the corium side up in a glass-bottom microwell dish . The spinal column sections were placed in the SIF-containing microwell dishes with the DRG facing towards the objective. Tissues were continuously superfused with 37°C SIF solution.The isolated skin tissue and spinal column segments were maintained 1 hr on ice and 30 min at room temperature in synthetical interstitial fluid (SIF) solution, containing (mM): 125 NaCl, 26.2 NaHCO+ retrogradely labeled cell bodies. The used stimuli to activate the specific TRP channels were for TRPM3: PS (100 μM) + CIM0216 (1 μM); for TRPA1: MO (100 μM); and for TRPV1 capsaicin (1 μM). Compounds were diluted in SIF supplemented with 0.1% DMSO. A depolarizing high K+ solution in which all NaCl was replaced by KCl was applied at the end of each experiment to identify excitable cells. All solutions were applied at 37°C and administered to the immediate proximity of the recording field.Calcium imaging of the skin-nerve and in situ DRG preparations was performed on an inverted spinning disk confocal microscope , equipped with a 20x air objective (NA 0.8), a 488 nm laser light and a EMCCD camera . For image acquisition and instrument control, Andor iQ software was used. A z-stack of 11 frames  was captured consecutively during the entire measurement at a speed of 0.25 Hz. Agonists were diluted in SIF and applied using a heated perfusion system . Before image acquisition, the DRG samples were excited at 640 nm to detect WGA-6472 in B27 (Invitrogen) supplemented neurobasal A medium, containing 2 ng/ml GDNF (Invitrogen) and 10 ng/ml NT4 (Peprotech). Isolated DRG neurons were loaded with 2 µM Fura-2-acetoxymethyl ester  for 30 min at 37°C. Fluorescence was measured during alternating illumination at 340 and 380 nm (yielding the Fura-2 fluorescence ratio) and at 480 nm (to monitor GCaMP3 fluorescence) using a CellM (Olympus) fluorescence microscopy system.In a limited set of experiments, shown in Spinning disk confocal microscope recordings were first z-averaged in Fiji  and corrected for translational drift using a reference image that averaged the first 10 frames prior to tissue stimulation .0 values.After drift correction, we used custom-made routines in Igor (Wavemetrics) to subtract fluorescence background. The background was obtained by fitting a fourth-order polynomial surface to non-responsive areas, which were defined as those pixels where the coefficient of variation in time was below a threshold, which was automatically obtained following the Otsu algorithm. After background correction, we used the ‘constrained non-negative matrix factorization for micro-endoscopic data’ framework  to identify ROIs as contiguous areas with temporally correlated activity . The bac+) and unlabeled (WGA-AF647-) neurons were initially identified visually; for analysis, only cells with a fluorescence signal that exceeded five times the standard deviation of the background fluorescence in this channel were retained in as WGA-AF647+. Raw fluorescent traces were converted to F/F0, where F is the fluorescence at a certain time point of interest, and F0 is the baseline fluorescence before stimulation. Cells that did not respond to high K+ where excluded from all further analysis.These recordings were analyzed using a general analysis protocol (GA2) in NIS-elements . Retrogradely labeled  had to exceed the standard deviation of the dF/dt signal before stimulation.In situ hybridization was performed on 10-μm-thin cryosections of DRG neurons innervating the contralateral and ipsilateral hind paw. DRG tissue was isolated as described previously and immersed in 10% neutral buffered formalin immediately after isolation. Prior to isolation, retrograde labeling and inflammation were induced as described previously. RNA transcripts were detected using the RNAscope 2.0 assay according to the manufacturer’s instructions . Probes for mTrpv1 (cat number: 313331), mTrpm3 (cat number: 459911), mTrpa1 (cat number: 400211) and Pgp9.5 (cat number: 561861-C2) were purchased from Advanced Cell Diagnostics. The staining was performed using the RNAscope Fluorescent Multiplex Reagent Kit (cat number: 320850). Cells were stained with DAPI and mounted on the slide with Gold Antifade Mountant. A total of 5802 neurons were analyzed from 12 lumbar (L5) DRG neurons , isolated from six mice.+ if the mean fluorescence exceeded five times the standard deviation of the background fluorescence in this channel. Due this stringent criterium, it is likely that some retrogradely labeled neurons with modest WGA-AF647 fluorescence are included in the WGA-AF647- group. Individual dots in the green channel were detected using a rolling ball filter (1 µm) and spot detection (0.8 µm). To correct for dot clusters arising from the overlap of individual RNAscope-dots, we determined the average intensity of single dots in each slide, and calculated the theoretical number of individual dots in a cluster as the ratio of the cluster intensity and the average intensity of single dots, as outlined in the manufacturers instructions . Cells containing five or more RNAscope dots were considered as positive for the respective TRP channels.Slides were imaged using a Nikon NiE - Märzhäuser Slide Express two equipped with a Hamamatsu Orca Flash 4.0 in combination with a Plan Apo 40x (NA 0.95), and custom made JOBS-GA2 protocol for sample detection. For analysis, a GA3 script in NIS-Elements 5.20.00 was used. Cells were segmented manually based on the Pgp 9.5 signal. Cells were considered as WGA-AF647versus CFA).Data analysis was performed using Origin software (OriginPro 2019b). Shapiro–Wilk test was used to test the normality of the data, determining whether parametric or non-parametric tests were applied. The specific parametric and non-parametric tests that were used are specified in the text and legends. Summary data for parametric datasets are shown as mean + SEM. We used bootstrapping with 10,000 bootstrap resamples to calculate 95% confidence intervals for non-parametric datasets. No statistical methods were used to predetermine the number of animals used in this study; since no a priory data were available for the novel imaging approaches used in this work, a power calculation was not feasible. However, our sample sizes are similar to those generally employed in other studies in the field, and potential limitations due to insufficient power are discussed in the text. Imaging of the DRG and skin preparations and subsequent data analysis were performed by researchers that were blinded for the treatment . They also use calcium imaging in ex vivo preps (whole ganglia and skin) to examine the function of TRPM3 and two other TRP channels important for temperature and pain signaling (TRPV1 and TRPA1) using pharmacology. Overall, their approach is technically innovative and the concept that TRPM3 is required for increased TRPV1 and TRPA1 activity is intriguing.Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Kenton Swartz as the Senior Editor and Reviewing Editor. Although the work is of interest, we regret to inform you that the findings at this stage are too preliminary for further consideration at eLife.Thank you for choosing to send your work, "Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue", for consideration at eLife is to only invite revisions if the additional work can be done within 2 month. We encourage you to consider the reviewers comments carefully, and if you choose to address all of their comments, we would be willing to reconsider your work as a new submission.As you will see, both reviewers thought your study was exciting and both thought your skin preparation had real potential. However, both reviewers thought that more needed to be done in terms of controls, quantification and providing more details, in particular for the new preparation. Although there was considerable enthusiasm for your work, the policy at Reviewer #1:In their current manuscript, Mulier et al. investigate the molecular mechanisms that underlie the development of increased sensitivity to heat pain after inflammation. Their interest focuses on the ion channel Trpm3 that this group has previously shown is thermosensitive and contributes to heat sensation. Here, they ask whether the expression of Trpm3 in sensory neurons is altered during peripheral inflammation and whether its function contributes to hyperalgesia. They authors provide evidence that Trpm3 message is upregulated in the sensory neurons innervating inflamed tissues using a combination of retrograde tracing and single molecule florescent in situ hybridization (FISH). They also use calcium imaging in ex vivo preps (whole ganglia and skin) to examine the function of TRPM3 and two other Trp channels important for temperature and pain signaling (TRPV1 and TRPA1) using pharmacology.Overall, their approach is technically innovative and the ideas interesting. While it is clear Trp channels contribute to heat and pain sensitization, it is still unclear their precise roles and interactions. Thus, the concept that TRPM3 is required for increased TRPV1 and TRPA1 activity is really intriguing. However, the manuscript title and claims that the upregulation of TRPM3 is necessary and sufficient to cause nociceptor hyperexcitability are too bold. The data show a correlation between elevated TRPM3 expression and functional TRP channel responses in inflamed tissue, but a causal relationship is not well-established. Moreover, the robustness of the initial premise that TRPM3 is upregulated in inflamed tissue is somewhat debatable. How TRPM3 might be affecting TRPV1 or TRPA1 function is really unclear. Finally, I have some issues regarding how the RNAscope and skin calcium imaging experiments were quantified.eLife. I also recommend reducing the strength of the claims and that including additional analyses would be helpful.Given the effect sizes are pretty small, these issues need addressing before publication in Figure 1:– In Figure 1B, is the Pgp9.5 labeling via antibody or RNAscope? This is really unclear from the figure legend and methods. No product number was listed in Methods.