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. 2021 Mar 17;41(11):2457-2474.
doi: 10.1523/JNEUROSCI.1551-20.2020. Epub 2021 Jan 21.

TRPM3 Channels Play Roles in Heat Hypersensitivity and Spontaneous Pain after Nerve Injury

Songxue Su  1   2 , Yevgen Yudin  1 , Nawoo Kim  1 , Yuan-Xiang Tao  3 , Tibor Rohacs  4
Affiliations

TRPM3 Channels Play Roles in Heat Hypersensitivity and Spontaneous Pain after Nerve Injury

Songxue Su et al. J Neurosci. .

Abstract

Transient receptor potential melastatin 3 (TRPM3) is a heat-activated ion channel in primary sensory neurons of the dorsal root ganglia (DRGs). Pharmacological and genetic studies implicated TRPM3 in various pain modalities, but TRPM3 inhibitors were not validated in TRPM3-/- mice. Here we tested two inhibitors of TRPM3 in male and female wild-type and TRPM3-/- mice in nerve injury-induced neuropathic pain. We found that intraperitoneal injection of either isosakuranetin or primidone reduced heat hypersensitivity induced by chronic constriction injury (CCI) of the sciatic nerve in wild-type, but not in TRPM3-/- mice. Primidone was also effective when injected locally in the hindpaw or intrathecally. Consistently, intrathecal injection of the TRPM3 agonist CIM0216 reduced paw withdrawal latency to radiant heat in wild-type, but not in TRPM3-/- mice. Intraperitoneal injection of 2 mg/kg, but not 0.5 mg/kg isosakuranetin, inhibited cold and mechanical hypersensitivity in CCI, both in wild-type and TRPM3-/- mice, indicating a dose-dependent off-target effect. Primidone had no effect on cold sensitivity, and only a marginal effect on mechanical hypersensitivity. Genetic deletion or inhibitors of TRPM3 reduced the increase in the levels of the early genes c-Fos and pERK in the spinal cord and DRGs in CCI mice, suggesting spontaneous activity of the channel. Intraperitoneal isosakuranetin also inhibited spontaneous pain related behavior in CCI in the conditioned place preference assay, and this effect was eliminated in TRPM3-/- mice. Overall, our data indicate a role of TRPM3 in heat hypersensitivity and in spontaneous pain after nerve injury.SIGNIFICANCE STATEMENT Neuropathic pain is a major unsolved medical problem. The heat-activated TRPM3 ion channel is a potential target for novel pain medications, but the pain modalities in which it plays a role are not clear. Here we used a combination of genetic and pharmacological tools to assess the role of this channel in spontaneous pain, heat, cold, and mechanical hypersensitivity in a nerve injury model of neuropathic pain in mice. Our findings indicate a role for TRPM3 in heat hyperalgesia, and spontaneous pain, but not in cold and mechanical hypersensitivity. We also find that not only TRPM3 located in the peripheral nerve termini, but also TRPM3 in the spinal cord or proximal segments of DRG neurons are important for heat hypersensitivity.

Keywords: TRPM3; heat; pain.

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Figures

Figure 1.
Figure 1.
