Cold- and TRPM8-dependent shaking in mammals and birds

Abstract

Removing cold water from wet fur or feathers can help endothermic animals to thermoregulate. The “wet dog shakes” (WDS) behavior has been largely characterized in mammals but to a much lesser extent in birds. Although it is known that TRPM8 is the main molecular transducer of low temperature in mammals, it is not clear if wetness-induced shaking in furred and feathered animals is dependent on TRPM8. Here, we show that a novel TRPM8 agonist induces WDS in rodents, and also in birds, similar to the shaking behavior evoked by water-spraying. Furthermore, the WDS onset depends on TRPM8, as we show in water-sprayed mice. Overall, our results provide multiple evidence for a TRPM8 dependence of WDS behaviors in all tested species. These suggest that a convergent evolution selected similar shaking behaviors to expel water from fur and feathers, with TRPM8 being involved in cold wetness sensing in both mammals and birds.

Shaking evoked by wet fur or plumage is reproduced by a synthetic agonist of the cold and menthol receptor TRPM8, in rats, mice, and chickens. In water sprayed mice, the onset of the shaking behavior depends on water temperature and TRPM8 expression.

Introduction

Among the thermoregulatory behaviors necessary for limiting heat loss and surviving in wet-cold conditions, one of the most spectacular is the shaking behavior mammals and birds use to rapidly expel a large amount of water from their coat. This behavior, called in mammals “wet dog shakes” (WDS), is a shudder motion consisting in vigorous and rapid rotations of the head and trunk around the spinal axis, with frequencies depending on body mass¹. A recent investigation on the identity of the neuronal and molecular mediators of mechanically-evoked WDS in mice showed that C-fiber low-threshold mechanoreceptors (C-LTMR) and Piezo2 ion channels, respectively, are the most likely candidates². While the mammalian WDS behavior has been the subject of biomechanics and pharmacological research^1–7, the analogous behavior in birds received little attention⁸ and it has not yet been investigated so far to what extent it can be triggered pharmacologically. By bringing together evidence from pharmacology, genetics and naturalistic stimulation, our study aimed to obtain a deeper understanding of the factors triggering WDS-like behaviors in mammals and birds, and of the role TRPM8 plays in these behaviors.

Studying WDS can also be a useful approach for understanding wetness sensing. By modulating the peripheral nervous system-dependent component of WDS, wetness sensing can be studied objectively and quantitatively in animal models. This allows genetic and pharmacological manipulations, which would constitute an advantage compared to human psychophysics⁹.

Icilin-induced WDS and jumping were previously shown in mice to be dependent on the cold and menthol receptor - the Transient Receptor Potential subfamily M (melastatin) member 8 (TRPM8) cation channel, as TRPM8 knock-out (Trpm8-/-) mice do not display icilin-induced WDS and jumping^10,11. In rats, the dependency of icilin-induced WDS on TRPM8 was demonstrated using TRPM8 antagonists^12–14. Nevertheless, the relationship between the pharmacologically induced behavior and the natural shaking of wet animals was not investigated. To what extent the shaking of rodents, when sprayed with cold water, is dependent on TRPM8, has not yet been clarified.

Thus far, the study of TRPM8-dependent shaking in rodents relied exclusively on the use of icilin. Because avian TRPM8 is insensitive to icilin¹⁵, the investigation of TRPM8 involvement in a putative bird shaking behavior has been hampered. A WDS-like shaking behavior in birds has been experimentally characterized only in one particular case (Anna’s hummingbirds, Calypte anna), perched and during flight⁸. Limited scientific evidence shows that some bird species shake to expel water from their plumage^16–19. We proceeded to confirm, and further investigate this behavior in chickens, by means of controlled experiments under laboratory conditions.

Thus, given the incomplete understanding of the sensory mechanisms that trigger shaking in wet mammals and birds, we embarked on a study to explore in detail the role of the cold sensor TRPM8 in these behaviors. For this purpose, we used a molecule from a novel class of TRPM8 agonists that we previously characterized^20,21, to overcome the limitations of icilin in activating avian TRPM8. We then set out to find what role TRPM8 plays in wetness-dependent WDS of mice under naturalistic stimulation. Overall, our study aims to offer a better understanding of how shaking to remove water is triggered across species.

Results

C-1 is a potent agonist of avian TRPM8

To identify a TRPM8 agonist able to elicit WDS-like behaviors in both birds and mammals, we started with pharmacological assays in heterologously expressed chicken (cTRPM8), rat (rTRPM8) and human (hTRPM8) TRPM8 orthologs using calcium imaging recordings with controlled temperature of the perfused solutions (as in Supplementary Fig. 1A). We considered a novel TRPM8 agonist, 1-di(propan-2-yl)phosphorylheptane, also known as Cryosim-1 (C-1^20,21), due to its good water solubility, which is advantageous for in vivo administration. C-1 belongs to a new class of “cooling compounds” featuring a phosphoryl group (Fig. 1A). This is structurally distinct from natural monoterpenoids, including menthol, and its synthetic derivatives, such as WS-12 (synonym acoltremon), one of the most potent and selective TRPM8 agonists, and from icilin, recognized as the most potent and efficacious mammalian TRPM8 agonist^22,23. We recorded calcium transients evoked by equal concentrations (10 μM) of three TRPM8 agonists (C-1, WS-12 and icilin) in HEK293T cells transfected with c/r/hTRPM8. As expected, icilin activated only the mammalian orthologs, while C-1 and WS-12 activated all three orthologs (Fig. 1B).

