A dedicated skin-to-brain circuit for cool sensation in mice

Abstract

Perception of external temperature is essential for maintaining homeostasis and avoiding thermal injury. Although molecular thermosensors such as transient receptor potential melastatin type 8 (TRPM8) have been identified, the neural circuits responsible for transmitting cool signals remain unclear. Here we show that a spinal circuit in mice conveys cool signals from the skin to the brain. Excitatory interneurons in the spinal dorsal horn expressing thyrotropin-releasing hormone receptor (Trhr⁺) act as a central hub for cool sensation. These Trhr⁺ neurons receive monosynaptic input from TRPM8⁺ sensory afferents and are selectively activated by innocuous cool stimuli. Ablating Trhr⁺ interneurons abolishes behavioral responses to cool, but not to warm or cold stimuli. We also identify a population of calcitonin receptor-like receptor-positive (Calcrl⁺) spinal projection neurons that receive convergent input from both TRPM8⁺ afferents and Trhr⁺ interneurons, and transmit cool-specific signals to the lateral parabrachial nucleus (lPBN). Our findings define a feedforward amplification circuit for cool sensation and reveal a modality-specific spinal pathway for thermal processing.

Fig. 1. Identification of cool-sensitive Calb1^Trhr neurons in the SDH.

a Workflow of using (1) an intersectional genetic strategy to ablate different neuron types in the superficial SDH, followed by two rounds (2 and 3) of behavioral screening assays. b Summary of the effect of ablating different neuron types in the SDH on innocuous and noxious cold sensitivities in mice. c Schematic showing the strategy used to label Calb1⁺ neurons in the SDH via dual-recombinases dependent reporter line. d Schematic illustrating the two-chamber recording system, with separate chambers for the DRG and the SC. Drug solution is administered to either the DRG chamber or the SC chamber. e Comparison of icilin-evoked EPSCs and APs by SC or DRG chamber perfusion, showing inward currents and action potential (AP) firing (left). The right pie chart displays the responding population of EPSCs and APs induced by the TRPM8 agonist (Icilin, 1 μM, DRG chamber perfusion) in Calb1⁺ neurons in lamina I–II, with n = 61 neurons from 14 mice. f Representative trace showing a Calb1⁺ neuron recorded upon administration of 1 μM icilin (blue) and/or 100 μM AMTB, a TRPM8 channel antagonist (red). g Quantification data of the inhibition of icilin-evoked EPSCs by the TRPM8 antagonist, AMTB. Data represent n = 15 neurons from 10 mice. Before vs AMTB, p < 0.0001; AMTB vs After, p < 0.0001; ^****p < 0.0001, two-way ANOVA with Tukey post hoc analysis. Box plots show the median (center line), 25th and 75th percentiles (box bounds), and minima and maxima (whiskers): Before = 547.38 (125.71–1001.50), AMTB = 0.00 (0.00–23.20), After = 560.37 (95.76–1000.12). h Characterization of icilin-activated Calb1⁺ neurons by single-cell RT-PCR. Experimental strategy (left). Gene expression profiling (Patch-to-profile) of Calb1⁺ neurons in lamina I–II from icilin-activated neurons (right). i Gene expression of icilin-activated Calb1⁺ neurons. j The temperature-controlled water-droplet test (TCWT) in control, Calb1^Abl, and Tac1^Abl mouse groups, control: n = 9, Calb1^Abl: n = 7; control: n = 8, Tac1^Abl: n = 8. Scoring system: 0, no response: 1, brief lifting: 2, one flinch: 3, multiple flinches or one lick: 4, prolonged licking or guarding. Control vs Calb1^Abl, p < 0.0001; Control vs Tac1^Abl, p < 0.6616; ns no significance, ^****p < 0.0001, two-tailed unpaired Student’s t-test. Data are presented as mean ± SEM. k Two major subpopulations of the icilin-activated Calb1⁺ neurons. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30 (applies to panels a, c, d, h, and k). Source data are provided as a Source data file.

Fig. 2. Innocuous cool temperature activates Trhr⁺ neurons in the SDH.