TRPM3 deficiency reduces hypersensitivity to thermal, but not to mechanical, stimuli in carrageen-induced inflammatory pain. A, B, Ca2+ imaging experiments on isolated DRG neurons from wild-type (A) and TRPM3−/− (B) mice. The applications of 12.5 μm PregS and 30 mm KCl are indicated by the horizontal lines. Data are shown as the mean ± SEM n = 69 for all PregS responsive neurons from one DRG isolation for wild-type mice, and n = 197 neurons for TRPM3−/− mice from a separate DRG neuron isolation on the same day. Three independent DRG neuron isolations both from wild-type mice and from TRPM−/− mice were performed, and PregS was applied in all three of them with similar results. C, D, Latencies to thermal stimuli after hind paw injection of carrageenan in the Hargreaves test of ipsilateral (C) and contralateral (D) sides of TRPM3-KO mice and wild-type littermates (TRPM3-WT; n = 9–11). The p values on the top indicate a difference between TRPM3-KO and TRPM3-WT mice; p values at the bottom indicate a difference from the baseline of TRPM3-WT mice. Data are expressed as the mean ± SD. Statistical significance was calculated with two-way ANOVA (genotype × time) and Tukey's post hoc test (Fgenotype(1,72) = 173.43, p < 1 × 10−15; Ftime(3,72) = 9.91, p = 1.5 × 10−5; Fgenotype × time(3,72) = 6.00, p = 0.001) for the ipsilateral side. E–H, Paw withdrawal frequencies to mechanical stimulation at 0.07 g (E, ipsilateral; F, contralateral) and 0.4 g (G, ipsilateral; H, contralateral) in von Frey filament test of TRPM3-WT and TRPM3-KO mice (n = 9-11), p values on top show a difference from baseline in TRPM3-KO mice; p values in the bottom show difference from baseline for TRPM3- WT mice. Bars represent the mean ± SD. Statistical significance was calculated with two-way ANOVA (genotype × time) and Tukey's post hoc test (Fgenotype(1,76) = 0.17, p = 0.68; Ftime(3,76) = 89.9, p < 1 × 10−15; Fgenotype × time(3,76) = 0.36, p = 0.78 for the values of ipsilateral side at 0.07 g. Fgenotype(1,76) = 0.15, p = 0.70; Ftime(3,76) = 98.6, p < 1 × 10−15; Fgenotype × time(3,76) = 0.10, p = 0.96 for the values of ipsilateral side at 0.4 g.
Figure 2.
Figure 2.
TRPM3 deficiency impairs carrageenan-induced pronociceptive signal transduction protein expressions in L3–L5 DRGs and the corresponding dorsal horn. A, Sections of c-Fos (left) and pERK (right) immunostaining in DRGs of TRPM3-WT and TRPM3-KO mice at 4 h after carrageen or vehicle injection. Scale bars, 50 μm. B, Sections of c-Fos (left) and pERK (right) immunostaining in dorsal spinal dorsal horn of TRPM3-WT and TRPM3-KO mice at 4 h after carrageenan or vehicle injection. Scale bars, 50 μm. C, The numbers of c-Fos-positive cells and pERK-positive cells in the DRG sections were counted (n =11–13 sections, from three independent DRG preparations). Data are expressed as the mean ± SEM and scatter plots. Statistical significance was calculated with two-way ANOVA (genotype × treatment) and Tukey's post hoc test (Fgenotype(1,42) = 10.07, p = 0.003; Ftreatment(1,42) = 119.29, p = 7.60 × 10−14; Fgenotype × treatment(1,42) = 8.18, p = 0.007 for c-Fos expression; Fgenotype(1,42) = 7.77, p = 0.008; Ftreatment(1,42) = 95.04, p = 2.38 × 10−12; Fgenotype × treatment(1,42) = 6.77, p = 0.01 for pERK expression). D, The numbers of c-Fos-positive cells and pERK-positive cells in the dorsal horn sections were counted (n = 11–13 sections, from three independent spinal cord preparations). Statistical significance was calculated with two-way ANOVA (genotype × treatment) and Tukey's post hoc test (Fgenotype(1,43) = 4.09, p = 0.049, Ftreatment(1,43) = 115.29, p = 9.50 × 10−14; Fgenotype × treatment(1,43) = 4.00, p = 0.05 for c-Fos expression; Fgenotype(1,36) = 4.55, p = 0.04, Ftreatment(1,42) = 116.05, p = 8.19 × 10−13; Fgenotype × treatment(1,36) = 3.31, p = 0.08 for pERK expression). Data are expressed as the mean ± SEM and scatter plots.
Figure 3.
Figure 3.