– To accompany Figure 1B, it would help to show an in situ image representing differences between control and CFA-treated TRPM3 neurons. Only control example images are shown– In Figure 1B, why does the TRPV1 in situ staining look much sparser than expected based on published gene expression analyses? Typically, Trpv1 ISH labels many small diameter neurons quite strongly, where are these cells?– In Figure 1C, the method of FISH quantification is non-standard. Typically for single molecule FISH, the number of puncta per cell are quantified (as detailed in the RNAscope paper cited in the Results section), not the overall fluorescence intensity of an ROI. These data would be more accurate if reanalyzed using a standard "puncta counting" approach.– Furthermore, difference between animals (not cells) should be shown.– Overall, the data in Figure 1 are not very convincing without a re-analysis (as described in the above point). A secondary method to confirm the FISH results would greatly strengthen the data, such as using FACS isolation and qRT-PCR analysis of TRP genes in WGA-positive neurons.2+ imaging appear to have elevated TRP gene expression and responses compared to the contralateral WGA-negative neurons? While I can see that the WGA labeling is not perfect, it seems a bit odd that the entire ipsilateral DRG is inflamed, not just the paw-innervating neurons.– Why do the ipsilateral WGA-negative neurons in the Figure 1C FISH and the Figure 2D CaFigure 2:– The data for WGA-negative cells should also be shown– In general, normalization to high potassium is still not sufficient to make very detailed comparisons between cells without using a ratiometric calcium probe. Normally, this isn't an issue and many in vivo studies rely on GCaMP, but here the effect sizes are small enough to warrant caution.– In Figure 2D-F, data from the Iso treatment experiments should additionally be shown for WGA-negative neurons . This is needed to demonstrate that the TRPM3 antagonist doesn't affect TRPA1 and TRPV1 responses at baseline in the neurons that don't innervate the inflamed tissue. This seems to be a particularly critical control.– In the bar plots, the Y-axes scales are each different and some have break lines. Things should be shown on the same scale to make comparisons across graphs easier– I recognize performing new experiments under the current conditions would be tough, but it is strange they don't look at heat responses, since this is what the title and Abstract of the paper are aboutFigures 3 and 4:– Figure 3 really is an example of the skin imaging technique, which is I think very cool. However, I am not convinced about the quantification in Figure 4. They admit that they cannot normalize to the maximum response. I don't see how they can account for the natural variation between different skin preparations . Overall, I'm not convince this shows what the authors claim- the effects are very small and the spread of the data quite large.At the end of the section, the authors write: "Taken together, these results indicate that inflammatory heat hyperalgesia is associated with increased functional expression of all three heat-activated TRP channels at the level of the DRG cell bodies." But they only show increased expression of TRPM3 in Figure 1.Reviewer #2:The authors have recently demonstrated that heat-induced pain in naïve mice depends on a trio of heat-activated TRP channels, TRPM3, TRPA1 and TRPV1. However, with tissue inflammation, genetic ablation or pharmacological inhibition of only TRPV1 or only TRPM3 inhibition can fully suppress inflammatory heat hyperalgesia. Here the authors seek to dissect the relative contributions of TRPM3, TRPA1 and TRPV1 to nociceptor sensitization during tissue inflammation.In this manuscript, the authors use RNAscope to demonstrate that there is an increase in TRPM3 expression 1 day following CFA injection in the hind paw, specifically in the neurons that innervate the hind paw; there is no difference in TRPA1 or TRPV1 expression. Using whole DRG calcium imaging and a novel ex vivo skin calcium imaging approach, they reveal that neurons are more sensitive to TRPV1 and TRPA1 agonists following CFA hind paw injections and that this sensitivity is dependent on TRPM3 activity; V1 and A1 hypersensitivity can be blocked with the M3 antagonist isosakuranetin. Based on these experiments, TRPM3 antagonists may be viable therapeutic candidates for heat hypersensitivity. The paper is generally well written and accessible to a broad audience.• The authors claim that M3-dependent TRPA1 hypersensitivity occurs following CFA but neither the amplitude of TRPA1 responses  nor the percentage of neurons responding to TRPM3+TRPA1 are very supportive of this claim. Furthermore, the increase in A1 sensitivity does not appear to be specific to paw-innervating (i.e. WGA-labeled) neurons, because the same increase (based on a few outliers) occurs in non-paw innervating unlabeled neurons. While the skin imaging data are more convincing, the authors should dampen their claim that TRPM3 expression increases TRPA1 sensitivity following injury.• Since TRPV1-GCaMP3 mice are being used for both DRG and skin calcium imaging, how is it possible that not all cells respond to TRPV1 agonists? In other words, how are M3 only, A1 only, or M3+A1 responders detectable using this genetic line since only TRPV1 positive neurons would express GCaMP3? It seems that this would limit the authors' ability to detect changes in A1 sensitivity following injury. If this population of TRPV1-lineage neurons includes all of the TRPM3 and TRPA1-expressing neurons in the adult, then this should be made even more clear in the manuscript.• The authors should discuss whether there is potential cross desensitization/sensitization between agonist in both the DRG and the peripheral skin calcium imaging experiments, since cross-interactions between agonists could limit the strength of their conclusions.• More information/citations should be provided as to the dose/concentration chosen for the TRPM3 antagonist isosakuranetin, particularly since a single concentration was used of this single antagonist.• The authors are commended for ex vivo imaging of sensory afferent terminals in the skin. Depending on the scientific question, this approach could have many benefits over imaging exclusively at the level of the DRG. Since this is the first description of this method, the authors should further clarify the methods in detail and address the following questions:How large is the bath containing the skin? How does buffer exit the bath? It appears that agonists are being presented to the tissue for 30-45 s. with an equivalent washout period between compounds . Is this sufficient time for the agonists to both access sensory terminals deep in the skin and then be washed away from the tissue? More details are needed on whether there is potential desensitization from one agonist to the next if insufficient time is allowed for agonist washout. More details on this method, the regions of interests chosen and how the data are quantified in a blinded manner should be provided.• The authors should comment on the use of a saphenous  preparation use in the context of the current injury model (i.e. 10 µL injection of CFA to the plantar surface of the paw)?I don't think that the use of the contralateral paw is the best control since there could be segmental effects, and separate animal control would be better. That said, this would require the entire data set to be re-generated and I'm not going to recommend this. But this is something that I think that the authors should note going forward and avoid using the contralateral paw as the control.