a Strategy used to label Trhr^iCre neurons in the SDH using Cre-dependent eYFP virus. b RNAscope in situ hybridization analysis of Trhr mRNA expression in Trhr^eYFP neurons. n = 40 sections from 4 mice. Scale bar, 100 µm. c RNAscope in situ hybridization analysis of Calb1 and Vglut2 expression in Trhr^eYFP neurons. Green indicates Trhr^eYFP, purple indicates Calb1 mRNA, red indicates Vglut2 mRNA, n = 21 sections from 3 mice. Scale bars, 100, 25 µm (magnification). d Schematic illustrating the in vivo two-photon Ca²⁺ imaging strategy, AAV9-CAG-FLEX-jGCaMP7f was injected into the SC of Trhr^iCre mice, enabling imaging of Trhr⁺ neurons during stimulation of the hindpaw with various stimuli, including water at different temperatures (4 °C, 10 °C, 15 °C, 20 °C, 30 °C, 40 °C, and 50 °C) and pinch. e A representative field of view of Trhr⁺ neurons under baseline and 15 °C water stimulation to the hindpaw (top). Relative ΔF/F0 heatmap from Trhr⁺ neurons responded to 15 °C water stimulus (white dashed line indicates stimulus onset) (bottom). f Traces represent the average ΔF/F0 responses across neurons from (e), with shading indicating ± SEM: pink (15 °C), orange (50 °C), blue (4 °C), and green (pinch). n = 57 neurons from 7 mice. BSL baseline, black dashed line indicates stimulus onset. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30 (applies to a, and d).

Fig. 3. Ablation of Trhr⁺ neurons within the SDH leads to deficits in cool sensation.

a Intersectional genetic ablation strategy of Trhr^Lbx1 (Trhr^Lbx1-Abl) neurons in the SDH. b Ablation efficiency of Trhr^Lbx1 neurons in the lumbar SC after intraperitoneal DTx injection. n = 3 mice in each group. Arrowheads indicate non-neuronal cells expressing Trhr. Scale bar, 25 µm. p < 0.0001; ^****p < 0.0001, two-tailed Student’s unpaired t-test. c Deficit of Trhr^Lbx1-Abl mice in innocuous cool sensing ability in TCWT at 15 °C, p < 0.0001; ^****p < 0.0001, two-tailed Student’s unpaired t-test. d Trhr^Lbx1-Abl mice in cold sensing in TCWT at 0 °C, ns no significance, two-tailed Student’s unpaired t-test. e Deficit of Trhr^Lbx1-Abl mice in the two-temperature choice assay, p < 0.0001; ^**p < 0.01, ^****p < 0.0001, two-way ANOVA with Šídák’s multiple comparisons analysis. f Deficit of Trhr^Lbx1-Abl mice in the gradient temperature assay, p < 0.0001; ^****p < 0.0001, ^####p < 0.0001, two-way ANOVA with Šídák’s multiple comparisons analysis. Control: n = 6; Trhr^Lbx1-Abl: n = 8 in (c–f). g Schematic of the spared nerve injury (SNI) model. h, i Responses of control and Trhr^Lbx1-Abl mice to cold allodynia (h) and mechanical allodynia (i) after SNI. BSL baseline, control: n = 6, Trhr^Lbx1-Abl: n = 8, ns no significance, two-way ANOVA with Šídák’s multiple comparisons analysis. j Schematic of the optogenetic stimulation and behavior recording setup using Trhr^iCre;Lbx1^Flpo;Rosa26^ds-ReaChR (Trhr^Lbx1-ReaChR) mice. k Representative images demonstrating the response of the ipsilateral hindpaw in control and Trhr^Lbx1-ReaChR mice during optogenetic activation. Arrows indicate the ipsilateral hindpaw of mice during light-off and light-on. Dashed lines indicate the floor surface. l Quantification of the number of hindpaw lifting and licking (out of five trials) following optogenetic activation of Trhr⁺ neurons. n = 6 mice in each group. Within Lift, p = 0.0005; Within Lick, p > 0.9999; ^***p < 0.001, two-way ANOVA with Šídák’s multiple comparisons analysis. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30 (applies to panels a, c, e, f, g, h, i, and j). Data are presented as mean ± SEM in b–f, h, and i. Source data are provided as a Source data file.

Fig. 4. Trhr⁺ neurons receive monosynaptic inputs from TRPM8⁺ sensory afferents.

