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The TRPM2 ion channel is required for sensitivity to warmth

Abstract

Thermally activated ion channels are known to detect the entire thermal range from extreme heat (TRPV2), painful heat (TRPV1, TRPM3 and ANO1), non-painful warmth (TRPV3 and TRPV4) and non-painful coolness (TRPM8) through to painful cold (TRPA1)1,2,3,4,5,6,7. Genetic deletion of each of these ion channels, however, has only modest effects on thermal behaviour in mice6,7,8,9,10,11,12, with the exception of TRPM8, the deletion of which has marked effects on the perception of moderate coolness in the range 10–25 °C13. The molecular mechanism responsible for detecting non-painful warmth, in particular, is unresolved. Here we used calcium imaging to identify a population of thermally sensitive somatosensory neurons which do not express any of the known thermally activated TRP channels. We then used a combination of calcium imaging, electrophysiology and RNA sequencing to show that the ion channel generating heat sensitivity in these neurons is TRPM2. Autonomic neurons, usually thought of as exclusively motor, also express TRPM2 and respond directly to heat. Mice in which TRPM2 had been genetically deleted showed a striking deficit in their sensation of non-noxious warm temperatures, consistent with the idea that TRPM2 initiates a ‘warm’ signal which drives cool-seeking behaviour.

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Figure 1: Around 10% of somatosensory neurons demonstrate a novel heat-sensitive response.
Figure 2: Properties of the novel heat-sensitive ion channel in autonomic neurons.
Figure 3: Deletion of TRPM2 shifts adult male mouse thermal preference towards warmer temperatures.

References

  1. Cesare, P. & McNaughton, P. A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin. Proc. Natl Acad. Sci. USA 93, 15435–15439 (1996)

    CAS  ADS  Article  Google Scholar 

  2. Caterina, M. J. et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824 (1997)

    CAS  ADS  Article  Google Scholar 

  3. Jordt, S. E., McKemy, D. D. & Julius, D. Lessons from peppers and peppermint: the molecular logic of thermosensation. Curr. Opin. Neurobiol. 13, 487–492 (2003)

    CAS  Article  Google Scholar 

  4. Basbaum, A. I., Bautista, D. M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139, 267–284 (2009)

    CAS  Article  Google Scholar 

  5. Vriens, J. et al. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Neuron 70, 482–494 (2011)

    CAS  Article  Google Scholar 

  6. Cho, H. et al. The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons. Nature Neurosci. 15, 1015–1021 (2012)

    CAS  Article  Google Scholar 

  7. Vriens, J., Nilius, B. & Voets, T. Peripheral thermosensation in mammals. Nature Rev. Neurosci. 15, 573–589 (2014)

    CAS  Article  Google Scholar 

  8. Caterina, M. J. et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 288, 306–313 (2000)

    CAS  ADS  Article  Google Scholar 

  9. Davis, J. B. et al. Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia. Nature 405, 183–187 (2000)

    CAS  ADS  Article  Google Scholar 

  10. Moqrich, A. et al. Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 307, 1468–1472 (2005)

    CAS  ADS  Article  Google Scholar 

  11. Huang, S. M., Li, X., Yu, Y., Wang, J. & Caterina, M. J. TRPV3 and TRPV4 ion channels are not major contributors to mouse heat sensation. Mol. Pain 7, 37 (2011)

    Article  Google Scholar 

  12. Miyamoto, T., Petrus, M. J., Dubin, A. E. & Patapoutian, A. TRPV3 regulates nitric oxide synthase-independent nitric oxide synthesis in the skin. Nature Commun . 2, 369 (2011)

    ADS  Article  Google Scholar 

  13. Bautista, D. M. et al. The menthol receptor TRPM8 is the principal detector of environmental cold. Nature 448, 204–208 (2007)

    CAS  ADS  Article  Google Scholar 

  14. Nagy, I. & Rang, H. Noxious heat activates all capsaicin-sensitive and also a sub-population of capsaicin-insensitive dorsal root ganglion neurons. Neuroscience 88, 995–997 (1999)

    CAS  Article  Google Scholar 

  15. Woodbury, C. J. et al. Nociceptors lacking TRPV1 and TRPV2 have normal heat responses. J. Neurosci. 24, 6410–6415 (2004)