Fig. 1. The main properties of the novel agonist C-1 on recombinant TRPM8 from mammals and birds.

A The distinct structures of the synthetic TRPM8 agonists WS-12 (acoltremon), icilin, and C-1. B Average (± SEM) calcium imaging traces (ΔF/F₀) of HEK293T cells expressing one of c/r/hTRPM8, stimulated with the three agonists: C-1, WS-12, icilin, all at 10 μM (129/110/93 cells for c/r/hTRPM8, respectively). C The menthol-insensitive Y745H mutant of hTRPM8 is also C-1-insensitive (100 μM). Cooling from 32 to 17 °C, and then from 32 to 25 °C, functionally confirmed the expression of hTRPM8-Y745H. Menthol (100 μM) was applied to confirm the differences in menthol sensitivity (averaged traces ± SEM, 114 cells expressing hTRPM8 and 126 cells expressing hTRPM8-Y745H). D Concentration-response curves for c/r/hTRPM8 at 25 (i) and 32 °C (ii). Each ΔF/F₀ data point for C-1 (0.1–100 μM) is averaged from 3–4 experiments, with 17–74 cells per experiment. The average ΔF/F₀ for each ortholog is normalized to the response of cTRPM8 to 10 μM C-1. Data represented by filled symbols were fitted with Hill equations; empty symbols show desensitized responses at 100 μM C-1. The EC₅₀ values show that cTRPM8 is the most sensitive ortholog to C-1. E i. Representative examples of I-V relationships for cTRPM8 expressed in HEK293T cells when superfused with WS-12 (10 μM), C-1 (10 μM) and C-1 plus AMTB (1 μM) at 25 °C. Trace ‘3’ represents an outwardly rectifying I-V curve (before maximum current was reached) and trace ‘4’ a linear relationship, at peak current. ii. The current time-courses at −100 and 100 mV from the same recording. Numbers indicate the data points corresponding to the I-V traces. F Representative inward currents recorded at −80 mV in HEK293T cells expressing either cTRPM8 (left) or rTRPM8 (right) during cooling ramps alone (from 32–33 to 18 °C) and together with C-1 (10 μM), WS-12 (10 μM), or AMTB (1 μM). The maximum current recorded before the beginning of the cooling ramp is marked with ‘1’. Currents recorded while the cooling ramp and a potent agonist were simultaneously applied show a local minimum (marked with ‘3’, between the two maxima at ‘2’ and ‘4’). cTRPM8 displayed a current dip to C-1, while rTRPM8 to WS-12. The bar plots represent data pooled from experiments as above, showing the average current densities and corresponding temperatures at which the peak currents to cooling alone (“cool”), C-1 (alone and with cooling) and WS-12 (alone and with cooling) were recorded. The blue horizontal bars mark the data for cooling during agonist application. ***, p < 0.001; **, p < 0.01; *, p < 0.05; one-way repeated measures ANOVA, followed by Tukey’s post-hoc test, n = 5 for each ortholog; ###, p < 0.001; #, p < 0.05, two-way repeated measures ANOVA, n = 5 for each ortholog. All data points and bar plots represent means ± SEM.

Tyrosine 745 is known to play an important role in the interaction of menthol with its binding pocket: the Y745H mutant of hTRPM8 is menthol-insensitive but nonetheless cold-sensitive²⁴. Here we show that cooling elicited similar responses in both wild-type (WT) and Y745H hTRPM8, while C-1 and menthol induced large calcium transients only in WT hTRPM8. This shows that C-1 binds to the channel in the same manner as most other TRPM8 agonists²⁴ (Fig. 1C). Of the three TRPM8 orthologs, only cTRPM8 showed a sustained response to C-1 (Fig. 1B), leading to a larger area under the ΔF/F₀ curve (AUC, Supplementary Fig. 1B).

To compare the C-1 sensitivity of the three orthologs, the ΔF/F₀ responses to a range of concentrations (0.1–100 μM C-1) were normalized to the ΔF/F₀ response of cTRPM8 to 10 μM C-1 and fitted with Hill equations. As expected for TRPM8, the EC₅₀ values for each ortholog were lower at 25 °C than at 32 °C. At both temperatures, cTRPM8 had the lowest EC₅₀ values, 0.25 μM and 1.33 μM, while hTRPM8 the highest values, 2.43 μM and 4.73 μM, respectively. Rat TRPM8 showed intermediate EC₅₀ values (Fig. 1D, Supplementary Table 1).