The effect of systemic isosakuranetin administration in naive mice and in WT and TRPM3−/− mice with peripheral nerve injury-induced neuropathic pain. A, Paw withdrawal latencies to thermal stimuli at different doses of isosakuranetin in naive mice (n = 9–12); one-way ANOVA and Tukey's post hoc test (Ftreatment(4,46) = 22.39, p = 2.60 × 10−10). Data are expressed as the mean ± SEM and scatter plots. B, The effect of 2 mg/kg isosakuranetin on paw withdrawal latency to thermal stimuli in the Hargreaves test of ipsilateral TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery (n = 9–14). The p values were calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,87) = 32.57, p = 1.56 × 10−7; Fgenotype(1,87) = 75.63, p = 1.89 × 10−13; Ftreatment(1,87) = 13.42, p = 0.0004; Fsurgery × genotype(1,87) = 13.04, p = 0.0005; Fsurgery × treatment(1,87) = 0.40, p = 0.53; Fgenotype × treatment (1,87) = 11.07, p = 0.001. C, D, The effect of isosakuranetin on mechanical sensitivity in von Frey test of ipsilateral hindpaws of TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery at 0.07 g (C) and 0.4 g (D; n = 10–14). Statistical significance was calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,88) = 280.24, p < 1 × 10−15; Fgenotype(1,88) = 3.24, p = 0.08; Ftreatment(1,88) = 28.83, p = 6.35 × 10−7; Fsurgery × genotype(1,88) = 0.0002, p = 0.99; Fsurgery × treatment(1,88) = 14.17, p = 0.0003; Fgenotype × treatment(1, 88) = 0.02, p = 0.88 for the values at 0.07 g; Fsurgery(1,88) = 376.92, p < 1 × 10−15; Fgenotype(1,88) = 0.39, p = 0.53; Ftreatment(1, 88) = 52.32, p = 1.65 × 10−10; Fsurgery × genotype(1,88) = 1.43, p = 0.23; Fsurgery × treatment(1,88) = 41.77, p = 5.53 × 10−9; Fgenotype × treatment(1,88) = 0.17, p = 0.68 for the values at 0.4 g). E, F, The effect of isosakuranetin on paw withdrawal responses to noxious cold (E) and innocuous cold (F) in cold plate test of ipsilateral of TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery (n = 7–9). The p values were calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,53) = 92.31, p = 3.35 × 10−13; Fgenotype(1,53) = 0.40, p = 0.53; Ftreatment(1,53) = 9.06, p = 0.004; Fsurgery × genotype(1,53) = 0.09, p = 0.76; Fsurgery× treatment(1,53) = 15.04, p = 0.0003; Fgenotype × treatment(1,53) = 0.22, p = 0.64 for the values at the 0°C plate; Fsurgery(1,55) = 217.21, p < 1 × 10−15; Fgenotype(1,55) = 1.30, p = 0.26; Ftreatment(1, 55) =7.59, p = 0.008; Fsurgery × genotype(1,55) = 0.55, p = 0.46; Fsurgery × treatment(1,55) = 16.63, p = 0.0001; Fgenotype × treatment(1,55) = 0.0004, p = 0.98; for the values at the 5°C plate). Data are expressed as the mean ± SEM and scatter plots.
Figure 4.
Figure 4.