    CAS  Article  Google Scholar 

  16. Lawson, J. J., McIlwrath, S. L., Woodbury, C. J., Davis, B. M. & Koerber, H. R. TRPV1 unlike TRPV2 is restricted to a subset of mechanically insensitive cutaneous nociceptors responding to heat. J. Pain 9, 298–308 (2008)

    CAS  Article  Google Scholar 

  17. Hu, H. Z. et al. 2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3. J. Biol. Chem. 279, 35741–35748 (2004)

    CAS  Article  Google Scholar 

  18. Birren, S. J. & Anderson, D. J. A v-myc-immortalized sympathoadrenal progenitor cell line in which neuronal differentiation is initiated by FGF but not NGF. Neuron 4, 189–201 (1990)

    CAS  Article  Google Scholar 

  19. Wu, L.-J., Sweet, T.-B. & Clapham, D. E. International Union of Basic and Clinical Pharmacology. LXXVI. Current progress in the mammalian TRP ion channel family. Pharmacol. Rev. 62, 381–404 (2010)

    CAS  Article  Google Scholar 

  20. Caterina, M. J., Rosen, T. A., Tominaga, M., Brake, A. J. & Julius, D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398, 436–441 (1999)

    CAS  ADS  Article  Google Scholar 

  21. Vennekens, R. & Nilius, B. Insights into TRPM4 function, regulation and physiological role. Handb. Exp. Pharmacol. 179, 269–285 (2007)

    CAS  Article  Google Scholar 

  22. Togashi, K. et al. TRPM2 activation by cyclic ADP-ribose at body temperature is involved in insulin secretion. EMBO J . 25, 1804–1815 (2006)

    CAS  Article  Google Scholar 

  23. Naziroglu, M., Ozgul, C., Celik, O., Cig, B. & Sozbir, E. Aminoethoxydiphenyl borate and flufenamic acid inhibit Ca2+ influx through TRPM2 channels in rat dorsal root ganglion neurons activated by ADP-ribose and rotenone. J. Membr.Biol. 241, 69–75 (2011)

    CAS  Article  Google Scholar 

  24. Usoskin, D. et al. Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing. Nature Neurosci . 18, 145–153 (2015)

    CAS  Article  Google Scholar 

  25. Hara, Y. et al. LTRPC2 Ca2+-permeable channel activated by changes in redox status confers susceptibility to cell death. Mol. Cell 9, 163–173 (2002)

    CAS  Article  Google Scholar 

  26. Togashi, K., Inada, H. & Tominaga, M. Inhibition of the transient receptor potential cation channel TRPM2 by 2-aminoethoxydiphenyl borate (2-APB). Br. J. Pharmacol. 153, 1324–1330 (2008)

  27. Yamamoto, S. et al. TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nature Med . 14, 738–747 (2008)

    CAS  Article  Google Scholar 

  28. Uchida, K. et al. Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice. Diabetes 60, 119–126 (2011)

    CAS  Article  Google Scholar 

  29. Ariza-McNaughton, L. & Krumlauf, R. Non-radioactive in situ hybridization: simplified procedures for use in whole-mounts of mouse and chick embryos. Int. Rev. Neurobiol. 47, 239–250 (2002)

    CAS  Article  Google Scholar 

  30. Schmid, D., Messlinger, K., Belmonte, C. & Fischer, M. J. Altered thermal sensitivity in neurons injured by infraorbital nerve lesion. Neurosci. Lett. 488, 168–172 (2011)

    CAS  Article  Google Scholar 

  31. Jang, Y. et al. TRPM2 mediates the lysophosphatidic acid-induced neurite retraction in the developing brain. Pflugers Arch . 466, 1987–1998 (2014)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Tolkovsky for help and advice, R.-L. Yu for assistance with experiments, Y. Mori for Trpm2−/− mice and for the cDNA for mouse TRPM2, S. Birren for MAH cells, J. Wood for loan of the thermal-preference test apparatus, S. Skerratt for PF-4674114, and E. Smith, J. Btesh, D. Andersson, and T. Buijs for their comments on the manuscript. Initial experiments were performed in the Department of Pharmacology, University of Cambridge. Supported by a grant from the BBSRC (UK) to P.A.McN. and a Raymond & Beverley Sackler studentship to C.-H.T.