a Schematic of the HTB-based mono-transsynaptic retrograde tracing approach used to identify presynaptic inputs onto Trhr⁺ neurons. b Validation of labeling of Trhr⁺ starter cells in the SC. Representative image showing GFP⁺/tdTomato⁺ Trhr⁺ starter neurons (yellow) in the SDH. The inset highlights labeled starter neurons (arrows). Scale bar, 50 μm. Repeated in four mice. c Labeling of presynaptic DRG neurons synapsed onto Trhr⁺ spinal neurons. Images of tdTomato⁺ traced DRG neurons co-stained with markers including Trpm8, Calca, TRPV1, and IB4. Arrows indicate the double-positive neurons (left). Quantification shows the proportion of tdTomato⁺ DRG neurons expressing each marker in the total amount of tdTomato⁺ neurons (right), n = 4 mice. Scale bar, 50 μm. d Image for triple staining for Trpm8, Calca, tdTomato. Arrowheads indicate tdTomato⁺/Trpm8⁺/Calca⁺ neurons. Pie chart showing the proportion of Trpm8⁺ and Trpm8^- neurons among tdTomato⁺/Calca⁺ cells. n = 88 neurons from 3 mice. e Schematic illustrating the approach to express ReaChR in TRPM8⁺ DRG neurons using a dual-recombinase-dependent strategy. f Strategy for random recording of neurons in lamina I–II in Trpm8^CreER;Avil^Flpo;Rosa26^ds-ReaChR mice. g Gene expression profiling of recorded neurons in lamina I–II with icilin-evoked APs (icilin-activated neurons). h No failure in light-evoked APs (5, 10, and 20 Hz, 473 nm, blue boxes) among icilin-activated neurons (10/12 neurons). i Effect of the sodium channel blocker (TTX, 500 nM, SC chamber) and the AMPA receptor antagonist (NBQX, 20 μM, SC chamber) on light-evoked EPSCs. n = 8 neurons from 7 mice (data points shown were neurons expressing both Calb1 and Trhr), p = 0.0042; ns: no significance, ^*p < 0.05, ^**p < 0.01, two-way ANOVA with Tukey post hoc analysis. Box plots show the median (center line), 25th and 75th percentiles (box bounds), and minima and maxima (whiskers): TTX = 83.72 (15.70–201.71), Control = 101.88 (15.30–198.10), NBQX = 0.00 (0.00–5.00). j Schematic representation showing TRPM8⁺ DRG neurons forming synapses onto Calb1^Trhr neurons. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30 (applies to panels a, e, f, and j). Source data are provided as a Source data file.

Fig. 5. Identification of cool-sensitive spinoparabrachial neurons.

a Schematic illustrating the recording of CTB-labeled spinoparabrachial (SPB) neurons in Trhr^iCre mice, in which ChR2 is expressed in Trhr⁺ neurons in the SDH via intraspinal injection of AAV9-EF1α-DIO-hChR2(H134R)-EYFP. b Light-evoked APs (5, 10, and 20 Hz, 473 nm, blue boxes) in CTB-labeled SPB neurons. c Representative traces showing light-evoked EPSCs (473 nm, blue boxes) of CTB-labeled SPB neurons in the spinal slice before and after TTX (500 nM), 4-AP (100 μM) (left). Quantification showing the amplitude of light-evoked EPSCs in ACSF, TTX, and TTX & 4-AP treated, n = 7 neurons from 3 mice, p = 0.0001; ns: no significance, ^***p < 0.001, two-way ANOVA with Tukey post hoc analysis (right). Box plots show the median (center line), 25th and 75th percentiles (box bounds), and minima and maxima (whiskers): ACSF = 146.0 (25.0–205.0), TTX = 2.0 (0.0–15.0), TTX & 4-AP = 145.0 (17.0–179.0). d Latency to induction of light-evoked EPSCs. The trace under the ACSF condition shown in panel (c) is temporally expanded to highlight the onset latency. n = 7 neurons from 3 mice. e Light-evoked APs of CTB⁺ SPB neurons expressed Calcrl, identified via single-cell RT-PCR post-recording. n = 7 neurons from 3 mice. f Schematic illustrating the recording of CTB-labeled SPB neurons in Trpm8^CreER;Avil^Flpo;Rosa26^ds-ReaChR mice, in which ReaChR channels are expressed in TRPM8⁺ sensory neurons in the DRG. The neurons are harvested after recording, and single-cell RT-PCR is conducted to identify Calcrl⁺ SPB neurons. g Light-evoked APs (5, 10, and 20 Hz, 473 nm, blue boxes) of Calcrl⁺ SPB neurons. h Representative traces and quantification of light-evoked EPSCs (473 nm, blue boxes) of Calcrl⁺ SPB neurons in the spinal slice before and after TTX (500 nM), AMPA receptor antagonist (NBQX, 20 μM), n = 6 neurons from 6 mice, p < 0.0001; ^**p < 0.01, ^****p < 0.0001, two-way ANOVA with Tukey post hoc analysis. Box plots show the median (center line), 25th and 75th percentiles (box bounds), and minima and maxima (whiskers): ACSF = 126.70 (87.15–147.55), TTX = 77.44 (59.55–101.45), NBQX = 0.00 (0.00–2.00). i Schematic representation showing TRPM8⁺ DRG neurons forming synapses onto Trhr⁺ interneurons and Calcrl⁺ SPB neurons. And Trhr⁺ interneurons forming synapses onto Calcrl⁺ SPB neurons. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30 (applies to panels a, f, and i). Source data are provided as a Source data file.