The effect of systemic administration of 0.5 mg/kg isosakuranetin in WT and TRPM3−/− mice with peripheral nerve injury-induced neuropathic pain. A, B, The effect of isosakuranetin on paw withdrawal latency to thermal stimuli in the Hargreaves test of ipsilateral (A) and contralateral (B) hindpaws of TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery (n = 8–10); three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,66) = 21.16, p = 2.0 × 10−5; Fgenotype(1,66) = 70.08, p = 5.73 × 10−12; Ftreatment(1,66) = 3.64, p = 0.06; Fsurgery × genotype(1,66) = 20.61, p = 2.0 × 10−5; Fsurgery × treatment(1,66) = 0.45, p = 0.50; Fgenotype × treatment(1,66) = 11.98, p = 0.0009 for the values of ipsilateral side). C–F, The effect of isosakuranetin on mechanical sensitivity in von Frey test of ipsilateral (C, E) and contralateral (D, F) hindpaws of TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery at 0.07 g (C, D) and 0.4 g (E, F; n = 8–10); three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,66) = 372.0, p < 1 × 10−15; Fgenotype(1,66) = 0.04, p = 0.85; Ftreatment(1,66) = 0.15, p = 0.70; Fsurgery × genotype(1,66) = 0.26, p = 0.61; Fsurgery × treatment(1,66) = 0.004, p = 0.95; Fgenotype × treatment(1,66) = 0.17, p = 0.68 for the values at the ipsilateral side at 0.07 g. Fsurgery(1,66) = 339.24, p < 1 × 10−15; Fgenotype(1,66) = 0.29, p = 0.59; Ftreatment(1,66) = 0.19, p = 0.66; Fsurgery× genotype(1,66) = 0.29, p = 0.60; Fsurgery× treatment(1,66) = 1.17, p = 0.28; Fgenotype × treatment(1,66) = 0.002, p = 0.96 for the values at the ipsilateral side at 0.4 g). G, H, The effect of isosakuranetin on paw withdrawal responses to noxious cold (G) and innocuous cold (H) in cold plate test of ipsilateral of TRPM3-WT and TRPM3-KO mice on day 6 following CCI or sham surgery (n = 8–10); three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test Fsurgery(1,66) = 581.58, p < 1 × 10−15; Fgenotype(1,66) = 0.54, p = 0.47; Ftreatment(1,66) = 0.04, p = 0.84; Fsurgery × genotype(1,66) = 0.74, p = 0.39; Fsurgery × treatment(1,66) = 0.16, p = 0.69; Fgenotype × treatment(1,66) = 0.76, p = 0.39 at the 0°C plate; Fsurgery(1,66) = 369.32, p < 1 × 10−15; Fgenotype(1,66) = 0.57, p = 0.45; Ftreatment(1,66) = 0.11, p = 0.74; Fsurgery × genotype(1,66) = 0.13, p = 0.72; Fsurgery × treatment(1,66) = 0.02, p = 0.88; Fgenotype × treatment(1,66) = 0.42, p = 0.52 at the 5°C plate). Data are expressed as the mean ± SEM and scatter plots.
Figure 5.
Figure 5.
The effect of systemic primidone administration in naive mice and WT and TRPM3-KO mice with peripheral nerve injury-induced neuropathic pain. A, Paw withdrawal latencies to thermal stimuli at different doses of primidone in naive mice (n = 11–23). Statistical significance was assessed by one-way ANOVA and Tukey's post hoc test (Ftreatment(4,65) = 21.33, p = 2.85 × 10−11). Data are expressed as the mean ± SEM and scatter plots. B, C, The effect of primidone on paw withdrawal latencies to thermal stimuli in the Hargreaves test of ipsilateral (B) and contralateral (C) side in TRPM3-WT and TRPM3-KO mice on day 6 post CCI or sham surgery (n = 11–17). The p values were calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,108) = 61.08, p = 3.86 × 10−12; Fgenotype(1,108) = 162.57, p < 1 × 10−15; Ftreatment(1,108) = 10.16, p = 0.002; Fsurgery × genotype(1,108) = 21.15, p = 1.0 × 10−15; Fsurgery × treatment(1,108) = 0.16, p = 0.69; Fgenotype × treatment(1,108) = 11.59, p = 0.0009 for the values at the ipsilateral side; Fsurgery(1,109) = 0.47, p = 0.50; Fgenotype(1,109) = 36.50, p = 2.17 × 10−8; Ftreatment(1,109) = 10.13, p = 0.002; Fsurgery × genotype(1,109) = 0.18, p = 0.67; Fsurgery × treatment(1,109) = 0.03, p = 0.85; Fgenotype × treatment(1,109) = 10.76, p = 0.001 for the values at the contralateral side). D–G, The effect of primidone on paw withdrawal frequencies to mechanical stimulation in von Frey test at 0.07 g (D, ipsilateral; E, contralateral) and 0.4 g (F, ipsilateral; G, contralateral) of TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery (n = 11–16); three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test Fsurgery(1,92) = 757.02, p < 1 × 10−15; Fgenotype(1,92) = 0.005, p = 0.94; Ftreatment(1,92) = 2.09, p = 0.15; Fsurgery × genotype(1,92) = 2.67, p = 0.11; Fsurgery × treatment(1,92) = 4.96, p = 0.03; Fgenotype × treatment(1,92) = 0.95, p = 0.33 for the values of ipsilateral side at 0.07 g; Fsurgery(1,100) = 538.51, p < 1 × 10−15; Fgenotype(1,100) = 0.03, p = 0.85; Ftreatment(1,100) = 4.87, p = 0.03; Fsurgery × genotype(1,100) = 0.03, p = 0.85; Fsurgery × treatment(1,100) = 3.39, p = 0.07; Fgenotype × treatment(1,100) = 1.17, p = 0.28 for the values at the ipsilateral side at 0.4 g. H, I, The effect of primidone on paw withdrawal responses to noxious cold (H) and innocuous cold (I) in cold plate test at the ipsilateral side of TRPM3-WT and TRPM3-KO mice on day 6 post CCI or sham surgery (n = 7–10). P values were calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,60) = 145.35, p < 1 × 10−15; Fgenotype(1,60) = 0.007, p = 0.93; Ftreatment(1,60) = 0.10, p = 0.76; Fsurgery× genotype(1,60) = 0.70, p = 0.41; Fsurgery× treatment(1,60) = 0.10, p = 0.76; Fgenotype × treatment(1, 60) = 0.13, p = 0.72 in noxious cold testing; Fsurgery(1,60) = 340.86, p < 1 × 10−15; Fgenotype(1,60) = 0.11, p = 0.74; Ftreatment(1, 60) = 0.01, p = 0.92; Fsurgery × genotype(1,60) = 0.89, p = 0.35; Fsurgery × treatment(1,60) = 0.03, p = 0.87; Fgenotype × treatment(1,60) = 0.11, p = 0.74 in innocuous cold testing). Data are expressed as the mean ± SEM and scatter plots.
Figure 6.
Figure 6.
Systemic administration of isosakuranetin alleviates spontaneous pain in WT and TRPM3-KO mice following CCI surgery in the CPP test. CPP experiments were performed as described in the Materials and Methods section. A, Time spent in the chamber paired with vehicle or isosakuranetin (2 mg/kg) in TRPM3-WT and TRPM3-KO mice in the sham-operated and CCI-operated groups (n = 10–12). The p values were calculated using three-way ANOVA (condition × genotype × treatment) and Tukey's post hoc test (Fcondition(3,156) = 0.02, p = 0.99; Fgenotype(1,156) = 0.02, p = 0.89; Ftreatment(1,156) = 58.73, p = 1.80 × 10−12; Fcondition × genotype(3,156) = 0.02, p = 0.99; Fcondition × treatment(3,156) = 49.28, p < 1 × 10−15; Fgenotype × treatment(1,156) = 10.22, p = 0.002. B, CPP score was calculated as described in the Materials and Methods section for TRPM3-WT and TRPM3-KO mice in the sham-operated and CCI-operated groups (n = 10–12). The p values were calculated using two-way ANOVA (genotype × surgery) and Tukey's post hoc test (Fgenotype(1,39) = 5.00, p = 0.03; Fsurgery(1,39) = 65.33, p = 7.35 × 10−10; Fgenotype × surgery(1.39) = 2.54, p = 0.12). Data are expressed as the mean ± SEM and scatter plots. C, D, similar measurements in mice injected with 0.5 mg/kg isosakuranetin. C, Time spent in corresponding chamber paired with vehicle or isosakuranetin (0.5 mg/kg) in TRPM3-WT and TRPM3-KO mice in the sham-operated and CCI-operated groups (n = 8–10). The p values were calculated using three-way ANOVA (condition × genotype × treatment) and Tukey's post hoc test (Fcondition(3,128) = 0.09, p = 0.97; Fgenotype(1,128) = 0.03, p = 0.87; Ftreatment(1,128) = 14.31, p = 0.0002; Fcondition × genotype(3,128) = 0.05, p = 0.99; Fcondition × treatment(3,128) = 5.62, p = 0.001; Fgenotype × treatment(1,128) = 2.00, p = 0.16; Fcondition×genotype× treatment(3,128) = 6.57, p = 0.0004). D, CPP score was calculated as described in the Materials and Methods section for TRPM3-WT and TRPM3-KO mice in the sham-operated and CCI-operated groups (n = 8–10). The p values were calculated using two-way ANOVA (genotype × surgery) and Tukey's post hoc test (Fgenotype(1,32) = 4.25, p = 0.047; Fsurgery(1,32) = 6.03, p = 0.02; Fgenotype× surgery(1,32) = 2.85, p = 0.10). Data are expressed as the mean ± SEM and scatter plots.