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by Kenton Swartz as the Senior Editor and Reviewing Editor. The following individual involved in review of your submission has agreed to reveal their identity: Cheryl L Stucky (Reviewer #1).Thank you for submitting your article "Upregulation of TRPM3 in nociceptors innervating inflamed tissue" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as The editors and reviewers found the revised manuscript to be greatly improved, and we appreciate your sincere efforts to address the concerns that were raised. Overall, the conclusions better align with the results obtained, although we have some remaining concerns with conclusions concerning the RNAscope and skin prep results.RNA scope:The example images in Figure 1 are improved, however the new staining for Trpm3 looks to either be quite non-specific or to have high background? Either might affect the conclusions. In addition, the reviewers remain unconvinced that TRPM3 expression increases in response to inflammation. Some changes might seem big percent-wise but cell to cell variation and the biological meaning of such differences should also be considered: for example, what's the difference between an average of 20 and 30 puncta when the range of variation between cells is far greater? While these results may be suggestive, our consensus is the RNAscope results are not conclusive. We were much more convince by the increased functional responses to TRPM3 agonists. We request that you tone down conclusions concerning the RNAscope results throughout the manuscript. For example, in the Results section the following sentence:“Taken together, these results indicate that inflammation is associated with a significantly increased transcription of TRPM3, specifically in sensory neurons innervating the inflamed tissue, whereas no inflammation-related changes were found in the mRNA levels of TRPV1 and TRPA1.”could be easily improved by replacing 'indicate' with 'suggest' and "a significantly" with 'an'.Skin assay:The limitations of the assay are clearly spelled out in the Results section, but as analyzed the results are inherently qualitative. As such we suggest also toning down the conclusion here. For example, in the Results the following sentence:“Overall, these results represent, to our knowledge, the first direct observation of functional upregulation of all three heat-activated TRP channels in intact nerve endings in inflamed skin.”could be improved by inserting 'qualitative' before 'results' to remind the reader of the nature of the measurements. [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:In their current manuscript, Mulier et al. investigate the molecular mechanisms that underlie the development of increased sensitivity to heat pain after inflammation. Their interest focuses on the ion channel Trpm3 that this group has previously shown is thermosensitive and contributes to heat sensation. Here, they ask whether the expression of Trpm3 in sensory neurons is altered during peripheral inflammation and whether its function contributes to hyperalgesia. They authors provide evidence that Trpm3 message is upregulated in the sensory neurons innervating inflamed tissues using a combination of retrograde tracing and single molecule florescent in situ hybridization (FISH). They also use calcium imaging in ex vivo preps (whole ganglia and skin) to examine the function of TRPM3 and two other Trp channels important for temperature and pain signaling (TRPV1 and TRPA1) using pharmacology.Overall, their approach is technically innovative and the ideas interesting. While it is clear Trp channels contribute to heat and pain sensitization, it is still unclear their precise roles and interactions. Thus, the concept that TRPM3 is required for increased TRPV1 and TRPA1 activity is really intriguing.However, the manuscript title and claims that the upregulation of TRPM3 is necessary and sufficient to cause nociceptor hyperexcitability are too bold. The data show a correlation between elevated TRPM3 expression and functional TRP channel responses in inflamed tissue, but a causal relationship is not well-established.and sufficient to cause nociceptor hyperexcitability, and we agree that such a statement would be too bold at this point. Maybe it is because none of us is a native English speaker, but we did not interpret our statements that "TRPM3 is an important/key driver of nociceptor hyperexcitability excitability" to be synonymous to "TRPM3 is necessary and sufficient to cause nociceptor hyperexcitability".We feel that this is based on a misunderstanding. At no point in the manuscript did we intend to claim that the upregulation of TRPM3 is necessary What we meant is that TRPM3 is an important contributor. We felt that such a statement was justified based on the novel findings that (1) TRPM3 mRNA expression is specifically upregulated, (2) TRPM3 function is robustly increased, particularly in neurons co-expressing TRPV1 and TRPA1, and (3) inhibition of TRPM3 function restores TRPV1- and TRPA1-mediated responses to normal levels, along with earlier findings that TRPM3 KO or pharmacological inhibition largely abolished hypersensitivity. We have reworded the title to something more unambiguous: “Upregulation of TRPM3 in nociceptors innervating inflamed tissue.”, and also revised all other instances where it could be implied that TRPM3 upregulation is sufficient to cause neuronal hyperexcitability. We do not think that these changes change the conclusions or importance of our manuscript.Moreover, the robustness of the initial premise that TRPM3 is upregulated in inflamed tissue is somewhat debatable.We feel that our initial way of presenting the data, showing all individual data points, may have led the reviewers to underestimate the actual magnitude of the TRPM3 upregulation. As outlined in detail below, the effect size of the effect is very robust and highly significant.How TRPM3 might be affecting TRPV1 or TRPA1 function is really unclear.In our Discussion, we propose a scenario whereby increased molecular and functional expression of TRPM3 in neurons innervating inflamed tissue increases the excitability of nociceptors co-expressing TRPA1 and TRPV1, contributing to the augmented responses to agonist stimulation. As such, less TRPV1 or TRPA1 activity would be sufficient to cause neuronal action potentials, leading to enhanced neuronal responses to agonists. We also discuss how this could explain that both TRPM3 knockout and TRPV1 knockout mice do not develop inflammatory hyperalgesia. At this point, we do not have any evidence for a direct molecular interaction between TRPM3 and the two other channels, but also do not exclude such a possibility.Finally, I have some issues regarding how the RNAscope and skin calcium imaging experiments were quantified.As outlined below, these issues have been resolved with additional analyses and further clarification of our methods.Given the effect sizes are pretty small, these issues need addressing before publication in eLife.pretty small." For instance, concerning TRPM3, we observe a >60% increase in mRNA  and >300% increase in functional responses (P<10-6). We also reanalyzed the data sets according to the different suggestions of this reviewer , and the results remain very similar and highly significant.See below. We politely disagree that the effect sizes are "From this and later comments, including also one comment from reviewer #2, we now realize that the way of representing our data  was maybe not the best choice to visualize the magnitude of the effects . By including additional summary plots and analyses, we are convinced that the reviewer will appreciate the size and robustness of our main conclusions.I also recommend reducing the strength of the claims and that including additional analyses would be helpful.We have carefully revised all the claims that we make in the manuscript and modified them whenever they could be perceived as too strong and not fully supported by the data.Figure 1:– In Figure 1B, is the Pgp9.5 labeling via antibody or RNAscope? This is really unclear from the figure legend and Materials and methods. No product number was listed in Materials and methods.We apologize for this omission. Pgp9.5 labeling was also done via RNAscope. This is now indicated in the Materials and methods.– To accompany Figure 1B, it would help to show an in situ image representing differences between control and CFA-treated TRPM3 neurons. Only control example images are shownWe now provide better images, showing both the vehicle- and CFA-treated conditions.– In Figure 1B, why does the TRPV1 in situ staining look much sparser than expected based on published gene expression analyses? Typically, Trpv1 ISH labels many small diameter neurons quite strongly, where are these cells?Thank you for pointing this out. There is indeed a large number of neurons that are intensely labeled by the TRPV1 RNAscope label, much more prominent than for TRPM3 and TRPA1. However, we agree that the original image of the in situ staining did not show this clearly, due to inappropriate contrast. This is now much more evident in the provided images in the new Figure 1A. In the original manuscript, the strongly labeled cells could be appreciated from the summary data, showing cells with very high dot intensity values compared to TRPM3 and TRPA1. In the new manuscript, Figure 1—figure supplement 2 shows cells with up to 1000 TRPV1 RNAscope dots, consistent with the literature.