Author information

Authors and Affiliations

Authors

Contributions

C.-H.T. designed and performed experiments, analysed data and wrote the paper. P.A.M. designed experiments, analysed data, supervised the work and wrote the paper.

Corresponding author

Correspondence to Peter A. McNaughton.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks M. Caterina, Y. Mori and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Effect of altering the order of agonist application in DRG neurons

a, Method for detecting novel heat-sensitive somatosensory neurons. Representative traces showing increases of [Ca2+]i (F340/380 ratio, ordinate) in DRG neurons in response to the TRPV1-3 agonist 2-APB (250 μM), to the specific TRPV4 agonist PF-4674114 (V4 agonist, 200 nM), to the specific TRPV1 agonist capsaicin (1 μM), to the TRPM3 agonist pregnenolone sulphate (PS, 100 μM), to a heat ramp from 35 °C to 46 °C (temperature protocol shown at bottom), and to KCl (50 mM). Other details as in Fig. 1. From top: TRPV1-expressing neuron responding to 2-APB, capsaicin (1 μM) and heat (red, 30% of 500 neurons); TRPM3-expressing neuron responding to PS (blue, 100 μM) and heat (18%); TRPV1-and TRPM3-co-expressing neuron responding to 2-APB, PS, capsaicin, and heat (brown, 10%); neuron unresponsive to TRP channel agonists but showing a response to heat and therefore expressing a novel heat sensitive ion channel (black, 8% of total). No neuron responded to the specific TRPV4 agonist PF-4674114 (200 nM). b, Heat has a small effect on the fura-2 fluorescence ratio30, so we eliminated neurons in which an increase of fluorescence ratio was due simply to this physical effect by comparing the increase of fura-2 fluorescence ratio in neurons with that in glial cells in the same culture. Maximum increases in F340/380 ratio in response to a heat ramp from 35 °C to 46 °C in wild-type glial cells (black bars, top, n = 60) and in wild-type DRG neurons not responding to known thermo-TRP agonists (black bars, bottom, from same images as the glial cells in top panel, n = 139). Vertical black dashed line in top panel shows mean + 3.09 s.d. (cumulative probability value of 99.9%) of the increase in the F340/380 ratio in glial cells; this value is taken as the maximum increase in F340/380 ratio caused by effect of heat on fura-2 and is used as the cut-off value for defining novel heat-sensitive neurons present in the same culture dish (vertical black dashed line in lower panel). Similar results from separate culture of Trpm2−/− glia (n = 40) and neurons (n = 76) shown in red. The proportion of novel heat-sensitive neurons was significantly reduced from 8% (41/500) in wild type to 0.4% (1/282) in Trpm2−/− (P ≤ 0.0001; Fisher’s exact test). The increases in F340/380 ratio of novel heat-sensitive neurons above the cut-off value in response to heat (smallest increase = 0.019854) are all higher than that of the single heat-responding Trpm2−/− neuron (0.019084). c, Pie charts showing the percentage of novel heat-sensitive neurons responding to TRP ion channel agonists and to heat in wild-type DRG neurons (left) and DRG neurons from Trpm2−/− mice (right). Deletion of Trpm2 reduces the percentage of novel heat-sensitive neurons from 8% to 0.4%. Cell numbers for ac were 500 DRG neurons from one wild-type mouse on 3 coverslips and 282 DRG neurons from one Trpm2−/− mouse on 2 coverslips imaged. No further replicates of this particular experimental protocol were performed.

Extended Data Figure 2 Effect of starting the heat ramp at a lower temperature in DRG neurons.