The selective TRPM8 antagonist AMTB, a water-soluble compound with high potency for hTRPM8 (IC₅₀ ~ 0.59 μM against the Ca²⁺ increase evoked by the EC₈₀ of icilin in HEK-293 cells expressing hTRPM8²⁵) abolished (at 1 μM) the calcium transients elicited by C-1 (10 μM) in all three orthologs, and the inhibition was substantially reversible only for cTRPM8 (Supplementary Fig. 1C-H). BCTC, a well-characterized TRPV1 and TRPM8 antagonist²², had a lower inhibitory effect compared to AMTB, particularly on cTRPM8 (Supplementary Fig. 1C-H).

The properties of heterologously expressed c/r/hTRPM8 were also investigated using whole-cell patch clamp recordings under calcium-free conditions. All three orthologs showed current-voltage (I-V) relationships with TRP-characteristic outward rectification, which often became linear when the channels were maximally activated (Fig. 1E, Supplementary Fig. 2A-D). AMTB inhibited the outward and inward whole-cell currents activated by C-1 (Supplementary Fig. 2E). When comparing the currents elicited by C-1 (100 μM) and WS-12 (10 μM) in the same cells, cTRPM8 exhibited larger currents to C-1 than to WS-12, while the opposite was true for the rat and human orthologs (Fig. 1E, Supplementary Fig. 2F).

In further experiments, currents were recorded at −80 mV, while the temperature was decreased from ~32 to ~18 °C (cooling ramp of ~37 s) during stimulation by agonists and antagonists. Cooling alone evoked small but measurable inward currents in r/hTRPM8-expressing HEK293T cells (Fig. 1F, Supplementary Fig. 3A-B). Cold-evoked inward currents were particularly small in HEK293T cells expressing cTRPM8, in line with results of previous studies²⁶. To confirm that heterologously expressed cTRPM8 is cold-sensitive, we performed additional recordings of outward and inward currents during voltage ramps from −100 to 100 mV. These experiments revealed mostly outward cold-sensitive cTRPM8 currents (Supplementary Fig. 3C-E). As expected, in all orthologs, C-1 and WS-12 enhanced the inward currents elicited by the cooling ramp, while AMTB abolished them (Fig. 1F, Supplementary Fig. 3). The maximum 1/Q₁₀ coefficients for the temperature-dependent inward current increased in the presence of agonists to values between 139 and 820, depending on the ortholog-agonist pairing (Supplementary Table 1). Interestingly, the current decreased when approaching the minimum temperature, during exposure to C-1 for cTRPM8, and to WS-12 for r/hTRPM8, producing a “W”-shaped current. The local current minimum coincided with the temperature minimum during the cooling ramp, suggesting that the TRPM8 conductivity in the open state is limited by the temperature-dependent diffusion of ions (Fig. 1F, Supplementary Fig. 3A-B). The Q₁₀ coefficients for the temperature-dependent current decrease ranged from 1.2 to 1.3 for all TRPM8 orthologs (Supplementary Table 1). This also shows that 10 μM C-1 elicits a saturating effect on cTRPM8 at temperatures below ~22.5 °C.

Native TRPM8 is selectively activated by C-1 in cultured DRG neurons from rodents and chickens

The sensitivity of native TRPM8 to C-1 was investigated in cultured sensory DRG neurons from rat, WT and Trpm8-/- mice, and chicken. In rat DRG cultures, microscopic fields containing neurons likely to express TRPM8 were selected by a moderate cooling step (from 32 to 25 °C), then pharmacologically tested at a constant temperature of 24–25 °C (Fig. 2A). At 10 μM, C-1 elicited a response in 48 out of 456 neurons, while at 100 μM it activated a larger proportion, 170 out of 970 neurons (Fig. 2B). The large overlap in the sensitivities to C-1 and the well-established TRPM8 agonist WS-12 strongly suggests that C-1 activates natively-expressed rTRPM8. The neuron proportions responding to 10 μM C-1 and 5 μM WS-12 (Fig. 2B) were not statistically different (p > 0.05, chi-square test for homogeneity). At 100 μM, C-1 activated a significantly larger proportion of neurons than WS-12 (p < 0.05), indicating that 5 μM WS-12 failed to recruit all neurons activated by 100 μM C-1. Although this could be the consequence of the different potencies and efficacies of the two agonists at rTRPM8, potential off-target effects could also have affected these results.

Fig. 2. Native TRPM8 from rat and mouse DRG neurons is selectively activated by C-1.