– In Figure 1C, the method of FISH quantification is non-standard. Typically for single molecule FISH, the number of puncta per cell are quantified (as detailed in the RNAscope paper cited in the Results section), not the overall fluorescence intensity of an ROI. These data would be more accurate if reanalyzed using a standard "puncta counting" approach.This is a misunderstanding based on a too brief explanation of the analysis method in the original manuscript, for which we apologize. We did not analyze the overall fluorescence of a ROI, but only the summed intensity of all spots after subtraction of non-specific background. This quantification is equivalent to the number of spots but also corrects for the fact that brighter/larger spots likely arise from superimposed/overlapping RNAscope dots. However, we understand that a puncta counting approach is the standard, and now report the number of dots as the prime parameter. This does not alter any of our conclusions.– Furthermore, difference between animals (not cells) should be shown.We agree that it is important to also look at differences between animals. However, given that the number of dots per cell is distributed in a highly non-Gaussian manner, including many negative cells, important information would get lost if we would only show a mean or median value per animal. Therefore, we now include two analyses in the manuscript. First, we provide a statistical comparison of the number of dots/cell for all analyzed cells  from the different mice . In addition, we also calculated the percentage of positive neurons (with a cutoff of 5 RNAscope dots) for each condition per mouse . Both analyses point at a robust and highly significant increase in TRPM3 mRNA expression in the neurons innervating the inflamed paw.– Overall, the data in Figure 1 are not very convincing without a re-analysis (as described in the above point). A secondary method to confirm the FISH results would greatly strengthen the data, such as using FACS isolation and qRT-PCR analysis of TRP genes in WGA-positive neurons.As outlined above, we believe that the additional analyses and statistics provide convincing evidence for a robust increase in TRPM3 mRNA expression. In the text, we now additionally provide a clear indication of the size of the effects, including confidence intervals and P values. As we now write: "On average, TRPM3 mRNA levels in the ipsilateral, WGA-AF647+ DRG neurons were increased to 163%  of the levels in the WGA-AF647+ neurons on the contralateral side, and to 133%  of the levels in WGA-AF647- neurons on the ipsilateral side."While FACS isolation and qPCR could, in principle, be a valuable approach, our experiences indicate that there is an unequal survival and representation of the different subtypes of DRG after the dispersion of the neurons and FACS sorting. Moreover, the procedure to isolate and disperse the neurons may by itself, influence mRNA expression levels. Therefore, we believe that our quantitative in situ hybridization approach is at this point, superior. Furthermore, the increased functional responses to TRPM3 agonists  provide further independent support for the increased expression of TRPM3.2+ imaging appear to have elevated TRP gene expression and responses compared to the contralateral WGA-negative neurons? While I can see that the WGA labeling is not perfect, it seems a bit odd that the entire ipsilateral DRG is inflamed, not just the paw-innervating neurons.– Why do the ipsilateral WGA-negative neurons in the Figure 1C FISH and the Figure 2D CaWe used a relatively conservative fluorescence threshold for a neuron to be considered WGA-positive. Therefore, there is likely a subset of neurons that innervate the injured paw but contain only a low level of WGA-AF647, and are therefore nevertheless included in the WGA-negative group. This is now stated in the Materials and methods section. Moreover, the CFA-induced inflammation causes massive inflammation in the entire hind paw, which may include regions where little or no WGA-AF647 was present. Therefore, it can be expected that part of the ipsilateral, WGA-negative neurons also innervate the inflamed paw, which may explain why we see a mild increase in mRNA and functional responses in these cells. For these reasons, we consider the WGA-positive neurons on the contralateral side as a cleaner control. Finally, it cannot be excluded that inflammation has some effects on neighboring DRG neurons within the same ganglion that are not innervating the inflamed paw. We now briefly discuss these issues in the revised manuscript.Figure 2:– The data for WGA-negative cells should also be shownThese data are now included. In fact, Figure 2 has been completely revised, including two figure supplements.– In general, normalization to high potassium is still not sufficient to make very detailed comparisons between cells without using a ratiometric calcium probe. Normally, this isn't an issue and many in vivo studies rely on GCaMP, but here the effect sizes are small enough to warrant caution.-7) when compared to the WGA-positive contralateral neurons . Also for TRPA1 and TRPV1, we measured at least a doubling of the average response amplitude when compared to the WGA-positive contralateral neurons.From this comment, we realize that the way of representing our data  was not fully adequate to visualize the magnitude of the effects, and we have included additional plots to make the difference more evident. In the revised manuscript, we now show summary data in the main Figure 2: the percentage of responders  in Figure 2C, and the mean amplitudes (with indication of 95% CI) for the different groups in Figure 2D-F. Data from individual cells are still provided, but now in Figure 2—figure supplement 2. In addition, in the text we provide actual values  for the relative increase in response amplitude for the different agonists in the ipsilateral, WGA-positive neurons compared to controls. We are confident that, with this way of representing the data, it becomes obvious that the effect sizes are not small by any means. For instance, the response amplitude to TRPM3 agonist in the ipsilateral WGA-positive neurons was increased to 448% . This is needed to demonstrate that the TRPM3 antagonist doesn't affect TRPA1 and TRPV1 responses at baseline in the neurons that don't innervate the inflamed tissue. This seems to be a particularly critical control.These data are now included in Figures 2D-F and in the figure supplements.– In the bar plots, the Y-axes scales are each different and some have break lines. Things should be shown on the same scale to make comparisons across graphs easierThank you for the suggestions. We have made new summary plots in Figure 2 that make a direct comparison much easier.– I recognize performing new experiments under the current conditions would be tough, but it is strange they don't look at heat responses, since this is what the title and Abstract of the paper are abouthttps://www.nature.com/articles/srep15978), thereby significantly reducing the amplitude of heat-evoked signals. While we are currently considering alternative approaches that would allow measurement of heat-evoked responses in these preparations (e.g. using different activity indicators), we consider it beyond the scope of the present manuscript, which was aimed at showing changes in expression and function of TRPM3, TRPA1 and TRPV1.We note that heat is not mentioned in the title. The primary aim of the manuscript was to look at alterations in the expression and function of known heat-activated TRP channels. However, the use of pharmacological tools to probe for their activity is certainly more specific, because a heat stimulus will evoke a mixed response with potential contributions of all three channels and as well as additional specific and non-specific signals. We have tried extensively to use heat as a stimulus in the ex vivo preparations, but this was not at all straightforward. First, using heat as a stimulus in the ex vivo skin preparation during imaging generally caused local movements, which made it difficult to reliably track responses in the small nerve endings. Moreover, genetically encoded calcium indicators such as GCaMP3 show substantial temperature dependence, with both reduced calcium binding affinity (due to faster off rates) and lower fluorescence upon heating . Overall, I'm not convince this shows what the authors claim- the effects are very small and the spread of the data quite large.In this assay, we directly compared the skin from the two hind paws of individual mice , thereby accounting for natural differences in skin thickness and innervation density between animals. Skins were prepared by researchers with yearlong experience in using this preparation for electrophysiological recordings, and maintained in a standardized manner before recording. The size of the imaged area was identical for all experiments. The experiments and their analysis was performed without knowledge of whether the tissue was from the ipsi- or contralateral side. We included additional information on the technique in the Materials and methods section of the revised manuscript.increased reactivity to agonists