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Upregulation of TRPM3 in nociceptors innervating inflamed tissue

  1. Marie Mulier
  2. Nele Van Ranst
  3. Nikky Corthout
  4. Sebastian Munck
  5. Pieter Vanden Berghe
  6. Joris Vriens
  7. Thomas Voets  Is a corresponding author
  8. Lauri Moilanen
  1. Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Brain & Disease Research, Belgium
  2. Department of Cellular and Molecular Medicine, KU Leuven, Belgium
  3. VIB Bio Imaging Core and VIB-KU Leuven Centre for Brain & Disease Research, Belgium
  4. Department of Neuroscience, KU Leuven, Belgium
  5. Laboratory for Enteric NeuroScience (LENS), TARGID, Department of Chronic Diseases Metabolism and Ageing, KU Leuven, Belgium
  6. Laboratory of Endometrium, Endometriosis and Reproductive Medicine, G-PURE, Department of Development and Regeneration, KU Leuven, Belgium
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Cite this article as: eLife 2020;9:e61103 doi: 10.7554/eLife.61103

Abstract

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 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.

Introduction

Painful stimuli are detected by nociceptors in peripheral tissues and are transmitted as action potentials towards the central nervous system to elicit pain sensation (Viana et al., 2019; von Hehn et al., 2012; Vriens et al., 2014). Under pathological conditions, such as inflammation or tissue injury, nociceptors become sensitized to mechanical and thermal stimuli. Such nociceptor hypersensitivity gives rise to allodynia (the sensation of pain to a stimulus that is usually not painful), hyperalgesia (increased pain sensation to a stimulus that is usually painful) or to spontaneous pain without any clear stimulus (Viana et al., 2019; von Hehn et al., 2012; Jensen and Finnerup, 2014). The current analgesic drug therapies, including non-steroidal anti-inflammatory drugs, opioids, gabapentinoids, and antidepressants, are often limited in efficacy in a large number of pain patients and may have severe adverse effects that limit their use (Colloca et al., 2017; Skolnick, 2018; Voets et al., 2019). The need for better and safer analgesic drugs is painfully illustrated by the dramatic rise in opioid addiction and related deaths, known as the opioid crisis (Skolnick, 2018). The search for new analgesic drugs with novel mechanisms of action inevitably depends on a deep understanding of the cellular and molecular mechanisms underlying nociceptor sensitization (Colloca et al., 2017).

In this context, several members of the transient receptor potential (TRP) 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 (Basbaum et al., 2009). For instance, we recently demonstrated that heat-induced pain in mice depends on a trio of heat-activated TRP channels, TRPM3, TRPA1, and TRPV1 (Vandewauw et al., 2018). Robust neuronal and behavioral heat responses were observed as long as at least one of these three TRP channels was functional, but were fully abolished in Trpm3-/-/Trpv1-/-/Trpa1-/- triple knockout mice (Vandewauw et al., 2018). These results indicate triple redundancy of the molecular sensors for acute heat. Intriguingly, however, there apparently is no such redundancy for the development of heat hypersensitivity in inflammatory conditions. Indeed, genetic ablation or pharmacological inhibition of either only TRPV1 (Caterina et al., 2000; Davis et al., 2000; Gavva et al., 2005; Honore et al., 2005) or only TRPM3 (Alkhatib et al., 2019; Vriens et al., 2011; Straub et al., 2013) is sufficient to fully suppress inflammatory heat hyperalgesia. However, the precise mechanisms and the relative contributions of the heat-activated TRP channels to nociceptor sensitization under inflammatory conditions remain incompletely understood.

To 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.

Results

Increased expression of TRPM3 mRNA in sensory neurons innervating inflamed tissue

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.

First, we addressed whether tissue inflammation is associated with increased expression of mRNA encoding TRPM3, TRPA1, and TRPV1, in particular in sensory neurons that innervate the inflamed tissue. Sensory neurons innervating the hind paws of mice have their cell bodies in the dorsal root ganglia, primarily at the lumbar levels L3-L6. These ganglia also contain cell bodies of sensory neurons that innervate other parts of the body, including viscera, necessitating an approach to specifically label neurons that have endings in the hind paw. Therefore, we injected the retrograde label WGA-AF647 intraplantar in both hind paws seven days prior to tissue isolation, which resulted in effective labeling of cell bodies of sensory neurons that innervate the injected territory (Figure 1A and Figure 1—figure supplement 1). We induced an inflammatory response in the ipsilateral hindpaw by injecting CFA into the plantar surface 24 hr before tissue collection; at the same time, the contralateral hindpaw was injected with vehicle (Figure 1—figure supplement 1). We used single-molecule fluorescent RNA in situ hybridization (RNAscope) (Wang et al., 2014), to quantify the levels of mRNA encoding TRPM3, TRPA1 and TRPV1 in the ipsi- and contralateral DRGs (Figure 1A), and compared the levels of mRNA, both between retrogradely labeled (WGA-AF647+) 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 (Figure 1B and Figure 1—figure supplement 2). On average, TRPM3 mRNA levels in the ipsilateral, WGA-AF647+ DRG neurons were increased to 163% (95% confidence interval [CI], 122% to 222%; p=0.008) of the levels in the WGA-AF647+ neurons on the contralateral side, and to 133% (CI, 108–163%; p=0.005) 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 (Figure 1C).

Figure 1 with 2 supplements see all
RNA expression of heat-activated TRP channels in sensory neurons innervating inflamed and control hind paws.

