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The kainate receptor GluK2 mediates cold sensing in mice

Nature Neuroscience volume 27, pages 679–688 (2024)Cite this article

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Abstract

Thermosensors expressed in peripheral somatosensory neurons sense a wide range of environmental temperatures. While thermosensors detecting cool, warm and hot temperatures have all been extensively characterized, little is known about those sensing cold temperatures. Though several candidate cold sensors have been proposed, none has been demonstrated to mediate cold sensing in somatosensory neurons in vivo, leaving a knowledge gap in thermosensation. Here we characterized mice lacking the kainate-type glutamate receptor GluK2, a mammalian homolog of the Caenorhabditis elegans cold sensor GLR-3. While GluK2 knockout mice respond normally to heat and mechanical stimuli, they exhibit a specific deficit in sensing cold but not cool temperatures. Further analysis supports a key role for GluK2 in sensing cold temperatures in somatosensory DRG neurons in the periphery. Our results reveal that GluK2—a glutamate-sensing chemoreceptor mediating synaptic transmission in the central nervous system—is co-opted as a cold-sensing thermoreceptor in the periphery.

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Fig. 1: GluK2 KO mice respond normally to mechanical and heat stimuli.
Fig. 2: GluK2 KO mice are defective in cold sensing.
Fig. 3: GluK2 KO mice are defective in cold nociception.
Fig. 4: Calcium imaging reveals that GluK2 is important for sensing cold rather than cool temperatures in dissociated DRG neurons.
Fig. 5: Patch-clamp recording reveals that GluK2 is important for cold sensing in dissociated DRG neurons.
Fig. 6: GluK2 is important for sensing cold rather than cool temperatures in DRG neurons in vivo.

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All the data generated or analyzed in this study are included in the figures, texts and Supplementary Information files. Additional data supporting the findings of this study are available upon reasonable request.

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Acknowledgements

We thank A. Contractor for providing GluK2-floxed mice; S. Tomita for providing GluK2 KO mice; and J. Feng, Z. Xie, H. Hu and M. Zhang for technical assistance and discussion. This work was supported by the NINDS (to X.Z.S.X. and B.D.) and NIGMS (to X.Z.S.X.).

Author information

Author notes

  1. These authors contributed equally: Wei Cai, Wenwen Zhang, Qin Zheng.

Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Wei Cai, Wenwen Zhang, Tong Pan & X. Z. Shawn Xu

  2. Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA

    Wei Cai, Wenwen Zhang, Tong Pan & X. Z. Shawn Xu

  3. Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Qin Zheng

  4. The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Qin Zheng & Xinzhong Dong

  5. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Chia Chun Hor, Mahar Fatima & Bo Duan

  6. Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Xinzhong Dong

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Contributions

W.C. performed most behavioral tests, conducted in vitro DRG imaging experiments and analyzed the data. W.Z. performed water droplet behavior tests, whole-cell recordings and RNAscope in situ hybridization, and analyzed the data. Q.Z. and X.D. performed in vivo DRG imaging experiments, and analyzed the data with W.C. and W.Z. C.C.H. assisted W.C. and W.Z. with behavioral tests. T.P. assisted W.C. with in vitro DRG imaging experiments. M.F. analyzed RNA-seq data and assisted W.Z. with RNAscope in situ hybridization. B.D. and X.Z.S.X. supervised the project. W.C., W.Z., B.D. and X.Z.S.X. wrote the paper with assistance from all other authors.

Corresponding authors

Correspondence to Bo Duan or X. Z. Shawn Xu.

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Nature Neuroscience thanks Diana Bautista, Ryan Pak, Raul Ramos, Nick Villarino and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mice show no response to water droplet stimuli near the temperature of their paw’s skin surface, and there is no observed difference between males and females.

(a, b) Water droplet stimuli at 27 °C are applied to mouse hindpaw using a syringe. Each mouse was tested three times, and the resulting score is calculated as the average of these tests. Error bars: SEM. P > 0.05 (not statistically significant; one-way ANOVA with Tukey test). Sample sizes in (a): control n = 15, TRPM8 KO n = 15, GluK2 KO n = 9, GluK2/TRPM8 dKO n = 14 (mice). Sample sizes in (b): control n = 10, TRPM8 KO n = 15, GluK2 cKOPirt n = 11, GluK2 cKOPirt /TRPM8 dKO n = 13 (mice). (c, d) No notable difference was observed between males and females. n.s. not statistically significant (P > 0.05; one-way ANOVA with Tukey test). Data in (a) and (b) are grouped separately as male and female cohorts for each genotype and analyzed separately. Sample sizes in (c): control male n = 8, control female n = 7, TRPM8 KO male n = 7, TRPM8 KO female n = 8, GluK2 KO male n = 5, GluK2 KO female n = 4, GluK2/TRPM8 dKO male n = 5, GluK2/TRPM8 dKO female n = 9 (mice). Sample sizes in (d): control male n = 5, control female n = 5, TRPM8 KO male n = 6, TRPM8 KO female n = 9, GluK2 cKOPirt male n = 5, GluK2 cKOPirt female n = 6, GluK2 cKOPirt /TRPM8 KO male n = 5, GluK2 cKOPirt /TRPM8 KO female n = 8 (mice). Error bars: SEM. Data are presented as mean ± SEM.