of all three channels in the inflamed skin".Although there is significant spread, we have to politely but strongly disagree that the effects are very small or not convincing. The average increase is >200% for all three channels! We now provide actual values  for the relative increase in the ipsilateral versus contralateral side in the manuscript, allowing a more direct assessment of the magnitude of the effect. The results are statistically significant, and precise P-values are indicated in the figure. We now also show results from individual experiments in Figure 4—figure supplement 1. With these robust and statistically significant outcomes, we believe it is fully justified to make the claims that we made, namely "At the end of the section, the authors write: "Taken together, these results indicate that inflammatory heat hyperalgesia is associated with increased functional expression of all three heat-activated TRP channels at the level of the DRG cell bodies." But they only show increased expression of TRPM3 in Figure 1.Functional expression" of ion channels/transporters is a term that we and others frequently use to denote the effective function of these proteins  at the membrane. Also here, we meant to point at the increased functional responses, which are evident for all three channels . We have changed this to "increased functionality" to avoid any misunderstanding."Reviewer #2:The authors have recently demonstrated that heat-induced pain in naïve mice depends on a trio of heat-activated TRP channels, TRPM3, TRPA1 and TRPV1. However, with tissue inflammation, genetic ablation or pharmacological inhibition of only TRPV1 or only TRPM3 inhibition can fully suppress inflammatory heat hyperalgesia. Here the authors seek to dissect the relative contributions of TRPM3, TRPA1 and TRPV1 to nociceptor sensitization during tissue inflammation.In this manuscript, the authors use RNAscope to demonstrate that there is an increase in TRPM3 expression 1 day following CFA injection in the hind paw, specifically in the neurons that innervate the hind paw; there is no difference in TRPA1 or TRPV1 expression. Using whole DRG calcium imaging and a novel ex vivo skin calcium imaging approach, they reveal that neurons are more sensitive to TRPV1 and TRPA1 agonists following CFA hind paw injections and that this sensitivity is dependent on TRPM3 activity; V1 and A1 hypersensitivity can be blocked with the M3 antagonist isosakuranetin. Based on these experiments, TRPM3 antagonists may be viable therapeutic candidates for heat hypersensitivity. The paper is generally well written and accessible to a broad audience.• The authors claim that M3-dependent TRPA1 hypersensitivity occurs following CFA but neither the amplitude of TRPA1 responses  nor the percentage of neurons responding to TRPM3+TRPA1 are very supportive of this claim. Furthermore, the increase in A1 sensitivity does not appear to be specific to paw-innervating (i.e. WGA-labeled) neurons, because the same increase (based on a few outliers) occurs in non-paw innervating unlabeled neurons. While the skin imaging data are more convincing, the authors should dampen their claim that TRPM3 expression increases TRPA1 sensitivity following injury.As outlined in response to reviewer #1, we realize that the original way of representing our data  was maybe not the best choice to visualize the magnitude of the effects . We have, therefore made important changes to Figure 2, including four figure supplements, to make this aspect clearer for the reader.+ ipsilateral neurons is more than doubled following CFA, which is now also easier to appreciate from the revised Figure 2E.The percentage of neurons that show a TRPA1 response is doubled in the retrogradely labeled ipsilateral neurons, and the percentage of neurons that respond both to TRPM3 and to TRPA1 agonism  and M3+A1+V1 groups) increases from ~4% to about 16% in this group. This can now be better appreciated from the new Figure 2C and Figure 2—figure supplement 4. Moreover, the average response amplitude to TRPA1 agonists in WGA-AF647+ neurons, which amounts to 229%  when compared to the WGA-AF647+ contralateral neurons. Note that this difference remains highly significant (P=3×10-5) if we delete the two highest values, so it is certainly not the case that two outliers drive the difference.In addition, we now also provide a value in the text  representing the average increase in response amplitude in ipsilateral WGA-AF647- ipsilateral neurons, although (at least for TRPM3 and TRPV1) less pronounced than in the WGA-AF647+ neurons. We used a relatively conservative fluorescence threshold for a neuron to be considered WGA-positive. Therefore, there is likely a subset of neurons that innervate the injured paw but contain only a low level of WGA-AF647, and are therefore included in the WGA-negative group. In addition, it cannot be excluded that there is some cross-talk at the level of the DRG between neurons that innervate the inflamed tissue and neighboring DRG neurons. This aspect is briefly discussed in the revised manuscript.It is true that we also observed increased responses for all three TRP channels in the WGA-AF647• Since TRPV1-GCaMP3 mice are being used for both DRG and skin calcium imaging, how is it possible that not all cells respond to TRPV1 agonists? In other words, how are M3 only, A1 only, or M3+A1 responders detectable using this genetic line since only TRPV1 positive neurons would express GCaMP3? It seems that this would limit the authors' ability to detect changes in A1 sensitivity following injury. If this population of TRPV1-lineage neurons includes all of the TRPM3 and TRPA1-expressing neurons in the adult, then this should be made even more clear in the manuscript.This is a misunderstanding, and we apologize for not explaining this issue better. The TRPV1-cre driver line, which was used to generate the TRPV1-GCaMP3 mice, expresses cre in all TRPV1-lineage neurons, i.e., all neurons that express TRPV1 at any time in their development. In the studies of Mishra et al. , it was shown that all sensory neurons involved in thermosensation inflammatory heat hyperalgesia express TRPV1 at an early developmental stage, but that a significant proportion (roughly 50%) of these TRPV1-lineage neurons lose expression of TRPV1 in adult mice. Since these neurons have expressed the cre recombinase in an early stage, they express GCaMP3 even though they are no longer TRPV1 positive. This population includes the M3 only, A1 only or M3+A1 responders, as well as TRPM8-responding cells, as nicely demonstrated in Mishra et al., 2011.To make this point clearer, we have now included an analysis of combined GCaMP3 and Fura-2 measurements of isolated neurons from the TRPV1-GCaMP3 mice as Figure 2—figure supplement 1. These results show that ~60% of the GCaMP3-positive and 0% of the GCaMP3-negative neurons respond to capsaicin. The ~40% GCaMP3-positive neurons that do not respond to capsaicin are those neurons that expressed TRPV1 during development but lost expression at the stage where we did our analysis. These indeed include neurons that respond to TRPM3 and TRPA1 agonism. In addition, we found that ~40% of the GCaMP3-positive and 0% of the GCaMP3-negative neurons respond to MO. This indicates that all TRPA1-positive neurons are indeed included within the analyzed set of neurons. This finding is also fully in line with the earlier findings showing that the ablation of TRPV1-lineage neurons fully abolished the expression of TRPA1 . Finally, we found that about 60% of the GCaMP3-positive but also ~10% of the GCaMP3-negative neurons respond to PS. This suggests that most, but not all, TRPM3-expressing DRG neurons are within the set of TRPV1-lineage neurons. This is also in line with our RNAscope-data, showing a higher percentage of TRPM3-positive DRG neurons than of TRPV1-positive DRG neurons.• The authors should discuss whether there is potential cross desensitization/sensitization between agonist in both the DRG and the peripheral skin calcium imaging experiments, since cross-interactions between agonists could limit the strength of their conclusions.In preliminary experiments, as well as in earlier work in isolated sensory neurons, we observed that capsaicin treatment leads to significant desensitization of sensory neurons to subsequent capsaicin/AITC/PS stimulation, whereas this is much less pronounced for AITC and not observed for PS. We, therefore, chose to use the stimulation order PS/ AITC/ capsaicin in all experiments, as also done in our earlier work . We have included a brief discussion of the issue of cross (de)sensitization in the revised manuscript. Whereas the full intricacies of the  direct or indirect interactions between the three channels remain to be uncovered, we don't think such interactions would significantly limit the strength of our conclusions. The primary novel finding, namely the increased activity of TRPM3, is certainly not compromised, as this channel was always probed first. Moreover, we believe that by using an identical stimulation protocol on all preparations, a direct comparison between the groups is certainly valid.