(A) Representative fluorescent images of processed contralateral (vehicle-treated) and ipsilateral (CFA-treated) L5 DRG. Shown are RNAscope stainings with specific probes for TRPM3 (left), TRPA1 (middle) or TRPV1 (right) (green). Retrogradely labeled neurons were identified based on the WGA-AF647 staining (magenta), and the blue color represents the nuclear marker DAPI. Retrogradely labeled neurons in the boxed areas are shown at double magnification, along with RNAscope staining for the neuronal marker Pgp9.5 (yellow), which was used to delineate neuronal cell bodies. (B,D,F) Quantification of the number of RNAscope dots per DRG neuron for TRPM3, TRPA1 and TRPV1, comparing retrogradely labeled (red) and unlabeled (black) sensory neurons, from the contralateral and ipsilateral L5 DRG. Values are presented as mean along with the 95% confidence interval. The data of the individual cells are shown in Figure 1—figure supplement 2. Statistical comparisons between groups were made using Kruskal-Wallis ANOVA with Dunn’s posthoc test. (C,E,G) Fraction of DRG neurons that showed a positive RNAscope signal (≥5 dots) for the three tested channels. Values are presented as mean ± SEM along with data points from the individual mice. Statistical comparisons between groups were made using one-way ANOVA with Holm–Šidák post-hoc test. Data are from six mice. The total numbers of analyzed neurons were for TRPM3: 752 ipsilateral and 1299 contralateral; for TRPA1: 954 ipsilateral and 947 contralateral; for TRPV1: 1054 ipsilateral and 995 contralateral.

In contrast, we did not observe significant inflammation-related changes in mRNA levels of TRPA1 or TRPV1 (Figure 1D–G and Figure 1—figure supplement 2). For TRPA1, mRNA levels in the ipsilateral WGA-AF647+ DRG neurons amounted to 120% (CI, 63% to 147%; p=0.56) of the level in the contralateral WGA-AF647+ DRG neurons and to 83% (CI, 40% to 139%; p=0.81) of the level in ipsilateral WGA-AF647- neurons (Figure 1D). In the case of TRPV1, mRNA levels in the ipsilateral WGA-AF647+ neurons amounted to 89% (CI, 53% to 140%; p=0.52) of the level in the contralateral WGA-AF647+ DRG neurons and to 126% (CI, 87% to 166%; p=0.15) of the level in ipsilateral WGA-AF647- neurons (Figure 1F). Likewise, the fraction of TRPA1- or TRPV1-positive DRG neurons did not differ significantly between WGA-AF647+ and WGA-AF647-DRG neurons on the ipsilateral and contralateral sides (Figure 1E,G).

Taken together, these results suggest that inflammation is associated with an 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.

Increased TRP channel functionality in sensory neurons innervating inflamed tissue

Next, we investigated whether sensory neurons innervating inflamed tissue exhibit altered functionality of heat-sensitive TRP channels. In a first analysis, we focused on the functional expression of TRP channels in the neuronal cell bodies. Since procedures to isolate and culture DRG neurons significantly alter the expression levels of many ion channels (Wangzhou et al., 2020), we developed an assay where the DRG was imaged as a whole in situ, using spinning-disk confocal imaging (Figure 2). For these experiments, we made use of a mouse line that expresses the genetically encoded calcium sensor GCaMP3 in TRPV1-lineage neurons (TRPV1-GCaMP3 mice), which include all the neurons involved in thermosensation and inflammatory thermal hyperalgesia (Mishra and Hoon, 2010; Mishra et al., 2011; Usoskin et al., 2015). It is important to note that not all GCaMP3-positive DRG neurons from TRPV1-GCaMP3 mice functionally express TRPV1 at the developmental stage where we did our analysis. Indeed, in line with earlier studies (Mishra et al., 2011), we found that TRPV1-lineage neurons identified based on their GCaMP3 fluorescence include all DRG neurons that functionally express TRPV1 and TRPA1, but also a subset of DRG neurons that no longer expressed TRPV1 (Figure 2—figure supplement 1). Retrograde labeling using WGA-AF647 (see Figure 1—figure supplement 1) was used to identify the neurons that innervate the mouse paw. The efficiency of the retrograde labeling was similar in the ipsi- and contralateral side, with 25.8 ± 9.7% and 27.7 ± 5.7% of the neurons being WGA-AF647+, respectively. Changes in GCaMP3 fluorescence were monitored upon application of specific agonists for TRPM3 (the combination of pregnenolone sulfate; PS and CIM0216), TRPA1 (mustard oil; MO) and TRPV1 (capsaicin), and of a depolarizing high K+ solution resulting in TRP channel-independent Ca2+-influx via voltage-gated Ca2+ channels (Figure 2B). The order of agonist application (TRPM3-TRPA1-TRPV1) and application timing was kept constant for all experiments. The order of agonist application was based on earlier experiments in isolated DRG neurons showing that capsaicin treatment leads to significant desensitization of sensory neurons to subsequent stimulation, whereas this is much less pronounced for the used agonists of TRPA1 and TRPM3 (Vriens et al., 2011; Vandewauw et al., 2018).

Figure 2 with 4 supplements see all
Inflammation-induced changes in TRP channel activity in DRG cell bodies.

(A) Confocal images of the ipsi- and contralateral L5 DRG of a CFA-treated mouse showing GCaMP3 (green) and WGA-AF647 (magenta) fluorescence. (B) Representative examples, corresponding to cells indicated in panel (A) of changes in GCaMP3 fluorescence (ΔF/F0) in response to application of agonists of TRPM3 (PS + CIM0216; M3) TRPA1 (MO; A1) and TRPV1 (capsaicin; V1) and of a depolarizing high K+ solution (K+). (C) Percentage of neurons responding to the indicated agonists in WGA-AF647+ (red) and WGA-AF647- (black) DRG neurons from the ipsi- and contralateral sides. Data are shown as mean ± SEM from 9 mice. Statistical comparisons between groups were made using one-way ANOVA with Holm–Šidák post-hoc test. Cells that did not respond to high K+ stimulation were excluded from the analysis. (D-F) Peak amplitudes of responses to the TRPM3, TRPA1 and TRPA1 agonists, normalized to the response to the depolarizing high K+ solution, comparing retrogradely labeled (WGA-AF647+; red) and unlabeled (WGA-AF647-; black) neurons on the ipsi- and contralateral side. Where indicated (grey background), DRGs were pre-incubated with isosakuranetin (20 μM). Values are presented as mean along with the 95% confidence interval. The data of the individual cells and the number of cells in the different groups are shown in Figure 2—figure supplement 2. A comparison of the non-normalized data is provided in Figure 2—figure supplement 3. Statistical comparisons between groups were made using Kruskal-Wallis ANOVA with Dunn’s posthoc test. Data are from 9 mice in the absence of isosakuranetin and another set of 9 mice in the presence of isosakuranetin.