Figure 7.
Figure 7.
TRPM3 deficiency impairs pronociceptive signal transduction following CCI surgery in L3-L5 DRGs. A, Sections of c-Fos (left) and pERK (right) immunostaining in DRGs of TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery. Scale bars, 50 μm. B, The numbers of c-Fos-positive cells in the sections were counted (n = 11–12 sections from three independent preparations). The p values were calculated with two-way ANOVA (genotype × surgery) with Tukey's post hoc test (Fgenotype(1,41) = 4.77, p = 0.035; Fsurgery(1,41) = 90.40, p = 6.31 × 10−12; Fgenotype × surgery(1,41) = 3.15, p = 0.08). C, The numbers of pERK-positive cells in the sections were counted (n = 12 sections from three independent preparations). The p values were calculated with two-way ANOVA (genotype × surgery) with Tukey's post hoc test (Fgenotype(1,44) = 4.97, p = 0.03, Fsurgery(1,44) = 65.14, p = 3.19 × 10−10; Fgenotype × surgery(1,44) = 4.49, p = 0.04) Data are expressed as the mean ± SEM and scatter plots.
Figure 8.
Figure 8.
The effect of isosakuranetin and primidone on pronociceptive signal transduction protein levels in dorsal spinal cord on day 6 following CCI or sham surgery. A, B, Sections of c-Fos (left, A) and pERK (right, B) in L3–L5 dorsal spinal cord of TRPM3-WT and TRPM3-KO mice injected intraperitoneally with vehicle, 2 mg/kg isosakuranetin, or 1 mg/kg primidone. Scale bars, 50 μm. C, D, The number of c-Fos-positive cells (C) and pERK-positive cells (D) in the sections were counted (n = 7–11 sections from three independent preparations). The p values were calculated with three-way ANOVA (surgery × genotype × treatment) with Tukey's post hoc test (Fsurgery(1,77) = 359.66, p < 1 × 10−15; Fgenotype(1,77) = 9.68, p = 0.003; Ftreatment(2,77) = 11.27, p = 5.09 × 10−5; Fsurgery × genotype(1,77) = 10.22, p = 0.002; Fsurgery × treatment(2,77) = 8.24, p = 0.0006; Fgenotype × treatment(2, 77) = 1.03, p = 0.36 for c-Fos expression; Fsurgery(1,90) = 369.90, p < 1 × 10−15; Fgenotype(1,90) = 6.98, p = 0.01; Ftreatment(2,90) = 12.77, p = 1.32 × 10−5; Fsurgery × genotype(1,90) = 5.28, p = 0.02; Fsurgery × treatment(2,90) = 5.54, p = 0.005; Fgenotype × treatment(2, 90) = 1.29, p = 0.28 for pERK expression). Data are expressed as the mean ± SEM and scatter plots.
Figure 9.
Figure 9.