Identical experiment to that shown in Fig. 1a–c, except that the temperature ramp started from 30 °C. a, Agonist and heat-responsive neurons as in Fig. 1a. From top: TRPV1-expressing neuron responding to capsaicin (1 μM) and heat (red, 28% of 491 neurons); TRPM3-expressing neuron responding to PS (blue, 100 μM) and heat (31%); TRPV1-and TRPM3-co-expressing neuron responding to capsaicin, PS and heat (brown, 13%); neuron unresponsive to TRP channel agonists but showing a response to heat and therefore expressing a novel heat-sensitive ion channel (black, 7% of total). A small number of neurons (8%) responded to 2-APB (250 μM) but not to other agonists, and 14% of DRG neurons did not respond to any of the agonists nor to heat (not shown). No neuron responded to the specific TRPV4 agonist PF-4674114 (200 nM). b, Maximum increases in F340/380 ratio in response to a heat ramp from 30 °C to 46 °C in wild-type glial cells (black bars, top, n = 60) and in heat-sensitive wild-type DRG neurons not responding to known thermo-TRP agonists (black bars, bottom, from same images as the glial cells in top panel, n = 103). Vertical black dashed line in top panel shows mean + 3.09 s.d. (cumulative probability value of 99.9%) of the increase in the F340/380 ratio in glial cells; this value is taken as the maximum increase in F340/380 ratio caused by effect of heat on fura-2 and is used as the cut-off value for defining novel heat-sensitive neurons present in the same culture dish (vertical black dashed line in lower panel). Similar results from separate culture of Trpm2−/− glia (n = 60) and neurons (n = 73) shown in red. The proportion of novel heat-sensitive neurons was significantly reduced from 7% (103/491) in wild type, to 2% (73/522) in Trpm2−/− (P ≤ 0.0001; Fisher’s exact test). The mean increase in F340/380 ratio of novel heat-sensitive neurons above the cut-off values in response to heat was also significantly reduced from 1.237 ± 0.09207 in wild type (n = 36) to 0.7959 ± 0.03767 in Trpm2−/− (n = 8) (P = 0.0313; two-tailed unpaired t-test). c, Pie charts showing the percentage of novel heat-sensitive and TRPV1- or TRPM3-expressing neurons in wild-type DRG neurons (left) and DRG neurons from Trpm2−/− mice (right). Cell numbers for imaging in ac were 491 DRG neurons from one wild-type mouse on 3 coverslips and 522 DRG neurons from one Trpm2−/− mouse on 3 coverslips. No further replicates of this particular experimental protocol were performed.

Extended Data Figure 3 Diameters of novel heat-sensitive DRG neurons compared to neurons responding to other TRP agonists.

a, Diameters of 1,324 DRG neurons taken from experiment illustrated in Fig. 1a (dotted line). Subpopulations of neurons are shown as follows: those responding to capsaicin and thus expressing TRPV1 (red); to PS and thus expressing TRPM3 (blue); to both agonists and thus co-expressing TRPV1 and TRPM3 (brown); to 2-APB alone (green); novel heat-sensitive neurons (orange), and neurons responding neither to heat nor to any of these agonists (black). b, Diameter comparison of subpopulations of neurons. TRPV1-expressing neurons have the smallest mean diameter (18.58 ± 0.17 μm), TRPM3-expressing neurons are intermediate (21.75 ± 0.33 μm), and neurons expressing only the novel heat-sensitivity have the largest mean diameter (25.47 ± 0.48 μm). Significance from one-way ANOVA and multiple comparisons with Tukey’s multiple comparison test (****P ≤ 0.0001; NS, not significant). Data obtained from Fig. 1a–c; 1,324 DRG neurons from one wild-type mouse on 4 coverslips were analysed.

Extended Data Figure 4 The novel heat-sensitivity in DRG neurons is partially co-expressed with TRPV1 and TRPM3, and is enhanced by H2O2.