Extended Data Fig. 2 GluK2 cKOAvil mice respond normally to mechanical and heat stimuli and behave normally in the rotarod test.

(a) The von Frey test (control: n = 8; GluK2 cKOAvil: n = 11; TRPM8 KO: n = 20; GluK2 cKOAvil/TRPM8 KO: n = 24). (b) The pinprick test (control: n = 5; GluK2 cKOAvil: n = 6; TRPM8 KO: n = 10; GluK2 cKOAvil/TRPM8 KO: n = 12). (c) The rotarod test (control: n = 7; GluK2 cKOAvil: n = 11; TRPM8 KO: n = 19; GluK2 cKOAvil/TRPM8 KO: n = 24). (d) The Hargreaves test (control: n = 18; GluK2 cKOAvil: n = 14; TRPM8 KO: n = 18; GluK2 cKOAvil/TRPM8 KO: n = 14). (e) The hot plate test (control: n = 5; GluK2 cKOAvil: n = 6; TRPM8 KO: n = 10; GluK2 cKOAvil/TRPM8 KO: n = 12). Error bars: SEM. P values: all >0.05 (not statistically significant; one-way ANOVA with Tukey test). Data are presented as mean ± SEM.

Extended Data Fig. 3 GluK2 cKOPirt mice respond normally to mechanical and heat stimuli and behave normally in the rotarod test.

(a) The von Frey test (control: n = 12; GluK2 cKOPirt: n = 12; TRPM8 KO: n = 7; GluK2 cKOPirt/TRPM8 KO: n = 19). (b) The pinprick test (control: n = 14; GluK2 cKOPirt: n = 9; TRPM8 KO: n = 7; GluK2 cKOPirt/TRPM8 KO: n = 16). (c) The rotarod test (control: n = 8; GluK2 cKOPirt: n = 10; TRPM8 KO: n = 6; GluK2 cKOPirt/TRPM8 KO: n = 17). (d) The Hargreaves test (control: n = 12; GluK2 cKOPirt: n = 12; TRPM8 KO: n = 8; GluK2 cKOPirt/TRPM8 KO: n = 20). (e) The hot plate test (control: n = 8; GluK2 cKOPirt: n = 8; TRPM8 KO: n = 10; GluK2 cKOPirt/TRPM8 KO: n = 20). Error bars: SEM. P-values: all >0.05 (not statistically significant; one-way ANOVA with Tukey test). Data are presented as mean ± SEM.

Extended Data Fig. 4 Heat map of in vitro calcium imaging data (related to Fig. 4).

Dissociated DRG neurons were recorded by calcium imaging as described in Fig. 4. The data from all recorded DRG neurons from one representative mouse was presented as a heat map for each genotype. (a) Control. (b) GluK2 KO. (c) TRPM8 KO. (d) GluK2/TRPM8 dKO.

Extended Data Fig. 5 Quantification of area under curve and amplitude of in vitro calcium imaging data (related to Fig. 4).

(a) Area under curve distribution of cold-specific DRG neurons. (b) Amplitude distribution of cold-specific DRG neurons. (c) Area under curve distribution of cool-sensitive DRG neurons. (d) Amplitude distribution of cool-sensitive DRG neurons. Neurons with an amplitude lower than 10% (ΔR/R) (<20 a.u. for area under curve) were considered no response as it was difficult to resolve signals from noises in these cases. Neurons with an amplitude at least 20% ((ΔR/R) were included for analysis in Fig. 4 (see Methods). Control: 2361 neurons; GluK2 KO: 1119 neurons; TRPM8 KO: 1681 neurons; GluK2/TRPM8 dKO: 1063 neurons.

Extended Data Fig. 6 Quantification of AITC-responding DRG neurons and the effect of PTX (pertussis toxin) on cool-sensitive and cold-specific DRG neurons.

Calcium imaging was performed on dissociated DRG neurons using the protocol described in Fig. 4. AITC (100 μM) and menthol (100 μM) were applied acutely to DRG neurons during calcium imaging. PTX (100 ng/ml) was pre-incubated with DRG neurons for 6 hours prior to calcium imaging. KCl (50 mM) was added at the end of the experiment to validate the health of DRG neurons. (a) Sample traces of cool-sensitive, cold-specific and AITC-responding DRG neurons. (b) Quantification of cool-sensitive and cold-specific DRG neurons that responded to AITC. (c) The percentage cool-sensitive DRG neurons is not significantly affected by PTX. (d) The percentage cold-specific DRG neurons is greatly reduced by PTX. (e) The percentage AITC-responding DRG neurons is not significantly affected by PTX. The numbers of responding and non-responding neurons are indicated in each panel (from 7 mice). n.s.: no significant difference (p > 0.05; Chi-square test). **p = 0.000173, PTX vs mock group (Chi-square test).