Fig. 6. Calcrl⁺ SPB neurons mediate cool sensation.

a Schematic illustrating the method of ablation of Calcrl⁺ SPB neurons (Calcrl^SPB-Abl, top). In Calcrl^tdTomato mice, numerous CTB-labeled SPB neurons are observed. A schematic indicating the locations of tdTomato-positive and CTB⁺ SPB neurons in the dorsal horn, along with a summary figure including representative SC images from control (Scale bar, 25 μm, bottom left), demonstrates quantification of ablation efficiency in Calcrl^SPB-Abl mice (bottom right). Green indicates CTB-positive neurons, yellow indicates CTB⁺/Calcrl⁺ neurons, arrows indicate double-labeled neurons, and arrowheads indicate CTB⁺ but Calcrl⁻ neurons. Quantification of Calcrl⁺ SPB neurons in the SDH of control and ablated mice (right). Control: n = 30 sections from three mice; Calcrl^SPB-Abl: n = 36 sections from three mice; p = 0.0009; ^***p < 0.001, two-tailed Student’s unpaired t-test. b, c Assays used to assess cool and cold sensing ability: b Deficit of Calcrl^SPB-Abl mice in the two-temperature choice assay. p = 0.0004. c Deficit of Calcrl^SPB-Abl mice in the gradient temperature assay. p = 0.0001. d Deficit of Calcrl^SPB-Abl mice in TCWT at 15 °C, p < 0.0001. e TCWT at 0 °C, p = 0.2694. Control: n = 9; Calcrl^SPB-Abl: n = 9. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001, ^####p < 0.0001, ns: no significance. Two-way ANOVA with Šídák’s multiple comparisons analysis in (b, c); two-tailed Student’s unpaired t-test in (d, e). Created in BioRender. Lee, H. (2025) https://BioRender.com/hwvgy30 (applies to panels a, b, c, and d). Data are presented as mean ± SEM in (a–e). Source data are provided as a Source data file.

Fig. 7. Trhr⁺ interneurons amplify cool information transmission to Calcrl⁺ SPB neurons.

a Representative traces showing the EPSCs of Calcrl⁺ SPB neurons in response to 1 μm icilin in control and Trhr^Lbx1-Abl mice. b Concentration-response curves of Calcrl⁺ SPB neurons from control and Trhr^Lbx1-Abl mice, p < 0.0001. Data are presented as mean ± SEM. c Representative traces of icilin-evoked AP firing in control and Trhr^Lbx1-Abl mice. d Quantification of icilin-evoked neuronal responses in Calcrl⁺ SPB neurons: (top) proportion of responsive neurons, and (bottom) total spike count within a 10 s interval following icilin application. Control: n = 18; Trhr^Lbx1-Abl: n = 16 neurons from 7 mice in each group. For icilin-eAP neurons (top): within 0.1 μM, p = 0.0023; within 0.3 μM, p = 0.0022; within 1 μM, p = 0.0169; within 3 μM, p = 0.0381; within 5 μM, p = 0.0544; within 10 μM, ns. For spike count (bottom): within 0.1 μM, p > 0.9999, within 0.3 μM, p = 0.9766, within 1 μM, p < 0.0001, within 3 μM, p < 0.0001, within 5 μM, p < 0.0001, within 10 μM, p < 0.0001. ^*p < 0.05, ^**p < 0.01, ^****p < 0.0001, ns no significance. Two-sided Chi-square test (d, top), two-way ANOVA with Šídák’s multiple comparisons (b; d, bottom). Data are presented as mean ± SEM. e, f Heatmaps showing the activity of SPB neurons (in ΔF/F0) in response to 4 °C, 15 °C, 50 °C, and pinch stimuli in control (e) and Trhr^Lbx1-Abl (f) mice. Control: n = 44 neurons from 3 mice; Trhr^Lbx1-Abl: n = 62 neurons from 3 mice. g Quantification of SPB neuron response to various stimuli (4 °C, 15 °C, 50 °C, and pinch) in control and Trhr^Lbx1-Abl mice, categorized based on their response to a 15 °C stimulus. Neuron responses are classified as Cool-specific (responding to 15 °C only), Cool-polymodal (responding to 15 °C and additional stimuli), Cool-insensitive (responding only to other stimuli but not 15 °C), and No response. Control: n = 44 neurons from 3 mice; Trhr^Lbx1-Abl: n = 62 neurons from 3 mice, ^****p < 0.0001, Two-sided Chi-square = 25.5, df = 3. h A diagram illustrating the cool sensation circuit: Trhr⁺ interneurons in lamina I–II of the SC receive input from TRPM8⁺ sensory neurons and connect to Calcrl⁺ SPB neurons. Proper transmission of cool-related information to the brain depends on Trhr⁺ neuron function, with their ablation compromising signal relay. Created in BioRender. Lee (2025) https://BioRender.com/hwvgy30. Source data are provided as a Source data file.