a, Temperature ramp to 47 °C, as in Fig. 1a, but with TRPV1 blocked with AMG9810 (5 μM) and TRPM3 blocked with naringenin (10 μM). Criterion level for significant increase (dashed lines) taken from glial cells in same field of view (data not shown). Black bars: 46% of all wild-type DRG neurons (n = 580) responded to heat ramp from 34 °C to 46 °C with an increase in [Ca2+]i above criterion level in presence of blockers of TRPV1 and TRPM3 (dashed vertical line), while the percentage decreased to 17% in Trpm2−/− DRG neurons (red bars, n = 1,007) (P ≤ 0.0001; Fisher’s exact test). The mean increase in F340/380 ratio above the cut-off values (dashed lines) in response to heat was also significantly reduced from 1.619 ± 0.06133 in wild type (n = 265) to 1.027 ± 0.08394 in Trpm2−/− (n = 175) (P ≤ 0.0001; two-tailed unpaired t-test). In similar experiments with TRPV1 blocker BCTC (4 μM) and naringenin (10 μM), 37% of wild-type neurons responded to heat (data not shown, n = 554). b, Similar plot as in a, but data from the subgroup of novel heat-sensitive neurons not responding to agonists for known thermo-TRP channels. After exposure to heat in presence of blockers of TRPV1 and TRPM3, blockers were removed and neurons not responding to known TRP agonists were identified as in Fig. 1a. Data from same experiment as shown in a. Proportion of neurons expressing the novel heat-sensitive mechanism in isolation (that is, without co-expression of TRPV1 or TRPM3) was significantly lower (52/580, 9%) than all neurons expressing the novel heat-sensitive mechanism (46%, see a). The proportion of novel heat-sensitive neurons was significantly reduced in Trpm2−/− mice, from 9% to 0.6% (6/1,007, P ≤ 0.0001; Fisher’s exact test). c, Temperature ramp to 42 °C. Few novel heat-sensitive neurons respond to this low temperature in either wild type or Trpm2−/−. The total number of neurons was n = 173 for wild type and n = 211 for Trpm2−/−. d, Responses of the same neurons to the same temperature ramp to 42 °C following addition of H2O2 (400 μM). Enhancement of response in wild type (black bars) was largely abolished in Trpm2−/− (red bars). The proportion of novel heat-sensitive neurons after sensitization with H2O2 was significantly reduced from 11% (74/635) in wild type to 8% (48/601) in Trpm2−/− (P = 0.0356; Fisher’s exact test). The mean increase in F340/380 ratio of novel heat-sensitive neurons above the cut-off values in response to heat was also significantly reduced from 1.175 ± 0.1516 in wild type (n = 72) to 0.4485 ± 0.04329 in Trpm2−/− (n = 48) (P = 0.0002; two-tailed unpaired t-test). Cell numbers and replicates for a and b were 580 DRG neurons from one wild-type mouse on 5 coverslips and 1,007 DRG neurons from one Trpm2−/− mouse on 5 coverslips imaged for the protocol with AMG9810. 554 neurons from one wild-type mouse on 4 coverslips were imaged for the protocol with BCTC. No further replicates were carried out. The cell numbers and replicates for c and d were 635 DRG neurons from one wild-type mouse on 3 coverslips and 601 DRG neurons from one knockout mouse on 3 coverslips imaged. The experiment was replicated with similar results on 4 additional coverslips from one mouse.

Extended Data Figure 5 Most novel heat-sensitive DRG neurons are IB4-positive.

a, Increases in [Ca2+]i (F340/380 ratio image, intensity-coded in red, in mouse DRG neurons) in response to heat ramp to 46 °C (TRPV1 blocked with AMG9810, 5 μM, and TRPM3 blocked with naringenin, 10 μM), superimposed on differential interference contrast (DIC) transmission image obtained post-fixation. b, The same field following fixation and labelling with fluorescent IB4 (green). c, Superimposed calcium and IB4 images from a and b. Black arrows show neurons responding to heat and positive for IB4. White arrow indicates a neuron responding to heat and negative for IB4. Black arrowhead shows neuron insensitive to heat and positive for IB4. Scale bars 50 μm. d, Diameter histogram of 743 fixed DRG neurons subgrouped according to novel heat-sensitivity (yellow, red) and IB4 binding (yellow, green). 25% (184/743) of DRG neurons showed novel heat-sensitivity, and 74% of these novel heat-sensitive neurons were IB4-positive, whereas only 53% of heat-insensitive neurons were IB4-positive. The percentage of IB4-positive neurons is significantly higher in the heat-sensitive group than in the heat-insensitive group (P ≤ 0.0001; Fisher’s exact test). The diameters shown in d are not directly comparable with the live cell diameters shown in Extended Data Fig. 3 because of a shrinkage artefact on fixation. Cell numbers were 743 DRG neurons from one wild-type mouse on 4 coverslips imaged. No further replicates were performed.