A Representative examples of fluorescence traces in cultured DRG neurons from rat. The mild cooling step (from 32 to 25 °C) was followed by WS-12 (5 μM), C-1 (100 μM), and KCl (50 mM). B Venn diagrams illustrating the overlap between the neurons responding to WS-12 (5 μM) and C-1 (10 or 100 μM) and the cooling step. The experiments were performed on 456 neurons (10 μM C-1) and 970 neurons (100 μM C-1). The bar plots show the average peak amplitudes (ΔF/F₀) to C-1 and WS-12, as well as the neuron numbers for each of the three populations: neurons passing the threshold for C-1-only, for both agonists, and for WS-12-only. C Representative ratiometric imaging traces from DRG neurons of C57BL/6 J WT mice. WS-12 (5 μM), C-1 (100 μM), AITC (100 μM), capsaicin (1 μM, “Cap.”) and KCl (50 mM) were superfused at constant temperature (~25 °C). D The same experiment as in C, performed in DRG neurons from Trpm8-/- mice. E The average F₃₄₀/F₃₈₀ traces (± SEM) of all neurons responding to either WS-12 or C-1, from WT (n = 216 neurons) and Trpm8-/- mice (n = 67 neurons). F Venn diagrams showing the overlap of responses to WS-12 and C-1 in DRG neurons from both WT and Trpm8-/- mice. Experiments were performed on 1165 neurons from WT mice and 1227 neurons from Trpm8-/- mice. The bar plots illustrate the average peak amplitudes (ΔF₃₄₀/F₃₈₀) and neuron numbers for each population. The relative scaling of the Venn diagrams in B and F reflects the percentages of responding neurons from the total neuron populations investigated. All bar plots represent means ± SEM.

The moderate cooling step highlighted a limited population of cold-sensitive neurons. To avoid including the small responses to cooling of the calcium indicator itself²⁷, we set a threshold at four standard deviations above the mean of the normal distribution fitting the small responses. This resulted in 3.6% cold-sensitive neurons, pooled from all experiments. A large portion of these neurons were also sensitive to both C-1 and WS-12: 13 out of 16 neurons in the 10 μM C-1 experiments, and 28 out of 36 neurons in the 100 μM C-1 experiments (Fig. 2B). One cold-sensitive neuron was sensitive only to C-1. These results confirm the important role of TRPM8 in mediating cold sensitivity in rat DRG neurons.

To unambiguously determine the role of TRPM8 in eliciting calcium transients in response to C-1 and WS-12, we next used DRG neurons from WT (C57BL/6 J) and Trpm8-/- mice (Trpm8^EGFPf/EGFPf on C57BL/6 background), investigated with ratiometric calcium imaging at ~25 °C. Fura-2 loaded cells were sequentially stimulated with TRP channels agonists (5 μM WS-12 for TRPM8, 100 μM AITC for TRPA1 and 1 μM capsaicin for TRPV1, Fig. 2C-D). The averaged ratiometric signal of all neurons responding to either WS-12 or C-1 in WT mice (216 neurons out of 1165) and Trpm8-/- mice (67 neurons out of 1227), allowed us to visually discriminate the effect of the absence of Trpm8-/- on responses to the two agonists (Fig. 2E). We noticed that in Trpm8-/- mouse neurons the responses to WS-12 were still present (albeit fewer and with lower amplitude compared to WT). These responses were well synchronized between neurons, as revealed by the shape of the average response (Fig. 2E), suggesting the presence of a single TRPM8-independent responsible mechanism.

In neurons from Trpm8-/- mice, responses to WS-12 alone were displayed by 56 neurons (4.6%). Moreover, the average ΔF₃₄₀/F₃₈₀ amplitudes of neurons responding to WS-12-only were comparable between neurons from WT and Trpm8-/- mice (Fig. 2F). The majority of the neurons from WT mice that were sensitive to WS-12-only, and most of the neurons sensitive to WS-12 from Trpm8-/- mice, were capsaicin-sensitive (87% and 75%, respectively; full details shown in Supplementary Table 2). Furthermore, in HEK-293T cells expressing mouse TRPV1 (mTRPV1), WS-12 (5 μM) evoked large calcium transients that were inhibited by AMG-9810, a selective TRPV1 inhibitor (Supplementary Fig. 4A-B). Rat TRPV1 was less activated by larger WS-12 concentrations (10 and 100 μM), while C-1 (at 10 and 100 μM) was also unable to evoke significant calcium transients in cells expressing mTRPV1 (Supplementary Fig. 4C-D). This shows that, unexpectedly, WS-12 is a mTRPV1 activator and that in mouse DRG neurons, C-1 has a higher selectivity towards TRPM8 compared to WS-12.

Nevertheless, the total number of neurons responding to either C-1 or WS-12 was significantly lower in Trpm8-/- than in WT mice (chi-square test for homogeneity, p < 0.001 for both C-1 and WS-12): 139 (C-1) and 192 (WS-12) from 1165 neurons in WT mice versus 11 (C-1) and 64 (WS-12) from 1227 neurons in Trpm8-/- mice (Fig. 2F).