Both in the ipsi- and contralateral DRG, we observed calcium responses to the different agonist applications, which were indicative of neurons with different patterns of functional expression of one, two or all three heat-activated TRP channels (Figure 2B; Figure 2—figure supplement 4). We did not observe any significant differences in the fraction of neurons that responded to the different TRP channel agonists or in the response amplitudes between WGA-AF647+ and WGA-AF647- neurons in the contralateral DRG, indicating that the retrograde label by itself did not affect TRP channel activity at the level of the cell bodies (Figure 2C–F; Figure 2—figure supplements 24). Likewise, responses to the depolarizing high K+ solution did not differ significantly between groups, and these responses were to normalize the TRP channel-mediated responses (Figure 2D–F; Figure 2—figure supplement 2; non-normalized data are shown in Figure 2—figure supplement 3).

Importantly, responses to TRPM3 agonists were strongly increased in the ipsilateral WGA-AF647+ neurons. The fraction of these neurons that exhibited a positive response increased significantly, to 52% compared to around 20% in the different control groups (Figure 2C). Likewise, the response amplitude in the ipsilateral WGA-AF647+ neurons was increased to 233% ([CI], 157% to 330%; p=0.007) when compared to the ipsilateral WGA-AF647- neurons and to 448% ([CI], 265% to 755%; p=7 × 10−7) when compared to the WGA-AF647+ contralateral neurons (Figure 2D). These findings indicate a strong functional upregulation of TRPM3, specifically in neurons innervating the inflamed paw.

In the case of TRPA1, we found that the fraction of responding neurons in the ipsilateral WGA-AF647+ neurons was doubled and the average response amplitude was increased to 229% ([CI], 146% to 355%; p=2 × 10−5) when compared to the WGA-AF647+ contralateral neurons (Figure 2C,E). However, there were no significant differences in the fraction of responding neurons or the response amplitudes between WGA-AF647+ and WGA-AF647- neurons on the ipsilateral side, suggesting that increased responsiveness to TRPA1 agonists may not be limited to neurons innervating the hind paw but affects the entire ipsilateral DRG.

Finally, in the case of TRPV1, there was a trend toward more responders in the ipsilateral WGA-AF647+ neurons, but this did not reach statistical significance (Figure 2C). However, the amplitude in the ipsilateral WGA-AF647+ neurons was significantly increased, to 140% ([CI], 109% to 173%; p=0.02) when compared to the ipsilateral WGA-AF647- neurons and to 205% ([CI], 152% to 280%; p=3 × 10−4) when compared to the WGA-AF647+ contralateral neurons (Figure 2F). These findings indicate that the neurons that innervate the inflamed paw show enhanced responses to agonists of TRPM3, TRPA1, and TRPV1. Since these three channels show a partially overlapping expression profile (Vandewauw et al., 2018), we evaluated how tissue inflammation affects the functional co-expression of the three channels. Notably, in the ipsilateral WGA-AF647+ neurons, we observed a pronounced increase in the fraction of neurons that show functional responses to agonists for both TRPM3 and TRPV1, as well as in the fraction that respond to all three agonists (Figure 2—figure supplement 4). Since these neurons showed a robust increase in mRNA expression of TRPM3, but not of TRPV1 and TRPA1 (Figure 1), we considered the possibility that increased TRPM3 expression and activity provoked by inflammation may contribute to enhanced excitability of sensory neurons, resulting in enhanced responses to TRPA1 and TRPV1 agonists. To investigate this possibility, we tested the responses of DRG neurons to TRP channel agonists in the presence of the TRPM3 antagonist isosakuranetin at 20 µM (Straub et al., 2013). This concentration is in accordance with the free plasma concentration of 17.5 ± 6.3 µM that is reached in mice following systemic (i.p.) application of 10 mg/kg isosakuranetin, a dose at which effective inhibition of TRPM3-mediated pain was reported (Krügel et al., 2017; Straub et al., 2013). As expected, isosakuranetin effectively eliminated the responses to TRPM3 agonists, illustrating the suitability of this compound to study TRPM3 functionality in DRG (Figure 2D; Figure 2—figure supplements 24). On the contralateral side, isosakuranetin had no significant effect on the responses to MO or capsaicin (Figure 2E,F), in line with the reported selectivity of the antagonist for TRPM3 (Krügel et al., 2017; Straub et al., 2013; Jia et al., 2017). Moreover, isosakuranetin did not affect the response to the depolarizing high K+ solution, indicating that the compound does not Ca2+ via voltage-gated Ca2+ channels. Notably, isosakuranetin caused a significant inhibition of the response amplitudes to capsaicin and MO on the ipsilateral side. In fact, in the presence of isosakuranetin we no longer detected any significant differences in MO or capsaicin responses between ipsi- and contralateral, WGA-AF647+, and WGA-AF647- neurons (Figure 2E,F; Figure 2—figure supplements 24).

Taken together, these results indicate that inflammatory heat hyperalgesia is associated with the increased functionality of all three heat-activated TRP channels at the level of the DRG cell bodies. In particular, we report for the first time a strong enhancement of TRPM3-mediated responses, as well as large increase in the fraction of neurons that co-express TRPM3 with TRPV1 and TRPA1. Notably, pharmacological inhibition of TRPM3 not only suppressed the responses to TRPM3 agonists but also reduced the responses to TRPA1 and TRPV1 agonists in neurons innervating inflamed tissue. These findings are in line with a model where increased molecular and functional expression of TRPM3 in the context of tissue inflammation enhances the excitability of sensory neurons, which may contribute to augmented responses of these neurons to TRPA1 and TRPV1 agonists.