The effect of intraplantar primidone administration in naive mice and WT and TRPM3-KO mice with peripheral nerve injury-induced neuropathic pain. A, B, The effect of primidone on paw withdrawal latency to thermal stimuli in the Hargreaves test of the ipsilateral (A) and contralateral (B) paws in naive mice (n = 7–8). Statistical significance was calculated with one-way ANOVA with Tukey's post hoc test (Ftreatment(2,19) = 8.00, p = 0.003). Data are expressed as the mean ± SEM and scatter plots. C, D, The effect of primidone on paw withdrawal latency to thermal stimuli in the Hargreaves test in ipsilateral (C) and contralateral (D) side of TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery (n = 7–12). Statistical significance was calculated with three-way ANOVA (surgery × genotype × treatment) and Tukey's post hoc test (Fsurgery(1,70) = 67.64, p = 7.05 × 10−12; Fgenotype(1,70) = 113.01, p < 1 × 10−15; Ftreatment(1,70) = 11.63, p = 0.001; Fsurgery × genotype(1,70) = 24.08, p = 5.80 × 10−5; Fsurgery × treatment(1,70) = 0.07, p = 0.79; Fgenotype × treatment(1, 70) = 13.19, p = 0.0005 for the values at the ipsilateral side). E–H, The effect of primidone on paw withdrawal frequency to mechanical stimulation in the von Frey test at 0.07 g (E, ipsilateral; F, contralateral) and 0.4 g (G, ipsilateral; H, contralateral) of TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery (n = 7–10). Statistical significance was calculated with three-way ANOVA (surgery × genotype × treatment) with Tukey's post hoc test (Fsurgery(1,62) = 438.77, p < 1 × 10−15; Fgenotype(1,62) = 0.03, p = 0.86; Ftreatment(1,62) = 3.60, p = 0.06; Fsurgery × genotype(1,62) = 6.41, p = 0.01; Fsurgery × treatment(1,62) = 0.04, p = 0.84; Fgenotype × treatment(1, 62) = 0.0004, p = 0.98 for the values at the ipsilateral side at 0.07 g; Fsurgery(1,61) = 361.27, p < 1 × 10−15; Fgenotype(1,61) = 1.44, p = 0.23; Ftreatment(1,61) = 0.08, p = 0.78; Fsurgery × genotype(1,61) = 9.63, p = 0.99; Fsurgery × treatment(1,61) = 0.74, p = 0.39; Fgenotype × treatment(1,61) = 1.10, p = 0.30 for the values of ipsilateral side at 0.4 g). Data are expressed as the mean ± SEM and scatter plots.
Figure 10.
Figure 10.
TRPM3 is expressed both in the DRG and in the dorsal horn. Multiplex fluorescence in situ hybridization experiments using the RNAScope system were performed as described in the Materials and Methods section. A–C, Confocal images taken with a 40× objective of a representative dorsal horn section from the side of CCI operation at different individual fluorescence channels for the neuronal marker NeuN (A, white), the glial marker GFAP (B, red) and for TRPM3 (C, green). D, Merged image for the three channels plus the nuclear stain DAPI, insets on the right side show enlarged parts of the slide. E, Quantification of the proportion of TRPM3-positive cells among NeuN-positive cells (neurons) and GFAP-positive cells (glia), n = 6 different spinal cord preparations. The colocalization was assessed by the CellProfiler software. Data are shown as the mean ± SEM, for n = 6 different dorsal horn preparations, two to three sections each, the data from different sections from the same preparation were averaged and taken as one data point.
Figure 11.
Figure 11.