In cultured chicken DRG neurons, WS-12 (10 μM) was used to select microscopic fields rich in TRPM8-expressing neurons before stimulation with C-1 (10 μM). The temperature stimulus was initially regarded as unreliable for selecting TRPM8-expressing neurons, because cooling also elicited TRPM8-independent calcium transients in chicken sensory neurons, as previously shown²⁸. Therefore, the initial recordings in chicken DRG neurons were performed at a constant temperature of 24–25 °C (Fig. 3A). Considering the sensitivity of recombinant cTRPM8, we chose a concentration of 10 μM, for both C-1 and WS-12, in chicken DRG neuron assays (Fig. 3A–B). A substantial overlap was observed between C-1 and WS-12 sensitivities: C-1 elicited responses in 185 out of 1952 neurons (9.5%), while WS-12 did so in 164 neurons (8.4%), from which 149 responded to both agonists (7.6%, Fig. 3C). The difference between the proportion of neurons activated by C-1 and WS-12 was not statistically significant (p > 0.05, chi-square test for homogeneity).

Fig. 3. In chicken DRG neurons, C-1 selectively activates TRPM8 and a subpopulation of cold-sensitive neurons.

A Representative fluorescence traces of cultured chicken DRG neurons exposed to WS-12 (10 μM), C-1 (10 μM), and KCl (50 mM) at constant temperature (24–25 °C). B Other chicken DRG neurons were first exposed to a cooling step (from 32 to 20 °C), followed by the same stimuli as in A. C Venn diagram illustrating the overlap in the responses to WS-12 and C-1 from experiments as in A, on a total of 1952 neurons. The bar plot shows the average peak amplitudes (ΔF/F₀) and the neuron numbers for each of the three populations (C-1-only, C-1 and WS-12, and WS-12-only sensitive neurons). D Venn diagram illustrating the overlap in the responses to C-1, WS-12 and the cooling step, from experiments as in (B), on a total of 590 neurons. The bar plots show the average peak amplitudes (ΔF/F₀) and neuron numbers for the C-1 and cold, and WS-12 and cold sensitivities. The row represents the number of neurons sensitive to C-1-only, both agonists, and WS-12-only. The relative scaling of the Venn diagrams in (C, D) reflects the percentages of neurons sensitive to the tested stimuli from the total neuron populations. All bar plots represent means ± SEM.

To explore the cold sensitivity of cultured chicken DRG neurons, a different set of experiments was performed with an added cooling step (from 32 to 20 °C) before testing the TRPM8 agonists (WS-12, C-1, both at 10 μM) at ~25 °C (Fig. 3B). The cooling step limited to 20 °C and the set threshold (same criterion used for rat neurons) highlighted only the most cold-sensitive neurons (42 of 590 neurons, 7.1%), which were partially C-1- and WS-12-sensitive (59.5% and 54.8%, respectively, Fig. 3D). We presume that the other C-1-sensitive neurons had lower TRPM8 expression levels, and, therefore displayed subthreshold calcium transients to the cooling step. The finding of a large proportion of cold-sensitive neurons nonreactive to TRPM8 pharmacology aligns with results from other studies where cold-sensitivity was investigated using calcium imaging in chicken DRG neurons^26,28.

As an alternative to testing the temperature-sensitivity of TRPM8 in chicken DRG neurons using cooling, we also quantified the reduction in intracellular calcium following a mild warming step starting from the basal temperature of 20 °C, at which native cTRPM8 is likely to be active^26,28 (Supplementary Fig. 5A). A total of 36% of the warm-inhibited neurons displayed responses to C-1 (Supplementary Fig. 5A), indicating that C-1 activates a population of tonic cold-sensitive chicken DRG neurons expressing TRPM8.

Lastly, the appropriateness of AMTB for inhibiting TRPM8 in chicken DRG neurons was tested in protocols with repeated C-1 challenges, revealing that, while AMTB significantly inhibited the responses elicited by C-1, it also evoked large calcium transients by itself in a considerable population of chicken DRG neurons, particularly at 10 μM (Supplementary Fig. 5B-E). AMTB’s lack of selectivity in chicken DRG neurons made us decide not to use this TRPM8 antagonist for in vivo experiments in chickens.

C-1, icilin, and water-spraying evoke similar WDS behaviors in rats

The property of icilin to induce WDS in a variety of mammals was discovered long before the cloning and characterization of TRPM8. Most of the early WDS experiments with icilin were carried out in rats^29–31. The generation of Trpm8-/- mice was instrumental in demonstrating that WDS and jumping behaviors triggered by icilin are completely dependent on TRPM8^10,11. The icilin-induced WDS assay in rats (usually performed with 0.5 mg/kg icilin) became a standard method that allowed the in vivo validation of TRPM8 antagonists^12–14.