Extended Data Figure 6 Properties of novel heat-sensitive ion channel expressed in autonomic neurons.

a, Representative traces showing increases of [Ca2+]i (F340/380 ratio, ordinate) in sympathetic neurons from superior cervical ganglion (SCG) in response to a mixture of capsaicin (TRPV1 agonist,1 μM) and pregnenolone sulphate (TRPM3 agonist, 100 μM); a mixture of 2-APB (TRPV1-3 agonist, 250 μM) and PF-4674114 (TRPV4 agonist, 200 nM); heat to 47 °C (temperature protocol shown at bottom); and KCl (50 mM). Similar results were obtained with parasympathetic neurons from pterygopalatine ganglion (PPG, data not shown). Trace is same as shown in Fig. 2a. b, Similar histograms as in Extended Data Figs 1b and 2b, but for SCG glial cells and neurons from wild-type mice (black bars) and Trpm2−/− mice (red bars). 58% of wild-type SCG neurons (n = 436) showed novel heat-sensitivity with increases in F340/380 ratio above the criterion level obtained from glial cells (n = 80) in same culture (black vertical dashed line). In similar experiments on PPG neurons (n = 484), 47% showed novel heat-sensitivity (not shown). Red bars and red dashed line show results from SCG glia (n = 80) and neurons (n = 430) from Trpm2−/− mice. The proportion of novel heat-sensitive neurons was significantly reduced by deletion of Trpm2, from 58% (252/436) in wild type to 12% (53/430) in Trpm2−/− (P ≤ 0.0001; Fisher’s exact test). The mean increase in F340/380 ratio of heat-sensitive neurons above the cut-off values in response to heat was also significantly reduced, from 1.629 ± 0.1928 in wild type (n = 252) to 0.5050 ± 0.1270 in Trpm2−/− (n = 53) (P = 0.0086; two-tailed unpaired t-test). c, Heat-evoked Ca2+ increase in SCG neurons is reduced but not abolished by removal of extracellular Na+ (replaced with choline) and is abolished by removal of extracellular Ca2+ (remaining small increase in F340/380 ratio is due to temperature sensitivity of fura-2, see b). Similar results were seen in 133 SCG neurons. d, Heat-evoked Ca2+ increase in SCG neurons is blocked by TRPV agonist 2-APB (25 μM). Similar results were seen in 130 SCG neurons. e, The Ca2+ increase in PPG neurons is not affected by TRPV channel blocker ruthenium red (50 μM). Similar results were seen in 75 PPG neurons. f, The Ca2+ increase in PPG neurons is not affected by the Na channel blocker tetrodotoxin (2 μM). Similar results were seen in 35 PPG neurons. g, The Ca2+ influx in PPG neurons is reduced but not eliminated by the L-type Ca2+ channel blocker verapamil (100 μM). Similar results were seen in 30 PPG neurons. Cell numbers and replicates for a were 166 SCG neurons from three wild-type mice on 3 coverslips imaged. Cell numbers and replicates for b were 436 SCG neurons from two wild-type mice on 4 coverslips and 430 SCG neurons from two Trpm2−/− mice on 4 coverslips imaged. Similar results as those shown for wild-type obtained with 15 further coverslips of SCG neurons from 9 wild-type mice and 7 coverslips of PPG neurons from 6 wild-type mice. Cell numbers and replicates for c were 133 SCG neurons from 3 wild-type mice on 5 coverslips that showed similar responses. Cell numbers and replicates for d were 130 SCG neurons from 3 wild-type mice on 2 coverslips hat showed similar responses. Similar results were also obtained for DRG neurons (4 coverslips from 1 mouse). Cell numbers and replicates for e were 75 PPG neurons from 3 wild-type mice on 4 coverslips that showed similar responses. Cell numbers and replicates for f were 35 PPG neurons from 3 wild-type mice on 4 coverslips that showed similar responses. Cell numbers and replicates for g were 30 PPG neurons from 3 wild-type mice on 2 coverslips that showed similar responses.

Extended Data Figure 7 The heat-induced membrane current in autonomic neurons is not gated by membrane voltage.