Optical measurements suggest increased TRP channel activity in cutaneous nerve endings in inflamed skin

Whereas these results indicate increased functionality of heat-activated TRP channels in the cell bodies of sensory neurons innervating the inflamed paw, they do not provide information regarding changes at the level of the sensory nerve endings. To address this issue, we developed an approach that allows direct measurement of TRP channel activity in intact cutaneous peripheral nerve endings in mouse hind paw skin (Figure 3A). In this assay, a skin flap of the dorsal surface of the hind paw and the innervating saphenous nerve (but lacking the DRG cell bodies) of TRPV1-GCaMP3 mice were excised and fixed in an organ bath, corium side up. Note that the plantar skin tissue is significantly thicker, and therefore less amenable for imaging nerve endings using this approach. Calcium signals in nerve endings expressing GCaMP3 were visualized from the epidermal side using a spinning-disk confocal microscope, while TRP channel agonists or a depolarizing high K+ solution were locally applied from the dermal side (Figure 3A). We used a gravity-driven perfusion system with an outlet positioned on one border of the recording field, and constant suction on the opposite border, allowing rapid exchange of agonists. We adapted a published algorithm to automatically detect contiguous regions of interest (ROIs) exhibiting synchronous activity, which we interpret as individual branches of sensory nerve endings (Zhou et al., 2018). As illustrated in Figure 3B,C and Figure 3—Video 1, this approach revealed distinct calcium responses in localized regions, indicative of sensory endings that functionally express TRPM3, TRPA1, and TRPV1.

Figure 3 with 1 supplement see all
Optical measurement of TRP channel activity in peripheral sensory nerve endings.

(A) Schematic illustration of the optical imaging setup. Sensory nerve fibers (red) innervating the dermal and epidermal skin layers are visualized using 488 nm laser light and an inverted spinning disk confocal microscope (20x objective). To avoid the barrier effect of the epidermis, solutions (at 37°C) were applied to the internal side of the sample from above. A total thickness of 20–30 μm was captured. (B) The first image depicts the summed raw fluorescence of the entire imaging experiment. The next five images represent normalized fluorescence (ΔF/F0) at baseline (before the first stimulus), upon stimulation with TRPM3, TRPA1 and TRPV1 agonists, and with the depolarizing high K+ solutions. Three automatically detected ROIs, corresponding to the traces in panel C, are indicated. Scale bar is 50 μm. See Figure 3—Video 1. (C) Time course of normalized GCaMP3 fluorescence (F/F0) from three different ROIs (top: ROI 1; middle: ROI 2; bottom: ROI 3) depicted in panel B, with indication of the application periods of TRP channel agonists.

We used this novel approach to compare TRP channel activity in the skin of CFA- and vehicle-treated paws (Figure 4A,B). To minimize cross-(de)sensitization between stimuli, we used the same order of agonist applications as used in the DRG preparation (Figure 2), and allowed a wash-out period of 5 min between stimuli (Figure 4A,B). Since we measure responses from GCaMP3-positive nerve endings in the skin, not from clearly identifiable individual cells as in the DRG assay, a quantitative comparison of responses between ipsi- and contralateral paw posed several problems. First, the individual regions of interest that were automatically identified based on proximity and correlated activity represent only segments of individual sensory nerve endings, not the entire ending. Therefore, quantification of the percentage of nerve endings that respond to the specific TRP channel agonists was not feasible. Second, nerve endings in the skin did not always show robust responses to a depolarizing high K+ solution, even when they showed robust responses to one or more TRP channel agonists (Figure 4B), making a normalization as was done for the neuronal cell bodies unreliable. The lack of consistent responses to a depolarizing high K+ solution may reflect that not all nerve endings contain voltage-gated Ca2+ channels or that inactivation of voltage-gated channels occurred due to the preceding TRP channel activation. Therefore, to quantify the functional expression of the three TRP channels, we determined the total area of automatically detected ROIs that showed a correlated response to each of the specific agonists, normalized this active area to the total imaged area (Figure 4A), and made a paired comparison between equivalent skin areas of the ipsi- and contralateral paws of the same animal. This analysis indicated increased reactivity to agonists of all three channels in the inflamed skin (Figure 4C–E and Figure 4—figure supplement 1). The responsive area was increased to 274% ([CI], 138% to 481%; p=0.04) for TRPM3 agonism, to 197% ([CI], 122% to 281%; p=0.02) for TRPA1 agonism and to 256% ([CI], 156% to 359%; p=0.02) for TRPV1 agonism, when compared to the contralateral side.

Figure 4 with 1 supplement see all
Increased TRPM3 activity in peripheral sensory nerve endings during inflammation.

(A) Normalized fluorescence at baseline (before the first stimulus), and upon stimulation with TRPM3, TRPA1 and TRPV1 agonists of the ipsi- and contralateral skin of a CFA-treated mouse. Scale bar is 50 μm. Boxed areas, magnified on the right, illustrate automatically detected ROIs. (B) GCaMP3 fluorescence, expressed as ΔF/F0, for the ROIs indicated in panel (A). (C-E) Responsive areas to the indicated agonists in the contralateral (black) and ipsilateral (red) skin. The paired Wilcoxon Signed Rank Test was used for a paired comparison of the responsive area in the ipsi- and contralateral paw skin of 11 mice, measured in the absence of isosakuranetin. The ipsi- and contralateral skin of another set of 6 mice was compared following pre-incubation with isosakuranetin (20 μM; grey background). Since the control and isosakuranetin-treated skin preparations originate from different mice, and considering the substantial inter-animal variability in skin thickness and innervation, a full statistical comparison between these data sets was not performed. (F) Percentage of the total imaged area responding to the indicated (combinations of) agonists in the contra- and ipsilateral paws, and following isosakuranetin pre-incubation. Further statistical comparison is provided in Figure 4—figure supplement 1.

We also evaluated how tissue inflammation affects the functional co-expression of the three channels in the nerve terminals in the skin. Like in the DRG cell bodies, we observed a pronounced increase in the surface of nerve endings that showed responses to more than one agonist (Figure 4F and Figure 4—figure supplement 1). Notably, the increase in TRPM3-mediated responses was primarily observed in nerve endings that also responded to TRPV1 and/or TRPA1 agonists (Figure 4F and Figure 4—figure supplement 1). Overall, these qualitative 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.