In rats, a complete WDS bout begins with a rotation of the head and continues towards the tail, often with the fur covering distant body segments rotating in opposite directions (Fig. 4A, Supplementary Movie 1). This is performed with the front paws off the ground and the straightened spine leaning forward at approximately 45°. To compare the effect of C-1 with the known WDS response to icilin, Wistar rats were injected intraperitoneally (i.p.) with either 33 mg/kg C-1 or 1 mg/kg icilin. The C-1 dose was selected based on results from a pilot study, to reliably evoke a robust number of WDS bouts within 30 min. The icilin dosage was close to the saturating dose for evoking WDS in rats within 30 min^7,14. In all rats injected with either drug, WDS bouts were frequently followed by grooming (Fig. 4B). WDS bouts were counted over 5-min intervals (Fig. 4C) and for the entire session (30 min, Fig. 4D). Both C-1 and icilin elicited robust WDS, with a similar number of bouts (~120 within 30 min). Although behavioral events were recorded for 30 min, WDS were still observed up to 1 h after injection. Injections of vehicles alone had virtually no effect (Fig. 4C). WS-12 was also tested in 4 rats, at doses up to 33 mg/kg (i.p.), without eliciting any WDS. To better understand these results, we analyzed the solubility of these compounds, characterized by their logP or logD at physiological pH. The predicted logD (at pH 7.4) is 2.23 for icilin, 2.66 for C-1, 2.79 for M8-Ag (another TRPM8 agonist reported to elicit sporadic WDS in rats³²), while for WS-12 it is 5.02 (as predicted by ACD/PhysChem Suite), suggesting that a logD in the range of 2 to 3 is important for eliciting WDS after i.p. administration.

Fig. 4. In rats, C-1 and icilin elicit comparable WDS behaviors.

A The typical rotational movements during a WDS bout in a rat. Often during a vigorous bout, the head rotates in the opposite direction relative to the fur on the rear of the body. B A representative ethogram (1 min), starting at 10 min after C-1 was administered. Grooming often followed the WDS bouts. This was typical for all recorded animals. C The average time-course of WDS bouts in rats that received i.p. C-1 (33 mg/kg) or icilin (1 mg/kg), and their respective vehicles (Ringer solution and PEG). The data points represent the number of WDS bouts counted over 5 min intervals (n = 6 for each experiment). D The total number of WDS bouts from experiments in (C). C-1 and icilin elicited similar numbers of WDS bouts over 30 min (n.s., p > 0.05, two-sample t-test, n = 6 for each agonist). All bar plots represent means ± SEM.

To investigate the natural response of rats to water droplets, similar to rain, we introduced a water spraying procedure, using water at two different temperatures. The aim of these experiments was to separate the cooling-induced effects from mechanically-induced ones, since, during rain, an animal is exposed to both a decrease in skin temperature and the impact of droplets.

Rats were sprayed 12 times in quick succession with a pump bottle delivering 0.9 ml of water per spray, within a spraying duration of ~5 s. The dorsal surface temperature of the rats was measured using infrared thermal imaging (Fig. 5A). For cold water-spraying, most of WDS bouts were displayed within the first 30 s after spraying, and this behavior alternated with grooming episodes (as shown in Fig. 5B, Supplementary Fig. 6). Immediately after spraying, the dorsal surface temperature of rats reached 34.8 ± 1.5 °C for warm and 12.9 ± 1.0 °C for cold water-spraying (mean ± SEM, n = 5). The difference in rats’ dorsal surface temperature between warm and cold spraying decreased to non-significant levels after 2 min (Fig. 5C). The number of WDS bouts counted in the first 60 s after spraying revealed a strong and significant difference between warm and cold spraying (Fig. 5D). In the first minute, warm spraying failed to evoke any WDS bouts (Fig. 5D). Altogether, these results show that in water-sprayed rats the evoked shaking behaviors are strongly temperature-dependent.

Fig. 5. In rats, water spraying evokes WDS in a temperature-dependent manner.

A Color-coded radiometric images recorded immediately after the same rat was sprayed with cold (left) and warm water (right), in separate experiments. The white polygonal contours demarcate the areas used for measuring the average temperature of the rat’s dorsal surface after spraying. The color gradients on the right side of each image indicate the temperature range for each image. B A representative ethogram of a rat sprayed with cold water, showing the behaviors recorded during the first 30 s from starting the spraying. C The average time-course of the rats’ dorsal surface temperature radiometrically measured before and after warm and cold water-spraying. Comparisons between warm and cold spraying at the same time points (***, p < 0.001; **, p < 0.01; *, p < 0.05; n.s., p > 0.05) and between the initial temperature (30 s before spraying) and the temperature immediately after cold water-spraying (###, p < 0.001), two-way repeated measures ANOVA followed by Tukey’s post-hoc test, n = 5. D The average time-course of WDS bouts in sprayed rats counted over 1-min intervals (n = 5). All data points represent means ± SEM. ***, p < 0.001, two-way repeated measures ANOVA followed by Tukey’s post-hoc test, n = 5.

