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The coding of cutaneous temperature in the spinal cord

Nature Neuroscience volume 19, pages 1201–1209 (2016)Cite this article

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Abstract

The spinal cord is the initial stage that integrates temperature information from peripheral inputs. Here we used molecular genetics and in vivo calcium imaging to investigate the coding of cutaneous temperature in the spinal cord in mice. We found that heating or cooling the skin evoked robust calcium responses in spinal neurons, and their activation threshold temperatures distributed smoothly over the entire range of stimulation temperatures. Once activated, heat-responding neurons encoded the absolute skin temperature without adaptation and received major inputs from transient receptor potential (TRP) channel V1 (TRPV1)-positive dorsal root ganglion (DRG) neurons. By contrast, cold-responding neurons rapidly adapted to ambient temperature and selectively encoded temperature changes. These neurons received TRP channel M8 (TRPM8)-positive DRG inputs as well as novel TRPV1+ DRG inputs that were selectively activated by intense cooling. Our results provide a comprehensive examination of the temperature representation in the spinal cord and reveal fundamental differences in the coding of heat and cold.

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Figure 1: In vivo two-photon calcium imaging in the spinal cord.
Figure 2: Representation of cold intensity in the spinal cord.
Figure 3: Rapid adaptive responses to cooling.
Figure 4: Neurons encode the change in temperature for cold.
Figure 5: The representation of heating in the spinal cord.
Figure 6: Neurons encode absolute temperatures for heat.
Figure 7: Spinal responses to temperature are mediated by specific DRG inputs.
Figure 8: Broadly tuned thermal responding neurons in the spinal cord.

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Acknowledgements

We thank L. Luo for his generous support during the entire project, Z. Shen for initial experiments and G. Kamalani for assistance; and B. Barres (Stanford University) and D. Julius (University of California, San Francisco) for Mgfap-cre and Trpa1 knockout mice. We are grateful to X. Gao, C. Guenthner, B. Weissbourd and members of the Chen laboratory for comments on the manuscript. This work was supported by grants from the intramural research program of NIDCR (M.A.H.), and the Whitehall Foundation, Terman Fellowship and start-up funding from Stanford University (X.C.).

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Authors and Affiliations

  1. Department of Biology, Stanford University, Stanford, California, USA

    Chen Ran & Xiaoke Chen

  2. National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, USA

    Mark A Hoon

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Contributions

C.R. and X.C. designed the study. C.R. conducted imaging experiments. C.R. and X.C. analyzed data. M.A.H. provided TRPM8- and TRPV1-DTR mice, and performed in situ hybridization experiments. C.R. and X.C. wrote the paper with help from M.A.H. X.C. supervised the research.

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Correspondence to Xiaoke Chen.

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Integrated supplementary information

Supplementary Figure 1 Temperatures recorded on the surface of the skin and under the skin are similar.

Stimulation temperature recorded on the surface of the skin (black) and under the skin (red). The data are averaged from 4 measurements and are presented as mean (dark color) ± s.e.m. (light color).

Supplementary Figure 2 Properties of spinal responses to cooling.

(a) The percentage of total neurons at different depths below the surface that are activated by cooling to 29 °C (yellow) and 5 °C (blue) (25 μm, n = 2 mice; 35 μm, n = 5 mice; 45 μm, n = 5 mice; 55 μm, n = 5 mice; 65 μm, n = 4 mice; 75 μm, n = 3 mice; 85 μm, n = 2 mice.) (b) Heat maps (middle) and example traces (bottom) of neuronal responses to cooling (top) in the spinal cord before (left) and after (right) application of NBQX (an AMPA and kainite receptors antagonist, 60 μM) and APV (an NMDA receptor antagonist, 150 μM). Scale bars, 10 s and 10% ΔF/F. (c) Top: An example FOV illustrating OGB (green) labeling in the spinal cord of a Mgfap-cre; Ai14 (cre-dependent tdTomato, red) mouse. Scale bar, 100 μm. Bottom: example calcium traces of cold response from astrocytes (red) and neurons (green). Scale bars, 10 s and 10% ΔF/F.

Supplementary Figure 3 Cooling-response curves at different ATs.

(a) Neuronal responses to cooling stimuli with a series of ΔTs (3 °C, 6 °C, 10 °C and 16 °C) at two ATs (Top traces and heat maps: AT = 32 °C. Bottom traces and heat maps: AT = 27 °C). (b) Quantification of a (n = 5 mice). Scale bars, 10 s.

Supplementary Figure 4 Dorsoventral distribution and absolute temperature-coding property of heat-responding neurons.

(a) The percentage of total neurons that are activated by heating to 37 °C (orange) and 45 °C (red) at different depths below the surface (n = 2-5 mice). (25 μm, n = 2 mice; 35 μm, n = 5 mice; 45 μm, n = 5 mice; 55 μm, n = 5 mice; 65 μm, n = 4 mice; 75 μm, n = 3 mice; 85 μm, n = 2 mice.) (b) Quantification of the number of heat-responding neurons (squares, left Y axis) and the averaged peak ΔF/F (circles, right Y axis) in response to heating at different rates (n = 5 mice).

Supplementary Figure 5 Diphtheria toxin (DT) treatments of the TRPM8-DTR-GFP and TRPV1-DTR-GFP mice.

(a) The expression of TRPM8 (in situ hybridization) and TRPV1 (immunofluorescence) in the DRGs of wild type, TRPM8- and TRPV1-DTR mice after diphtheria toxin treatment. Similar observations were made in all mice (number specified below). Scale bar, 100 μm. (b) Quantification of a (Left: n = 2 mice for WT, n = 5 mice for TRPM8-DTR and n = 4 mice for TRPV1-DTR, right: n = 4 mice per genotype). (c) The expression of GFP (immunofluorescence) in the DRGs of saline- or DT-treated TRPM8- and TRPV1-DTR-GFP mice. Similar observations were made in all mice (number specified below). Scale bar, 100 μm. (d) Quantification of c (TRPM8-DTR-GFP: n = 2 mice per treatment group, TRPV1-DTR-GFP: n = 4 mice per treatment group).

Supplementary Figure 6 TRPA1 channel does not mediate spinal responses to cold.

(a) Heat maps of the activities of all cold-responding neurons in representative FOVs in WT (138 neurons) and Trpa1 knockout (146 neurons) mice. (b) The temperature−response relationship in WT (n = 12 mice, same data as in Fig. 2c) and Trpa1 knockout (n = 4 mice) mice. (c) The distribution of activation thresholds of cold-responding neurons in WT (same data as in Fig. 5e) and TRPA1 knockout mice. Scale bar, 10 s.

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Ran, C., Hoon, M. & Chen, X. The coding of cutaneous temperature in the spinal cord. Nat Neurosci 19, 1201–1209 (2016). https://doi.org/10.1038/nn.4350

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