In a separate set of experiments, we compared the TRP channel-mediated responses in the skin of the ipsi-and contralateral paws following pre-incubation with isosakuranetin. Under this condition, responses to TRPM3 agonists were strongly reduced to 12% ([CI], 3% to 45%; p=0.01; Mann-Whitney test) 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 (Figure 4C–F).

Discussion

In recent work, we demonstrated that three TRP channels (TRPM3, TRPV1, and TRPA1) 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 (Vandewauw et al., 2018). Notably, earlier work had also revealed an absolute requirement of both TRPV1 and TRPM3 for the development of inflammatory heat hypersensitivity, since genetic ablation or pharmacological inhibition of either channel individually fully abrogates heat hyperalgesia in the rodent CFA model (Caterina et al., 2000; Davis et al., 2000; Gavva et al., 2005; Honore et al., 2005; Alkhatib et al., 2019; Krügel et al., 2017; Vriens et al., 2011). These combined findings suggested that inflammation modulates the functional interplay between these heat-activated TRP channels leading to pathological heat hypersensitivity, but the underlying processes and mechanisms remained unclear. Here, by directly comparing the levels of mRNA expression and functional activity of TRPM3, TRPV1 and TRPA1 between sensory neurons innervating the healthy and inflamed hind paw in mice, we arrive at the following novel conclusions: (1) acute inflammation is associated with an increased TRPM3 expression at the mRNA level, specifically in DRG neurons innervating the inflamed paw; no significant changes were detected in the mRNA levels for TRPA1 or TRPV1; (2) inflammation is associated with increased functionality of TRPM3, TRPA1 and TRPV1 in DRG neurons, as evidenced by increased Ca2+ 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.

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 (Endres-Becker et al., 2007; Ji et al., 2002). However, some earlier studies found increased levels of TRPA1 mRNA in lumbar DRG following CFA treatment of the hind paw (Obata et al., 2005; Zhou et al., 2013). These results seem at odds with our current findings, which did not reveal a statistically significant increase in TRPA1 mRNA in neurons innervating the inflamed paw. It should be noted, however, that in these earlier studies, bulk mRNA from entire ganglia was analyzed, which inevitably includes not only neurons that innervate injured tissue, but also neurons from the same DRG that innervate healthy tissue, as well as non-neuronal cells present in DRG such as satellite glia (Shin et al., 2020). In contrast, our approach using quantitative in situ hybridization and retrograde labeling allowed us to specifically measure mRNA levels in the cell bodies of the sensory neurons that innervate the inflamed or control hind paw. On the other hand, given the significant variability in TRPA1 mRNA levels between individual DRG neurons observed in our RNA-scope experiments, and the relatively limited number of neurons that were identified as WGA-AF647+, 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.

We used two novel approaches to assess inflammation-induced alterations in the functionality of the three TRP channels, one at the level of the cell bodies and the other at the level of the sensory nerve endings of DRG neurons. First, we developed an acute ex vivo preparation of the relevant part of the spinal column, where the cell bodies of sensory neurons were imaged within an intact DRG, but lacking the peripheral input. This preparation avoids the potential loss of specific neuronal populations as well as rapid alterations in their functional properties that are inherent to the isolation and culturing of DRG neurons (Wangzhou et al., 2020). The use of the TRPV1-cre line to drive GCaMP3 expression allowed the specific recording from neurons involved in thermosensation and nociception (Mishra and Hoon, 2010; Mishra et al., 2011), whereas the use of WGA-AF647-labeling ensured the specificity for afferent neurons that innervated the hind paw tissue. With the use of specific agonists, we were able to directly and quantitatively compare the TRPM3-, TRPA1- and TRPV1-induced activity between cell bodies of neurons that innervated the inflamed versus the control paw. Interestingly, we found increased responses to agonists for all three channels and a particular increase in neurons that functionally co-expressed TRPM3 with TRPV1 and TRPA1. Given the stringent criterium we used to classify a cell as WGA-AF647+ (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 (Figure 1B) and TRP-mediated responses (Figure 2D–F) in the ipsilateral, WGA-AF647- DRG neurons. Increased activity of TRPV1 and TRPA1 in DRG neurons following CFA-induced inflammation is fully in line with earlier work (Zhou et al., 2013; Breese et al., 2005; Nicholas et al., 1999), but this study is, to our knowledge, the first to reveal the specific increase in the subpopulation of DRG co-expressing the three heat-sensing TRP channels. Secondly, we developed an ex vivo assay, where we used GCaMP3-based calcium imaging to monitor TRP channel-mediated responses in intact sensory nerve endings in the paw skin. The skin preparation that we used is identical to the saphenous skin–nerve preparation that has been widely studied using extracellular electrodes to measure propagated action potentials from the receptive fields of single sensory nerve endings in the skin, including responses evoked by TRP channel agonists such as capsaicin (Reeh, 1986; Zimmermann et al., 2009). This novel imaging approach allowed us to demonstrate for the first time that TRPM3, TRPA1, and TRPV1 show a pattern of partial spatial functional overlap in peripheral nerve endings in the skin. Moreover, similar to the findings in the neuronal cell bodies, our results suggest an increase in the surface of nerve endings that functionally express TRPM3 in the skin of the inflamed paw, particularly in endings that also respond to TRPV1 and TRPA1 agonists.

The potent TRPM3 antagonist isosakuranetin effectively eliminated TRPM3-mediated calcium responses, both in the DRG cell bodies and nerve terminals. Intriguingly, isosakuranetin also tempered the responses to capsaicin and MO in DRG neurons innervating the inflamed paw, restoring both the response amplitudes and the fraction of responding cells to the same level as in neurons innervating uninjured tissue. We can exclude the possibility that the reduced responses are due to a direct inhibitory effect of isosakuranetin on TRPV1 or TRPA1 activity since the responses to capsaicin and MO were unaffected by the TRPM3 antagonists in neurons innervating healthy tissue. Likewise, earlier studies have shown that isosakuranetin does not inhibit heterologously expressed TRPV1 or TRPA1 (Straub et al., 2013). Instead, these data raise the possibility that 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. This interpretation also provides a straightforward mechanism for the observation that heat hyperalgesia does not develop in TRPM3-deficient mice and is fully alleviated by TRPM3 antagonists (Alkhatib et al., 2019; Vriens et al., 2011). Since heat hyperalgesia is also strongly attenuated by pharmacological inhibition or genetic ablation of TRPV1 (Caterina et al., 2000; Davis et al., 2000; Gavva et al., 2005; Honore et al., 2005), we hypothesize that those DRG neurons that gain functional co-expression of TRPM3 and TRPV1 under inflammatory conditions play a central role in the development of heat hypersensitivity.