Extended Data Fig. 7 A small population of menthol/cold-sensitive DRG neurons is dependent on TRPM8 but not GluK2.

(a) In experiments described in Fig. 4,we also identified a small population of DRG neurons that were insensitive to cool temperatures but sensitive to menthol and cold temperatures. The number of responding neurons and total neurons assayed is indicated for each genotype. Wild-type littermate mice are used as control. Error bars: SEM. Control: n = 31 (mice); GluK2 KO: n = 15 (mice); TRPM8 KO: n = 19 (mice): GluK2/TRPM8 dKO: n = 10 (mice). **p = 1.21E-07 TRPM8 KO vs Control group (Chi-square test).

Extended Data Fig. 8 No significant differences are observed in the intrinsic properties and excitability of dissociated DRG neurons from TRPM8 KO and GluK2/TRPM8 dKO mice (related to Fig. 5).

(a) Whole-cell patch recording of action potential (AP) firing frequency induced by injected current steps. (b) AP firing frequency induced by 60 pA injected currents. (c-f) Quantification of the membrane resistance (c), membrane capacitance (d), time constant (e), and resting membrane potential (f) of recorded neurons. (g) Quantification of the first step current that induced AP (Rheobase) in recorded neurons. (h-j) Quantification of the threshold (h), amplitude (i) and half-width (j) of the first induced AP. Error bars: SEM. n.s.: no significant difference (P > 0.05; two-tailed Student’s t-test). Sample size: n = 130 neurons (TRPM8 KO; 11 mice); n = 84 neurons (GluK2/TRPM8 dKO; 6 mice). Data are presented as mean ± SEM.

Extended Data Fig. 9 Heat map of in vivo calcium imaging data (related to Fig. 6).

In vivo calcium imaging of DRG neurons was performed as described in Fig. 6. The data from all imaged DRG neurons from one representative mouse was presented as a heat map for each genotype. (a) Control: wild-type littermate. (b) GluK2 KO. (c) TRPM8 KO. (d) GluK2/TRPM8 dKO.

Extended Data Fig. 10 GluK2 mRNA (Grik2) expression pattern in the DRG determined by single-cell RNA-seq and RNAscope in situ hybridization.

(a) Quantification of single-cell RNA-seq data from published work (ref. 15) shows that GluK2 mRNA (Grik2) positive neurons partially co-localize with myelinated neuron populations (NF), but exhibit little co-localization with TRPM8 mRNA (Trpm8), TRPA1 mRNA (Trpa1) and TRPV1 mRNA (Trpv1) in mouse DRGs. The percentages of colocalization with each marker are indicated. The CGRP mRNA (Calca) positive DRG neuron population is not analyzed due to inconsistency. (b-i) Quantification of GluK2 mRNA (Grik2) positive neuron population colocalization with various markers in mouse DRGs, including NF200, CGRP, IB4, TRPM8 mRNA (Trpm8), TRPA1 mRNA (Trpa1) and TRPV1. Neurons with more than three positive signals (observed as puncta) within the periphery of the neurons (determined by the phase contrast image) were considered as positive. (b) Bar graph summarizing the percentages of colocalization of GluK2 mRNA (Grik2) positive neuron with each marker. n = 3 (mice). Error bars: SEM. Data are presented as mean ± SEM. (c) GluK2 mRNA (Grik2) is expressed in neurons as well as some glial cells. About 26% of DRG neurons express GluK2 mRNA (data was quantified with 3 mice). GluK2 mRNA-positive neurons (indicated by white dotted lines) and glial cells (represented by white long broken lines) are distinguished using bright field (BF, left) and fluorescent images (right) with DAPI staining, based on their morphology and nucleus characteristics. White arrows indicate GluK2 mRNA-positive neurons and white arrow heads indicate GluK2 mRNA-positive glial cells. Scale bar: 10 μm. (d-i) Representative RNAscope images show colocalization of GluK2 mRNA-positive neurons with NF200 (d), CGRP (e), IB4 (f), TRPM8 mRNA (g), TRPA1 mRNA (h), and TRPV1 (i) in DRG neurons. White dotted lines label GluK2 mRNA-positive neurons, while white arrows indicate overlayed neurons. Scale bar: 20 μm.

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Supplementary Information

Supplementary Figs. 1–3.

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Supplementary Table 1

Table 1a,b is related to Fig. 2e–h, Extended Data Fig. 1 and Supplementary Fig. 2c–f.

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Cai, W., Zhang, W., Zheng, Q. et al. The kainate receptor GluK2 mediates cold sensing in mice. Nat Neurosci 27, 679–688 (2024). https://doi.org/10.1038/s41593-024-01585-8

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