TRPM8 is required for C-1-triggered WDS in mice

To test whether C-1-triggered WDS are dependent on TRPM8, the behavior of WT (C56BL/6) and Trpm8-/- mice (Trpm8^-/- mice on C56BL/6 background) was compared. Wild-type and Trpm8-/- mice were injected i.p. with 33 mg/kg C-1 and their behavior was recorded for 30 min. We selected the same C-1 dose as used in rats, to allow an interspecies comparison. As we found no statistically significant sex differences, data from mice of both sexes were pooled. Following the injection, a crouched posture and an early-onset (<1 min) high-frequency grooming interspersed with WDS bouts were detected in WT mice (Fig. 6A-C, Supplementary Movie 2). In contrast, WDS bouts were almost absent in Trpm8-/- mice during the entire 30 min of the experiment, while the grooming duration was significantly shorter (Fig. 6C, D). Occasionally, escape-like jumping behavior was also seen in WT mice after C-1 administration.

Fig. 6. TRPM8 is required for C-1-elicited WDS and grooming in mice.

A Typical postures of WT (i) and Trpm8-/- mice (ii) after receiving C-1 (33 mg/kg, i.p.): WT mice adopted a crouched posture most of the time, while Trpm8-/- mice displayed a normal posture. B A representative ethogram (5 min) of a WT mouse behavior, starting at min 10 after C-1 injection. C The average time-course of WDS bouts (left Y-axis) and grooming duration (right Y-axis) in WT and Trpm8-/- mice after receiving C-1. Data points represent the average number of WDS bouts and grooming duration counted over 5-min intervals (for WDS bouts ***, p < 0.001; **, p < 0.01; *, p < 0.05, and for grooming ###, p < 0.001; ##, p < 0.01; #, p < 0.05, both WDS and grooming tested with a two-way mixed ANOVA followed by Tukey’s post-hoc test, n = 10 for both genotypes). D Total WDS bouts and grooming duration from experiments in C, counted over 30 min. In WT mice, i.p. C-1 elicited significantly more WDS bouts and grooming than in Trpm8-/- mice (for WDS bouts: ***, p < 0.001; for grooming: ###, p < 0.001, two-sample t-test, n = 10 for both genotypes). All bar plots represent means ± SEM.

TRPM8 is required for the temperature-dependent WDS latency in water-sprayed mice

The response of mice to water spray stimuli was investigated in both WT (C57BL/6 J) and Trpm8-/- mice (Trpm8^EGFPf/EGFPf on C57BL/6 background), using water at two different temperatures, as previously done in rats. Preliminary observations showed that C57BL/6 J mice shake in response to mechanical stimuli alone (dust sprinkling on their coat, Supplementary Movie 3); thus we did not expect a complete absence of shaking behaviors in Trpm8-/- mice, or in WT mice sprayed with neutral-warm water. However, we expected a temperature modulation of the WDS bouts number and latency. Therefore, one target temperature was chosen to be close to the initial temperature measured on the animals’ dorsal surface (~32 °C, hereafter referred to as “warm” spraying), thus likely capturing only the effects of the mechanical stimulation associated with spraying. The other target temperature was chosen to reflect significant cooling, without entering the painfully cold range for mice (below 12 °C³³, hereafter referred to as “cold” spraying). Spraying consisted of 4 rapid successive actuations of a pump bottle, each delivering ~0.85 ml of distilled water (Supplementary Movie 4), within a spraying duration of ~1.6 s, after which the dorsal fur of the mice was thoroughly wet (Fig. 7A, B). The measured dorsal surface temperatures were 32.0 ± 2.4 °C (warm, n = 26) and 14.5 ± 1.6 °C (cold, n = 26), while the average temperatures inside the spray bottle were ~58 and ~4 °C, respectively (Fig. 7A, C-E).

Fig. 7. TRPM8 is required for short latency shaking in cold water-sprayed mice.