In conclusion, the present findings provide the first evidence that TRPM3 expression and activity are increased in sensory neurons that innervate acutely inflamed tissue. In particular, we found a marked increase in sensory neurons that functionally co-express TRPM3 with TRPV1 and TRPA1, two other TRP channels implicated in heat sensing and inflammatory pain, and this was observed both in the peripheral nerve endings and in the DRG cell bodies. Strikingly, pharmacological inhibition of TRPM3 also reduced TRPV1- and TRPA1-mediated responses in nociceptors innervating the inflamed paw but not in the contralateral control neurons, suggesting that enhanced TRPM3 activity may contribute to neuronal hyperexcitability under inflammatory conditions. Therefore, these results provide a straightforward rationale for the development of TRPM3 antagonists to prevent or alleviate inflammatory pain.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent (M. musculus)C57BL/6JRjJanvier Labshttps://www.janvier-labs.com/en/fiche_produit/c57bl-6jrj_mouse/
Genetic reagent (M. musculus)TRPV1-GCaMP3This paperObtained by crossing Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J mice (Stock#: 029043) with Trpv1tm1(cre)Bbmmice (Stock#: 017769). Both strains were acquired from Jackson Laboratory. Crossings were made in house.
Peptide, recombinant proteinWGA-AF647 (wheat germ agglutinin- Alexa Fluor 647)Thermo Fisher ScientificCat#: W324660.8% in PBS; 10 µl per injection
Commercial assay or kitRNAscope 2.0 Fluorescent Multiplex Reagent KitAdvanced Cell DiagnosticsCat#: 320850; RRID:SCR_012481
Commercial assay or kitTRPV1 ProbeAdvanced Cell DiagnosticsCat#: 313331
Commercial assay or kitTRPM3 ProbeAdvanced Cell DiagnosticsCat#: 459911
Commercial assay or kitTRPA1 ProbeAdvanced Cell DiagnosticsCat#: 400211
Commercial assay or kitPgP9.5 ProbeAdvanced Cell DiagnosticsCat#: 561861-C2
Chemical compound, drugisosakuranetinExtrasyntheseCat#: 137420 µM
Chemical compound, drugCIM0216Sigma-AldrichCat#: 5343591 µM
Chemical compound, drugPregnenolone sulfateSigma-AldrichCat#: P162100 µM
Chemical compound, drugMustard oilSigma-AldrichCat#: W203408100 µM
Chemical compound, drugcapsaicinSigma-AldrichCat#: M20281 µM
Chemical compound, drugDAPIThermo Fisher ScientificCat#: P36931
Software, algorithmTurboreg algorithmhttps://imagej.net/TurboRegRRID:SCR_003070
Software, algorithmCNMF-E algorithmhttps://github.com/zhoupc/CNMF_ERRID:SCR_001622
Software, algorithmNIS softwareNikon InstrumentsRRID:SCR_014329
Software, algorithmOriginPro 2019bOriginlabsRRID:SCR_014212
Software, algorithmIgor Pro 8Wavemetrics
OtherCFA (complete freund’s adjuvant)Sigma-AldrichCat#: F5581(1 mg/ml) 10 µl per injection
OtherGlass-bottom microwell dishMatTekCat#: P35G-1.5–14 C
OtherGlass-bottom chamberFluorodish, WPICat#: FD35-100
OtherDAPIThermo Fisher ScientificCat#: P36931

Animals

C57BL/6J wild type mice (Janvier Labs, Le Genest-Saint-Isle, France) and TRPV1-GCaMP3 mice on a C57BL/6J background were used. TRPV1-GCaMP3 mice were generated by crossing Rosa26-floxed-GCaMP3 mice (Zariwala et al., 2012) with TRPV1-cre mice (Mishra et al., 2011). Mice were housed in a conventional facility at 21°C on a 12 hr light-dark cycle with unrestricted access to food and water. Both male and female mice between 8 and 12 weeks of age were used. Experiments were performed in concordance with EU and national legislation and approved by the KU Leuven ethical committee for Laboratory Animals under project number P075/2018 and P122/2018.

Reagents

Reagents were purchased from Sigma-Aldrich (Chemical Co., St. Louis, Missouri) unless otherwise indicated.

Retrograde labeling

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To specifically label afferents from the hind paw skin, 10 μl Alexa Fluor 647-conjugated wheat germ agglutinin (WGA-AF647, Thermo Fisher Scientific, Invitrogen, Eugene, Oregon, USA; 0.8% in sterile PBS) 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.

Paw inflammation

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Local inflammation was induced by injection of 10 µl complete Freund’s adjuvant (CFA, 1 mg/ml) into the plantar surface of the ipsilateral hindpaw of the studied mouse. The contralateral hind paw was injected with 10 μl vehicle (saline, Baxter, Lessen, Belgium). All ipsilateral mouse hind paws showed substantial edema 24 hr after the injection, which was not observed in the control paw.

Calcium imaging

Animals were euthanized using CO2 inhalation, and skin and DRG tissue were collected immediately.

Skin nerve preparation

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Sensory 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.

In situ DRG preparation

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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.

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 NaHCO3, 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 (20 μM; Extrasynthese, Genay Cedex, France). During image acquisition, skin tissue was fixed with the corium side up in a glass-bottom microwell dish (MatTek, 35 mm petri dish, Ashland, USA). 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.