Current–voltage difference relations of a PPG neuron with a voltage ramp starting from a negative potential (inset) show a similar linear heat-induced current to that obtained with reverse voltage ramp (see Fig. 2c). Trace obtained by subtracting current–voltage relations at 36 °C from that at 47 °C. Similar results were obtained in 3 cells on 3 coverslips.

Extended Data Figure 8 Responses to heat in adrenal-derived MAH and PC12 cell lines, and effects of factors causing differentiation to neuronal phenotype.

a, MAH cells. Black indicates maximum increase in F340/380 ratio in response to heat (47 °C, n = 254) when cultured in dexamethasone (5 μM). No cell responded to TRP agonists, but 27% of cells responded to heat with increase above mean criterion level obtained from glial cells in neuronal cultures (see Fig. 1b). Red indicates similar histogram after 12 days of culture in growth factors (bFGF, CNTF and NGF, see Methods, n = 170). No cell responded to TRP agonists; 9% responded to heat. The proportion of heat-sensitive cells was significantly reduced from 27% (69/254) in dexamethasone to 9% (16/170) in growth factors (P ≤ 0.0001; Fisher’s exact test). The 66% reduction in the proportion of heat-sensitive cells was not significantly different from the reduction in Trpm2 expression caused by differentiation of MAH cells (Table 1; P = 0.056; two-tailed unpaired t-test). The mean increases in F340/380 ratio above the cut-off values (dashed lines) in response to heat were 1.755 ± 0.1255 in dexamethasone (n = 69) and 1.420 ± 0.1474 in presence of growth factors (n = 16) (P = 0.2203; two-tailed unpaired t-test). b, PC12 cells. Black indicates culture in growth medium (10% horse serum plus 5% fetal bovine serum, n = 200). 93% of cells responded to heat with increase above mean criterion level obtained from glial cells in neuronal cultures (see Fig. 1b). Red indicates effect on heat responses of 12 days of culture in NGF (1% horse serum plus 100 ng ml−1 NGF, n = 108). The proportion of heat-sensitive cells was significantly reduced from 93% (186/200) in growth medium to 46% (50/108) in NGF (P ≤ 0.0001; Fisher’s exact test). We note that a significantly lower expression of mRNA for TRPM2 in differentiated PC12 cells has been reported31. The mean increase in F340/380 ratio above the cut-off values (dashed lines) in response to heat was significantly reduced from 3.753 ± 0.2431 in growth medium (n = 186) to 2.603 ± 0.3104 in NGF (n = 50) (P = 0.0213; two-tailed unpaired t-test). c, Temperature thresholds of PC12 cells cultured in growth medium. Top left, temperature protocol. Bottom left, temperature responses of three representative cells. Right, temperature thresholds calculated as in Fig. 1d. Cell numbers and replicates for a and b were 2 coverslips for each condition imaged. Cell numbers are given above. Replicates of 4 coverslips (MAH cells) and 3 coverslips (PC12 cells) for each condition gave similar results. Cell numbers and replicates for c were 165 PC12 cells on one coverslip imaged.

Extended Data Figure 9 Effect of deletion of TRPM2 on maximal calcium responses to heat in neurons expressing TRPV1 or TRPM3.

a, Maximum increases in F340/380 ratio in response to a heat ramp from 34 °C to 46 °C in neurons responding only to capsaicin (TRPV1-expressing) from wild-type (black) and Trpm2−/− (red) mice. The increase in F340/380 ratio in response to heat (above the increase caused by the effect of temperature on fura-2, vertical dotted lines, for method of calculation see Fig. 1b) is not significantly different between wild type and Trpm2−/− (P = 0.1168, two-tailed Mann–Whitney U-test). Details as in Fig. 1. b, Neurons responding only to pregnenolone sulphate (PS, TRPM3-expressing) from the same experiments as in a. The increases in F340/380 ratio in response to heat are significantly reduced by deletion of Trpm2 (from 6.389 ± 1.225 to 4.411 ± 1.582, P < 0.0001, two-tailed Mann–Whitney U-test). c, Neurons responding to both capsaicin and PS (TRPV1- and TRPM3-expressing). The increases in F340/380 ratio in response to heat are not significantly different between wild type and Trpm2−/− (P = 0.0633; two-tailed Mann–Whitney U-test). Cell numbers and replicates for ac were 1324 DRG neurons from one wild-type mouse on 4 coverslips and 981 DRG from one Trpm2−/− mouse on 4 coverslips imaged. Similar results obtained from 42 additional coverslips from 6 wild-type mice and 8 additional coverslips from one Trpm2−/− mouse.