The effect of intrathecal primidone administration on nociceptive behaviors following CCI surgery in WT and TRPM3-KO mice. A, B, The effect of intrathecal injection of primidone on paw withdrawal latency to thermal stimuli in the Hargreaves test on the ipsilateral (A) and contralateral (B) side in TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery (n = 7–12). Statistical significance was calculated with three-way ANOVA (surgery × genotype × treatment) with Tukey's post hoc test, Fsurgery(1,60) = 40.73, p = 2.80 × 10−8; Fgenotype(1,60) = 167.69, p < 1 × 10−15; Ftreatment(1,60) = 18.60, p = 6.08 × 10−5; Fsurgery × genotype(1,60) = 12.24, p = 0.0009; Fsurgery × treatment(1,60) = 1.55, p = 0.22; Fgenotype × treatment(1, 60) = 11.51, p = 0.001 for the values at the ipsilateral side; Fsurgery(1,60) = 1.11, p = 0.30; Fgenotype(1,60) = 91.89, p = 1.03 × 10−13; Ftreatment(1,60) = 11.57, p = 0.001; Fsurgery × genotype(1,60) = 0.07, p = 0.79; Fsurgery × treatment(1,60) = 0.0001, p = 0.99; Fgenotype × treatment(1, 60) = 9.39, p = 0.003 for the values at the contralateral side. C–F, The effect of intrathecal primidone on paw withdrawal frequency to mechanical stimulation in the von Frey test at 0.07 g (C, ipsilateral; D, contralateral) and 0.4 g (E, ipsilateral; F, contralateral) of TRPM3-WT and TRPM3-KO mice on day 6 after CCI or sham surgery (n = 7–12). Statistical significance was calculated with three-way ANOVA (surgery × genotype × treatment) with Tukey's post hoc test (Fsurgery(1,59) = 308.47, p < 1 × 10−15; Fgenotype(1,59) = 0.13, p = 0.72; Ftreatment(1,59) = 0.02, p = 0.88; Fsurgery × genotype(1,59) = 2.63, p = 0.11; Fsurgery × treatment(1,59) = 0.03, p = 0.86; Fgenotype × treatment(1, 59) = 0.21, p = 0.65 for the values of ipsilateral side at 0.07 g; Fsurgery(1,60) = 635.61, p < 1 × 10−15; Fgenotype(1,60) = 0.02, p = 0.88; Ftreatment(1,60) = 0.70, p = 0.41; Fsurgery × genotype(1,60) = 0.0009, p = 0.98; Fsurgery × treatment(1,60) = 0.15, p = 0.70; Fgenotype × treatment(1,60) = 1.50, p = 0.22 for the values of ipsilateral side at 0.4 g). Data are expressed as the mean ± SEM and scatter plots.
Figure 12.
Figure 12.
The effect of intrathecal administration of the TRPM3 agonist CIM0216 on nociceptive behavioral responses and pronociceptive signal transduction protein expressions in dorsal spinal cord. A, B, The effect of CIM0126 on paw withdrawal latency to thermal stimuli in the Hargreaves test of left and right sides (n = 10–19). Statistical significance was calculated with two-way ANOVA (genotype × treatment) with Tukey's post hoc test (Fgenotype(1,85) = 95.80, p = 1 × 10−15; Ftreatment(2,85) = 5.62, p = 0.005; Fgenotype × treatment(2,85) = 1.75, p = 0.18 for the values of left side; Fgenotype(1,83) = 83.02, p = 3.90 × 10−14; Ftreatment(2,83) = 6.09, p = 0.003; Fgenotype × treatment(2,83) = 0.21, p = 0.81 for the values of right side). C, Sections of c-Fos (left) and pERK (right) in L3–L5 dorsal spinal cord of TRPM3-WT and TRPM3-KO mice at 30 min after CIM0216 or vehicle injection. Scale bars, 50 μm. D, E, The numbers of c-Fos-positive cells (D) and pERK-positive cells (E) in the sections were counted (n = 8–15 sections from three independent preparations). Statistical significance was calculated with two-way ANOVA (genotype × treatment) followed by Tukey's multiple comparisons test (Fgenotype(1,44) = 18.27, p = 0.0001; Ftreatment(1,44) = 202.31, p < 1 × 10−15; Fgenotype×treatment(1,44) = 21.21, p = 4.0 × 10−5 for c-Fos expression; Fgenotype(1,31) = 4.79, p = 0.036, Ftreatment(1,31) = 162.50, p = 7.30 × 10−14; Fgenotype × treatment(1,31) = 4.79, p = 0.036 for pERK expression). Data are expressed as the mean ± SEM and scatter plots.
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