Calcium imaging of the skin-nerve and in situ DRG preparations was performed on an inverted spinning disk confocal microscope (Nikon Ti; Yokogawa CSU-X1 Spinning Disk Unit, Andor, Belfast, Northern Ireland), equipped with a 20x air objective (NA 0.8), a 488 nm laser light and a EMCCD camera (iXon3 DU-897-BV, Andor). For image acquisition and instrument control, Andor iQ software was used. A z-stack of 11 frames (total thickness of 20–30 μm) 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 (Multi Channel Systems, Reutlingen, Germany). Before image acquisition, the DRG samples were excited at 640 nm to detect WGA-647+ 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.

Isolated DRG neurons

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In a limited set of experiments, shown in Figure 2—figure supplement 1, we performed combined Fura-2 and GCaMP3 fluorescence measurements on isolated DRG neurons. Ganglia were excised, washed in neurobasal A medium (Invitrogen) supplemented with 10% fetal calf serum (basal medium), and then incubated for 45 min at 37°C in a mix of 1 mg/ml collagenase and 2.5 mg/ml dispase (Gibco). Digested ganglia were gently washed twice with basal medium and mechanically dissociated by passage through syringes fitted with increasing needle gauges. Neurons were seeded on poly-L-ornithine/laminin coated glass-bottom chambers (Fluorodish, WPI) and cultured overnight at 37°C in 5% CO2 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 (Alexis Biochemicals) 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.

Image processing and analysis

Spinning disk confocal microscope recordings were first z-averaged in Fiji (imageJ, 1.52i) and corrected for translational drift using a reference image that averaged the first 10 frames prior to tissue stimulation (Turboreg plugin, imageJ).

Skin nerve

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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 (CNMF-E, Matlab R2017b) to identify ROIs as contiguous areas with temporally correlated activity (Zhou et al., 2018). The background-subtracted fluorescence was normalized to the basal fluorescence of the reference image (first 10 frames prior to stimulation), yielding F/F0 values.

In situ DRG

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These recordings were analyzed using a general analysis protocol (GA2) in NIS-elements (NIS 5.20.00, Nikon Instruments Europe B.V.). Retrogradely labeled (WGA-AF647+) 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.

To determine responders in the skin-nerve and DRG recordings, two criteria had to be fulfilled: first, the peak F/F0 during stimulation had to exceed five times the standard deviation of the F/F0 value before stimulation. Second, the peak of the first derivative of the fluorescent signal (dF/dt) had to exceed the standard deviation of the dF/dt signal before stimulation.

In situ hybridization

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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 (Advanced Cell Diagnostics, Hayward, CA, United States). 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 (6 contralateral and six ipsilateral), isolated from six mice.

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-AF647+ 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 (https://acdbio.com/ebook/introduction/materialsmethod). Cells containing five or more RNAscope dots were considered as positive for the respective TRP channels.

Statistical analysis

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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 (vehicle versus CFA).

References

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Decision letter

  1. Kenton J Swartz
    Senior and Reviewing Editor; National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
  2. Cheryl L Stucky
    Reviewer; Medical College of Wisconsin, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Mulier and colleagues 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, the authors investigate 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 concept that TRPM3 is required for increased TRPV1 and TRPA1 activity is intriguing.

Decision letter after peer review:

Thank you for choosing to send your work, "Upregulation of TRPM3 drives hyperexcitability in nociceptors innervating inflamed tissue", for consideration at 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.

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 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.

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.

Given the effect sizes are pretty small, these issues need addressing before publication in eLife. I also recommend reducing the strength of the claims and that including additional analyses would be helpful.

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.

– Why do the ipsilateral WGA-negative neurons in the Figure 1C FISH and the Figure 2D Ca2+ 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.

Figure 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 (for a total of 4 conditions per side). 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 about

Figures 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 (size, thickness density of innervation, health, etc). 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 (seems as if just 2 outliers are driving this difference; Figure 2) 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 (Figure 3C). 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 (presumably dorsal hairy skin) 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.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Upregulation of TRPM3 in nociceptors innervating inflamed tissue" for consideration by 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).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

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 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.

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.

https://doi.org/10.7554/eLife.61103.sa1

Author response

[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.

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 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".

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.

See below. We politely disagree that the effect sizes are "pretty small." For instance, concerning TRPM3, we observe a >60% increase in mRNA (with P values as low as 10-5) and >300% increase in functional responses (P<10-6). We also reanalyzed the data sets according to the different suggestions of this reviewer (per animal instead of per cell; with or without normalization to high potassium responses; see the revised Figures 1 and 2, and their figure supplements), and the results remain very similar and highly significant.

From this and later comments, including also one comment from reviewer #2, we now realize that the way of representing our data (showing all individual data points and diamond plots) was maybe not the best choice to visualize the magnitude of the effects (although it is a recommended way of showing non-parametric data). 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 shown

We 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 (ipsilateral and contralateral, retrogradely labeled or not) from the different mice (Figure 1B, D, F and Figure 1—figure supplement 2). In addition, we also calculated the percentage of positive neurons (with a cutoff of 5 RNAscope dots) for each condition per mouse (Figure 1C, E, G). 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% (95% confidence interval [CI], 122% to 222%; P=0.008) of the levels in the WGA-AF647+ neurons on the contralateral side, and to 133% (CI, 108%-163%; P=0.005) 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 (Figures 2-4) provide further independent support for the increased expression of TRPM3.

– Why do the ipsilateral WGA-negative neurons in the Figure 1C FISH and the Figure 2D Ca2+ 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.

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 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 shown

These 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.

From this comment, we realize that the way of representing our data (showing all individual data points and diamond plots) 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 (based on data from individual mice) 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 (and 95% confidence intervals) 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% ([CI], 265% to 755%; P=7×10-7) when compared to the WGA-positive contralateral neurons (Figure 2D). Also for TRPA1 and TRPV1, we measured at least a doubling of the average response amplitude when compared to the WGA-positive contralateral neurons.