• More information/citations should be provided as to the dose/concentration chosen for the TRPM3 antagonist isosakuranetin, particularly since a single concentration was used of this single antagonist.Additional information is included in the revised manuscript. We now refer to papers that show that the concentration of 20 µM is in line with free plasma levels after systemic application of isosakuranetin in mice at a dose that inhibits TRPM3 activity in vivo.• The authors are commended for ex vivo imaging of sensory afferent terminals in the skin. Depending on the scientific question, this approach could have many benefits over imaging exclusively at the level of the DRG. Since this is the first description of this method, the authors should further clarify the methods in detail and address the following questions:How large is the bath containing the skin? How does buffer exit the bath? It appears that agonists are being presented to the tissue for 30-45 s. with an equivalent washout period between compounds . Is this sufficient time for the agonists to both access sensory terminals deep in the skin and then be washed away from the tissue? More details are needed on whether there is potential desensitization from one agonist to the next if insufficient time is allowed for agonist washout. More details on this method, the regions of interests chosen and how the data are quantified in a blinded manner should be provided.We included more details in the revised manuscript, including a clear time course of the agonist application periods for the experiments in Figure 4. As is shown there, we allowed 5 minutes in between stimuli.• The authors should comment on the use of a saphenous  preparation use in the context of the current injury model (i.e. 10 µL injection of CFA to the plantar surface of the paw)?The dorsal hairy skin turned out to be much better for imaging than the thicker and more irregular plantar side. We confirmed that CFA injection resulted in an inflammation of the entire paw. More details on these issues are now included in the revised manuscript.I don't think that the use of the contralateral paw is the best control since there could be segmental effects, and separate animal control would be better. That said, this would require the entire data set to be re-generated and I'm not going to recommend this. But this is something that I think that the authors should note going forward and avoid using the contralateral paw as the control.There could indeed be alterations on the contralateral side, although, at least behaviorally, effects on the contralateral side have not been observed in the CFA model after 24 h. Using separate animal controls would require at least twice as many animals, and probably many more because paired statistics would no longer be possible. Moreover, by using both paws from the same animal, we compensate for inter-animal variations in skin thickness and innervation. Therefore, for the CFA model, we believe that our approach is adequate and justified in our striving to reduce the use of animals to the minimum. We do, however, fully agree with the reviewer that separate animal controls would be more apt for more chronic pain models  where clear contralateral sensitization takes place.[Editors’ note: what follows is the authors’ response to the second round of review.]The editors and reviewers found the revised manuscript to be greatly improved, and we appreciate your sincere efforts to address the concerns that were raised. Overall, the conclusions better align with the results obtained, although we have some remaining concerns with conclusions concerning the RNAscope and skin prep results.RNA scope:The example images in Figure 1 are improved, however the new staining for Trpm3 looks to either be quite non-specific or to have high background? Either might affect the conclusions. In addition, the reviewers remain unconvinced that TRPM3 expression increases in response to inflammation. Some changes might seem big percent-wise but cell to cell variation and the biological meaning of such differences should also be considered: for example, what's the difference between an average of 20 and 30 puncta when the range of variation between cells is far greater? While these results may be suggestive, our consensus is the RNAscope results are not conclusive. We were much more convince by the increased functional responses to TRPM3 agonists. We request that you tone down conclusions concerning the RNAscope results throughout the manuscript. For example, in the Results section the following sentence:“Taken together, these results indicate that inflammation is associated with a significantly increased transcription of TRPM3, specifically in sensory neurons innervating the inflamed tissue, whereas no inflammation-related changes were found in the mRNA levels of TRPV1 and TRPA1.”could be easily improved by replacing 'indicate' with 'suggest' and "a significantly" with 'an'.We have toned down the conclusions regarding the RNAscope along the suggested lines.While we recognize the reviewers’ reservations, we do not think that the Trpm3 staining is non-specific or has high background. Compared to TRPA1 and TRPV1, TRPM3 is expressed in a higher proportion of the neurons, but never at very high levels (mainly <100 dots/cell). For TRPA1 and TRPV1 there are cells with very high expression levels  along with a large proportion of cells without any RNAscope signal. These findings are in line with functional studies in isolated DRG neurons, which show that a larger fraction of sensory neurons respond to TRPM3 agonists than to capsaicin, but also that the amplitude of TRPM3-mediated currents is an order of magnitude smaller than that of capsaicin-activated currents .Skin assay:The limitations of the assay are clearly spelled out in the Results section, but as analyzed the results are inherently qualitative. As such we suggest also toning down the conclusion here. For example, in the results the following sentence:“Overall, these results represent, to our knowledge, the first direct observation of functional upregulation of all three heat-activated TRP channels in intact nerve endings in inflamed skin.”could be improved by inserting 'qualitative' before 'results' to remind the reader of the nature of the measurements.We have toned down the conclusions regarding the skin assay along the suggested lines in several instances.While we acknowledge (and discuss in the manuscript) that quantification of calcium signals in nerve endings in the skin is not as straightforward as in cell bodies, we do not think that the results obtained with this approach are inherently purely qualitative. Our analysis pipeline provides quantitative values representing the area of nerve terminals responding to agonists, which in our opinion is a valid measure of channel activity in nerve endings.


Case Description: A 76-year-old lady, with a history of nonvalvular atrial fibrillation, presented with a new onset of progressive left lower and upper extremity weakness. She reported back pain and numbness in the left leg up to the knee along with numbness in the left arm up to the shoulder. A CT scan of the neck was suggestive of an epidural hematoma extending from C2-C3 level to C6-C7. As the patient was on apixaban at the time, surgical treatment was delayed for two days to decrease the risk of intraoperative bleeding. Nine days later, she was discharged. Her physical exam was almost unchanged from that on presentation, except for resolution of pain and minimal improvement in motor power in her left lower extremity from 1/5 to 2/5 distally.  With the rise in the use of direct oral anticoagulants (DOACs), more hemorrhagic complications are being encountered. Since the first description of a case of spontaneous spinal epidural hematoma (SSEH) related to the utilization of DOACs in 2012, there have been few reports describing a similar association. However, no cases so far have reported an association between SSEHs and apixaban.  