Extended Data Figure 10 Correlation between novel heat sensitivity and expression of mRNA for TRPM2.

a, Representative DIC transmission image of DRG neurons following in situ hybridization with the sense probe as negative control. Non-specific density was linearly dependent on cell diameter (see e). Mean + 3.09 s.d. (cumulative probability value of 99.9%) of density as a function of diameter with sense probe (5 μm bins) was used as threshold criterion for significant expression of Trpm2 in images of antisense hybridization. A similar analysis of non-specific density was carried out for glial cells. b, Representative DIC transmission image with antisense probe against Trpm2. Using the threshold criterion as function of diameter obtained from sense probe images (see a), 89% (1,121/1,250) of DRG neurons but 3% (4/120) of glial cells were positive for TRPM2 mRNA. c, Novel heat-sensitive DRG neurons determined using calcium imaging. Increases in [Ca2+]i (F340/380 ratio image, intensity-coded in red) in response to a heat ramp to 46 °C with TRPV1 blocked with AMG9810 (5 μM) and TRPM3 blocked with naringenin (10 μM). d, Superimposed image of novel heat-sensitive neurons (red) and in situ hybridization using antisense probe. Solid red arrows indicate novel heat-sensitive neurons also positive for TRPM2; solid black arrow shows neuron not responding to heat but positive for TRPM2; open black arrow shows cell negative for TRPM2 (that is, with density below the criterion level obtained from e). 42% (92/218) of DRG neurons positive for TRPM2 exhibited novel heat-sensitivity. However only 13% (2/16) of DRG neurons negative for TRPM2 from in situ hybridization exhibited novel heat-sensitivity. The percentage of novel heat-sensitive DRG neurons is significantly reduced in TRPM2 negative DRG neurons (P = 0.0188; Fisher’s exact test). Scale bars, 50 μm. e, Density of non-specific label in neurons obtained from hybridization with sense probe (see a) depends on cell size. Data used to calculate significance thresholds for neurons in b. Cell numbers and replicates for a was one coverslip exposed to sense probe used as negative control. Cell numbers and replicates for b were all neurons on 5 coverslips measured and one coverslip was measured for TRPM2-positive glial cells. Cell numbers and replicates for c and d was one coverslip was analysed as in a and b for the combined calcium imaging and in situ hybridization protocol. Cell numbers and replicates for e were 500 DRG neurons on one coverslip exposed to the sense probe used to determine the background threshold as a function of cell diameter. Similar in situ hybridization results were obtained on 16 additional coverslips.

Supplementary information

Novel heat-sensitive DRG neurons identified by calcium imaging

Calcium imaging of DRG neurons in an experiment similar to that shown in Fig. 1A. Video speeded up by factor of 20x. The F340/380 ratio is shown on a false-colour scale from blue (low) to red (high) then white (highest). Timing of application of TRP channel agonists (shown to left) is identical to that shown in Fig 1A. Neuron circled in early frames does not respond to any TRP agonist but is activated by a heat ramp to 46°C. Other novel heat-sensitive neurons are also visible. (MP4 24648 kb)

Behavioural responses of wild-type and TRPM2-/- littermates in a two-plate thermal choice test at temperatures of 33°C and 37°C

WT (bottom) and TRPM2-/- (top) adult male littermates in two-plate thermal preference apparatus. Video speeded up by factor of 81x. Plate temperatures, as shown, were reversed after 30 min. Two experiments are shown on same mice, 4h apart. WT mouse shows strong preference for 33°C over 38°C while thermal preference is absent or even slightly reversed in TRPM2-/- mouse. (MP4 6140 kb)

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Tan, CH., McNaughton, P. The TRPM2 ion channel is required for sensitivity to warmth. Nature 536, 460–463 (2016). https://doi.org/10.1038/nature19074

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