As to the normalization to high potassium, we have reanalyzed and compared the responses both with and without normalization. First, we found that there was no difference in absolute response amplitudes to the high potassium stimulus between the groups, as is now shown in Figure 2—figure supplement 3A. Next, we found that by and large the reported differences between the groups remain significant when normalization was not applied, as is now shown in Figure 2—figure supplement 3B, D. We note, however, that normalization to high potassium reduced the overall variation, and thus that most P-values are slightly higher when no normalization is applied. We are confident that, by showing both the normalized and non-normalized data, the readers can readily appreciate the robustness of the described effects.

– In Figure 2D-F, data from the Iso treatment experiments should additionally be shown for WGA-negative neurons (for a total of 4 conditions per side). 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 easier

Thank 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 about

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 (see e.g. https://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.

Figures 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 (size, thickness density of innervation, health, etc). 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 (we used a paired test, as indicated in the legend), 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.

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 (along with a 95% confidence interval) 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 "increased reactivity to agonists of all three channels in the inflamed skin".

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 (e.g. current, transport) at the membrane. Also here, we meant to point at the increased functional responses, which are evident for all three channels (Figures 2 and 4). 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 (seems as if just 2 outliers are driving this difference; Figure 2) 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 (showing all individual data points and diamond plots) was maybe not the best choice to visualize the magnitude of the effects (although it is a generally recommended way of showing non-parametric data). We have, therefore made important changes to Figure 2, including four figure supplements, to make this aspect clearer for the reader.

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 (which include both the M3+A1 (a very small subset) 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+ ipsilateral neurons is more than doubled following CFA, which is now also easier to appreciate from the revised Figure 2E.

In addition, we now also provide a value in the text (as well as the corresponding 95% confidence interval [CI]) representing the average increase in response amplitude in ipsilateral WGA-AF647+ neurons, which amounts to 229% ([CI], 146% to 355%; P=2×10-5) 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.

It is true that we also observed increased responses for all three TRP channels in the 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.

• 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. (Mishra and Hoon, 2010 and Mishra et al., 2011), 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 (Zariwala et al., 2012). 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 (Vandewauw et al., 2018; Vriens et al., 2011). We have included a brief discussion of the issue of cross (de)sensitization in the revised manuscript. Whereas the full intricacies of the (potential) 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 (Figure 3C). 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 (presumably dorsal hairy skin) 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 (e.g., PSNL neuropathic pain) 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 (>300 dots per cell; see Figure 1—figure supplement 2) 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 (see e.g. Vriens et al., 2011).

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.

https://doi.org/10.7554/eLife.61103.sa2

Article and author information

Author details

  1. Marie Mulier

    1. Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Writing - original draft, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8429-7136
  2. Nele Van Ranst

    1. Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Contribution
    Formal analysis, Investigation
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2242-4050
  3. Nikky Corthout

    1. VIB Bio Imaging Core and VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Neuroscience, KU Leuven, Leuven, Belgium
    Contribution
    Software, Formal analysis, Investigation, Writing - review and editing
    Competing interests
    No competing interests declared
  4. Sebastian Munck

    1. VIB Bio Imaging Core and VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Neuroscience, KU Leuven, Leuven, Belgium
    Contribution
    Supervision, Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5182-5358
  5. Pieter Vanden Berghe

    Laboratory for Enteric NeuroScience (LENS), TARGID, Department of Chronic Diseases Metabolism and Ageing, KU Leuven, Leuven, Belgium
    Contribution
    Methodology, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0009-2094
  6. Joris Vriens

    Laboratory of Endometrium, Endometriosis and Reproductive Medicine, G-PURE, Department of Development and Regeneration, KU Leuven, Leuven, Belgium
    Contribution
    Supervision, Writing - review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2502-0409
  7. Thomas Voets

    1. Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Contribution
    Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft
    Contributed equally with
    Lauri Moilanen
    For correspondence
    thomas.voets@kuleuven.vib.be
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5526-5821
  8. Lauri Moilanen

    1. Laboratory of Ion Channel Research (LICR), VIB-KU Leuven Centre for Brain & Disease Research, Leuven, Belgium
    2. Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium
    Present address
    Department of Pathology, Central Finland Health Care District, Jyväskylä, Finland
    Contribution
    Conceptualization, Formal analysis, Supervision, Investigation, Methodology, Writing - original draft, Writing - review and editing
    Contributed equally with
    Thomas Voets
    Competing interests
    No competing interests declared

Funding

Hercules Foundation (AKUL/09/50)

  • Pieter Vanden Berghe

KU Leuven (C1-TRPLe)

  • Thomas Voets

Fonds Wetenschappelijk Onderzoek (G0B7620N)

  • Thomas Voets

Belgian Foundation Against Cancer

  • Joris Vriens
  • Thomas Voets

Queen Elisabeth Medical Foundation for Neurosciences

  • Thomas Voets

Vlaams Instituut voor Biotechnologie

  • Sebastian Munck
  • Thomas Voets

Fonds Wetenschappelijk Onderzoek (G0B5316N)

  • Thomas Voets

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We acknowledge the Cell and Tissue imaging cluster (CIC; KU Leuven), where confocal microscopy was performed, and the Light Microscopy and Imaging Network (LiMoNe; CBD-VIB), where in situ hybridization slides were imaged. Calcium imaging at CIC was performed on an Andor Revolution Spinning Disk System supported by Hercules AKUL/09/50 to PVB. This research was further supported by grants from the VIB, KU Leuven Research Council (C1-TRPLe to TV), the Research Foundation-Flanders (FWO G0B7620N to TV), the Belgian Foundation Against Cancer (to JV and TV) and the Queen Elisabeth Medical Foundation for Neurosciences (to TV).

Ethics

Animal experimentation: Experiments were performed in concordance with EU and national legislation and approved by the KU Leuven ethical committee for Laboratory Animals under project number P075/2018 and P122/2018.

Senior and Reviewing Editor

  1. Kenton J Swartz, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States

Reviewer

  1. Cheryl L Stucky, Medical College of Wisconsin, United States

Publication history

  1. Received: July 15, 2020
  2. Accepted: August 20, 2020
  3. Version of Record published: September 3, 2020 (version 1)

Copyright

© 2020, Mulier et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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