Spinal hematomas represent surgical emergencies with earlier intervention portending better outcome. Based on the few case reports that point to DOACs as a potential culprit, it appears that a high suspicion index resulting in earlier SSEH diagnosis and intervention is crucial for improved neurological outcome and recovery. Prompt diagnosis remains a challenge, especially that SSEH can mimic cerebrovascular accidents. Since the first description of a case of SH associated with the use of a DOAC (rivaroxaban) in 2012 . As the A 76-year-old African American lady presented to the hospital with new onset weakness and back pain. Her medical history consisted of essential hypertension, atrial fibrillation, abdominal aortic aneurysm, sick sinus syndrome, asthma, and gout. She reported that her back pain started after she came back from the bathroom at 4 : 00 a.m. while trying to sit down on the bed. The patient described the pain as shooting in nature, severe in intensity, and radiating down her left arm with no clear exacerbating or relieving factors. Over the following few minutes, she developed progressive weakness in her left lower and upper extremities and later on became unable to move her left leg which led to her being transferred to the Medical Intensive Care Unit. She also developed numbness in her left leg up to the level of the knee and in her left arm up to the level of the shoulder. There was no history of any urinary or stool incontinence or retention or any previous similar episodes.The patient had been recently diagnosed with atrial fibrillation one month prior to presentation in an outside hospital and had since been maintained on apixaban 1 tablet 5 mg twice daily. Her other home medications included alprazolam, allopurinol, losartan, hydrochlorothiazide, and metoprolol. She reported full compliance with her medications prior to presentation. A dual chamber pacemaker was inserted 11 days before this visit in an outside hospital, and her other surgical history included a hysterectomy several years ago for uterine fibroids. She denied any history of smoking, alcohol abuse, or illicit substances usage. The patient did not have previous bleeding history.The patient's vital signs on presentation were within normal limits, and her neurological exam showed a patient alert and oriented to time, place, and person with intact speech. Her motor power was 5/5 in her right upper extremity proximally and 4/5 distally and 3/5 in her left upper extremity proximally and 1/5 distally. In her lower extremities, the motor power was 5/5 on the right and 1/5 all over on the left. She had decreased sensation to light touch in her left upper and lower extremities. She had no clonus, and Hoffman and Babinski signs were negative bilaterally. Her reflexes were 2+ all over and symmetric.Initial lab tests were significant for hypokalemia (3.1 mmol/L), leukocytosis (12700 WBCs per microliter), and slight anemia with a hemoglobin level of 9.3 g/dl with unknown baseline. Her other lab tests including liver function and renal function were within normal limits.Given her presentation of acute focal neurological deficit, a noncontrast head CT scan was ordered and showed signs of chronic vascular ischemic disease only. The patient continued to have severe back pain, and having a history of abdominal aortic aneurysm, an acute aortic dissection was suspected, and hence, a CT angiogram of the abdomen, pelvis, and thorax was ordered. The CT angiogram did not show any signs of aortic dissection. A CT scan of the neck was performed to investigate the cause of the left upper extremity weakness and numbness. It showed an epidural soft tissue density extending along the left aspect of the spinal canal causing compression and marked rightward deviation of the cord concerning for an epidural hematoma extending from C2-C3 level to C6-C7 .As the patient was on apixaban at the time, a decision was made to delay surgical treatment for 2 days to decrease the risk of intraoperative bleeding. A C2-C7 laminectomy, hematoma exclusion, and spinal fusion were then performed. On discharge, 9 days later, her physical exam was mostly unchanged from that on presentation except for the resolution of pain and improvement in motor power in her left lower extremity from 1/5 to 2/5 distally.Spinal hematomas represent surgical emergencies with earlier intervention portending better outcome . The acuX inhibitors [According to our literature review, 4 patients underwent surgical intervention within 24 hours of symptoms onset, and 3 of these 4 patients 75%) had excellent outcomes with unknown outcome in the third one [% had exchibitors . There whibitors . Among tThe number of cases reported is insufficient to reach any statistically significant result, but seems to be pointing towards the importance of early surgical intervention for neurologic recovery. With the development of new reversal agents and increased experience with the current ones, earlier surgical intervention is most likely to increase in frequency as surgeons become more comfortable operating on these patients leading hopefully to less neurologic deficit. This means that a high suspicion index leading to earlier diagnosis is key for timely intervention, and we hope that this article will help in raising awareness about this condition as a potential complication of DOACs use, especially with the significant and continuous increase in their utilization.


N = 30) and healthy controls (N = 25). Further, we used support vector machines to examine the optimal combination of channels of functional near-infrared spectroscopy that can classify cognitive alterations in CLD. Behavioral findings showed that CIT performance was significantly worse in the patient group and CIT significantly correlated with neurocognitive evaluation . The findings indicated that CIT can measure auditory cognitive capacity and its difference existing between patient group and healthy controls. Additionally, optimal subsets classified the 16-dimensional haemodynamic data with 78.35% classification accuracy, yielding markers of cognitive alterations in the prefrontal regions . The results confirmed the potential use of behavioral as well as haemodynamic responses to music perception as an alternative or supplementary method for evaluating cognitive alterations in chronic liver disease.Early detection and evaluation of cognitive alteration in chronic liver disease is important for predicting the subsequent development of hepatic encephalopathy. While visuomotor tasks have been rigorously employed for cognitive evaluation in chronic liver disease, there is a paucity of auditory processing task. Here we focused on auditory perception and examined behavioral and haemodynamic responses to a melodic contour identification task (CIT) to compare cognitive abilities in patients with chronic liver disease (CLD,  Chronic liver disease (CLD) is a progressive destruction of liver functions over a period more than 6 months leading to fibrosis and cirrhosis  that is designed to measure the selectivity of auditory attention, which is the core characteristics of auditory information processing. Our previous studies confirmed the validity of using melodic CIT to measure the various types of attention in moderate-to-severe traumatic brain injury patients. Jeong  validate2 (oxygenated hemoglobin) in the frontal lobe, which is known as an indicator of cognitive alterations in patients with chronic liver disease . These findings were suggestive of HbO2 obtained from the frontal area can reflect cognitive alterations along with progress of liver disease.We also examined HbO2 can be indicative of cognitive alterations in chronic liver diseases, and whether the regions of the prefrontal cortex, known to modulate attention and cognition, can be specified to characterize the alterations in chronic liver disease. For this analysis, we employed a support vector machine (SVM) combined with an fNIRS to classify cognitive alterations following liver diseases, a technique which has been increasingly used to classify clinical and healthy populations. Monden et al. . In addition, Ichikawa et al.  and examined the criterion validity of CIT, in correlation with standard neurocognitive tests. We also examined whether changes in HbOn et al.  previousa et al.  employedA total of 55 participants were included: 30 patients with chronic liver diseases , and 25 healthy controls  were matched by age and the level of education. Participants who had a <3 months of regular involvement in musical activities and/or professional training and who had a minimal ability to understand the spoken instruction were eligible to participate in the study. The diagnosis of liver disease was based on either a liver biopsy or the presence of portal hypertension and markers of hepatocyte synthetic dysfunction. We included patients who have been diagnosed as liver disease for more than 6 months. Participants with any of the following conditions were excluded: diabetes mellitus, systemic arterial hypertension, metabolic liver disease (hemochromatosis and Wilson's), personal history of stroke or cancer, use of neuropsychiatric drugs, neuropsychiatric disorders, current alcohol intake or smoking, rotating shift work, or acute inflammatory responses of infectious origin. Three melodic contours  were adopted from Jeong and Ryu . MelodicThe six test items were modulated in five different keys (G# to C major) and were presented using three instrument timbres , yielding a total of 90 test items. We selected the instruments based on a previous study that classified various musical instruments according to their spectral features of timbre, such as the harmonic structure, inharmonicity, and harmonic energy skewness . The computerized version of the CIT was designed to measure different types of auditory attention and the associated cognitive load changes . The tasCIT1 had no visual cues, however, in CIT2, a picture of an instrument that plays target contours was presented prior to presenting the item to inform which contour the participants selectively listened to. In CIT3, outlined boxes were additionally used to guide at which contours the participants selectively listened to and shifted from one to another instrument see . For exa2; deoxygenated hemoglobin, HHb) during cognitive, motor, and sensory stimulation. We used a 16-channel Spectratech OEG-16  for the measurements . Healthy adults voluntarily participated and were recruited via a headphone with controlled volume, while the visual cues specifying the target musical instrument were presented to the participants on a monitor. The participants underwent a practice session to become familiar with the direction identification task. When their accuracy was over 80%, the main experimental session was administered. A total of 18 test items were presented in each CIT (a blocked design) and the order of CIT was randomized across participants  was calculated for each of the 16 channels. Since we found that the fNIRS response peak was delayed by a few seconds compared to the stimulus onset  and 75 from HC. We used the data as inputs and the diagnosis  of the groups as outputs.The fNIRS raw data were collected throughout the experiment and were converted into concentration changes of hemoglobin using the modified Beer–Lambert law  eight channels from the right hemisphere (CH1–CH8), (2) eight channels from the left hemisphere (CH9–CH16), and (3) 16 channels from both hemispheres (CH1–CH16). The standardized HbO2 data from the eight channels from both hemispheres and the 16 channels have an 8-dimensional (28- 1 = 255 subsets) or 16-dimensional  feature vector, respectively. The obtained HbO2 data were trained and classified to diagnose groups. SVMs have been applied previously for classification problems in various domains, and have yielded high generalization capability . If the value was more than 0, the sample was classified as positive. If the value was <0, the sample was classified as negative.Once we trained the SVM model, the weight vector To evaluate the trained model, we first computed the confusion matrix shown in From the confusion matrix, the basic performance measures were calculated according to the following equations:Sensitivity is the probability to correctly predict the samples that are actually classed as positive. It is an indicator that assess the ability of a model to identify positive samples. Specificity also assess a model's ability to correctly to correctly predict the samples whose class is actually negative. Accuracy is the ability of a model to correctly identify classes for all samples. However, accuracy could lead to evaluation errors when the data is imbalanced. If one class is a majority class, the real performance of the other class cannot be reflected accurately. Moreover, sensitivity and specificity are not proper measures to study the balanced performance. For a balanced evaluation, the following measure was used.We assessed a modified classification performance measure using a MCC, which is used widely in biomedical research  8-dimension dataset obtained from the right hemisphere (CH1–CH8), (2) 8-dimension dataset obtained from the left hemisphere (CH9–CH16), and (3) 16-dimension dataset obtained from the bilateral hemispheres (CH1–CH16). For each of these three datasets, we trained the SVM and evaluated its classification accuracy using Matthew correlation coefficient (MCC) and the balanced accuracy (bACC).We analyzed behavioral responses to CITs and performed classifications between patients with chronic liver disease (CL) and healthy controls (HC) using HbOF = 65.566, p < 0.001], indicating that CIT performance for both groups worsened significantly as the CITs became more difficult. The pairwise post hoc comparison using the Bonferroni correction revealed that CIT1 was significantly better than CIT2 (p < 0.001), and that CIT2 was significantly better than CIT3 (p < 0.01). We also found a significant interaction between the Group and Task . Interestingly, the pairwise post-hoc comparison revealed that the CL group performed significantly better in the CIT2 than in the CIT3 (p < 0.001), while the HC group showed similar performances in the CIT2 and CIT3 (p > 0.05). There was a significant main effect of the Task  on the response time. The pairwise post hoc comparison using the Bonferroni correction revealed that only the CL group performed significantly worse in the CIT3 than CIT1 and CIT2 . A two-way mixed ANOVA  was performed to validate accuracy and reaction time. For accuracy, there was a significant main effect of the Task [2 obtained from the bilateral hemisphere yielded better classification performance than values obtained from the unilateral hemispheres. After an additional t-test, we found that classification performance of the bilateral subset was significantly higher than that of the right and left hemisphere subsets .Further, we classified the differences in haemodynamic responses to CIT1 and CIT2 between HC and CL in combination with an SVM and an exhausted feature selection method. We decided to exclude CIT3 since our behavioral findings clearly showed that CIT3 was too difficult for CL group and, thus, haemodynamic response to CIT3 might not indicate appropriate cognitive load in this group. With the analysis, we found a total of 15 subsets that classified the data more accurately into two groups among the 255 subtests . 2 in CH6, CH7, and CH10 were higher in the CL than in the HC, and that the HbO2 in CH13, CH14, and CH16 were lower in the CL than in the HC. Further, our findings suggest that the frontal areas of each hemisphere were intercommunicating, so it was necessary to include subsets from both hemispheres to yield better classification performance.Further, we created a matrix plot using 50 subsets and constructed features that correspond to the classification performance of each subtest. In this study, we examined the potential of melodic CITs to evaluate cognitive alterations in chronic liver disease. Our behavioral findings indicated that CITs can differentiate changes in auditory perception and cognition in patients with chronic liver disease from healthy controls. Correlation analysis using standard neurocognitive evaluations revealed that CITs had good criterion validity and potential for measuring cognitive alterations that occur in chronic liver disease. Then, we applied SVMs with 5-fold cross validation to the haemodynamic responses obtained during the CIT performance to classify cognitive alterations in patients with chronic liver disease. We exhaustively searched all subsets of the measurement channels and evaluated each classification performance by repeating the 5-fold cross validation method 20 times. Our findings yielded an optimal subset for the classification of the haemodynamic data with 78.35% accuracy. Our results indicated that the subsets obtained bilaterally can better classify the differences that exist between HC and patients with CL. Also, we found channel features that could specify between groups. Three channels  in the left dorsolateral prefrontal cortex (DLPFC), one channel (CH6) in the right orbitofrontal cortex (OFC), and two channels  in the right frontopolar area (FP) were important for the classification of CL from HC. From the behavioral findings, we found poorer overall CIT performance in the CL than in the HC group, and the difference was more obvious between CIT2 and CIT3. The findings indicated that overall auditory cognition deteriorated in the CL group. Given that CIT1 and CIT2 involve auditory selective attention and that CIT3 involves auditory alternating attention, the cognitive threshold of patients with CLD might be auditory alternating attention (or cognitive flexibility). The current findings resembled previous findings where selective attention . DST-Backward significantly correlated with accuracy only in the HC group—not for the CL group. DST measures auditory working memory (DST-Forward) and auditory executive function  in the CL group were lower as compared to the HC group, indicating that patient group consumed less cognitive resources during the CIT performances than healthy adult group. DLPFC regions modulate higher order cognitive systems, such as working memory, decision-making, and problem solving  were greater in the CL group as compared to HC group. The right frontopolar area, which corresponds to BA10, is involved in a variety of cognitive performances, ranging from simple to highly complex tasks , as well as the OFC (BA11). Note that BA11 is known for its role in emotional behaviors, especially in evaluating the emotional valence of external stimuli  and a skewed female-to-male ratio. Some previous studies have reported gender difference issues in patients with chronic liver disease . The patients/participants provided their written informed consent to participate in this study.GJ and EJ conceived and designed the experiments. DJ recruited participants and arranged individual experiments. EJ performed the experiments and edited the manuscript. GJ, Y-MK, DJ, and EJ analyzed, interpreted the data, and wrote the manuscript. GJ and EJ prepared tables and figures. All authors reviewed the manuscript.GJ was employed by the company Daehong Communications Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.