A The system used for recording the behavior and dorsal surface temperature of water-sprayed mice. Each WT and Trpm8-/- mouse was sprayed with warm and cold water in separate experiments. The measured dorsal surface temperatures were 32.0 ± 2.4 °C (warm, n = 26) and 14.5 ± 1.6 °C (cold, n = 26). The corresponding water temperatures inside the spraying bottles necessary to reach the above-mentioned temperatures on the mouse dorsal surface were ~58 and ~4 °C, respectively. B Representative image of a mouse after the spraying sequence. Following spraying, the fur on the head and back of the mouse showed marked hair clumping. C Color-coded radiometric images acquired immediately after the same mouse was sprayed with warm (top) and cold water (bottom). The white polygonal contours demarcate the areas used for measuring the average temperature of the mice’s dorsal surface after spraying. The color gradients on the right side of each image indicate the temperature range for each image. D A composite figure showing a representative ethogram of a WT mouse sprayed with cold water (first minute after spraying, upper part), synchronized with the thermal imaging measured temperature of the dorsal surface (“cold spray”, lower part). The “warm spray” trace was added for comparison to illustrate the temperature variation of the same mouse when sprayed with warm water. E The average time-course of dorsal surface temperature in WT and Trpm8-/- mice before and after warm and cold water-spraying. Comparisons between warm and cold spraying at the same time points (***, p < 0.001; *, p < 0.05 for WT, n = 12, and ###, p < 0.001; ##, p < 0.01; #, p < 0.05 forTrpm8-/-, n = 13), and comparison between the initial (60 s before spraying) temperature and the temperature recorded immediately after cold water-spraying (††† for WT and §§§ forTrpm8-/-, p < 0.001, n = 13 for each genotype), two-way repeated measures ANOVA followed by Tukey’s post-hoc test. All other differences were n.s. (p > 0.05). F WDS bouts counted over 1-min intervals during the first 5 min after spraying. In WT mice, cold spraying evoked significantly more WDS bouts than warm spraying in the same mice, during the first minute: **, p < 0.01 (WT, n = 13), and #, p < 0.05 (Trpm8-/-, n = 13), two-way repeated measures ANOVA followed by Tukey’s post-hoc test. All other differences were n.s. (p > 0.05). G The number of WDS bouts immediately after the spray. Trpm8-/-, but not WT mice, performed a lower number of cold-induced WDS bouts in the first 30 s of the test (*, p < 0.05; n.s., p > 0.05, paired-sample t-test, n = 13 for both genotypes). H The latency of the first WDS after spraying in WT and Trpm8-/- mice. WT mice had significantly shorter WDS latencies in response to cold than warm spraying, while the difference was not statistically significant in Trpm8-/- mice (***, p < 0.001; n.s., p > 0.05, paired-sample t-test, n = 13 for both genotypes). I The dorsal surface temperature measured immediately before the first WDS was significantly different between WT and Trpm8-/- mice for cold, but not warm spraying (***, p < 0.001; n.s., p > 0.05, two-sample t-test, n = 13 for each condition). All bar plots represent means ± SEM.

The post-spraying behavior consisted of WDS and grooming, while some animals also showed a startle-like response, by jumping off the ground with all four paws in response to single sprays. In a typical response, the frequency of WDS bouts decreased, while the grooming duration increased, as the dorsal surface temperature recovered after spraying (Fig. 7D). The dorsal surface temperature, measured immediately after cold spraying, was significantly lower than the baseline temperature (Fig. 7E) and it quickly recovered to baseline (in less than 1 min, Fig. 7D, E). As intended, there were no significant differences between the dorsal surface basal temperature and the temperature measured immediately after warm spraying (Fig. 7E). The dorsal surface temperature difference between warm and cold spraying was analyzed immediately, and at 1, 2, 5, 10, and 15 min after spraying. As expected, the largest significant difference was found immediately after spraying (Fig. 7E).

Because the dorsal surface temperature differences between warm and cold spraying decreased greatly over time, we counted WDS bouts in 1 min intervals during the first 5 min after spraying. The number of WDS bouts decreased over time in all conditions (Fig. 7F). During the first minute, WT mice sprayed with cold water showed a significantly greater number of WDS bouts than after warm water-spraying (Fig. 7F). In contrast, the WDS bout difference between cold and warm water sprayed animals in the first minute was not significant in Trpm8-/- mice. In these mice there was a significant difference solely in the second minute after spraying (Fig. 7F). Only in WT mice, a significant difference was found in the number of WDS bouts evoked by cold and warm water-spraying in the first 30 s (Fig. 7G).

Considering the short-lasting effect of cooling the fur and skin after spraying, we focused our analysis on the minimum duration necessary to record the WDS behavior, which is the latency of the first WDS event. This was measured from the onset of spraying. WT mice had an average latency of the first WDS event to cold spraying of 4.13 ± 0.84 s, less than half of the latency to warm spraying, 10.14 ± 1.32 s (p < 0.001, n = 13, Fig. 7H). Trpm8-/- mice showed no significant differences in WDS latency between warm and cold spraying, indicating that their WDS onset was not sensitive to temperature (Fig. 7H). A significant relationship between individual spray temperatures and WDS latencies was found for WT mice (p < 0.01), with a positive correlation between latency and dorsal surface temperature (r² = 0.33, Supplementary Fig. 7A).

Likely as a consequence of the different WDS latencies, combined with the rapid recovery of the dorsal surface temperature towards baseline, the temperature measured immediately before the first WDS event evoked by cold spraying was significantly lower in WT mice than in Trpm8-/- mice (17.2 ± 0.9 versus 22.8 ± 0.7 °C, p < 0.001, n = 13, unpaired t-test, Fig. 7I, left bar plots). The same comparison for warm spraying showed no statistically significant differences (Fig. 7I, right bar plots).

Most TRPM8-expressing free nerve terminals are located in the stratum spinosum and stratum granulosum of the epidermis³⁴. However, to understand whether and how subcutaneous (s.c.) temperature was affected by cold spraying, miniature temperature transponders were implanted s.c. above the thoracic spine in another group of mice. Although cold spraying significantly reduced both the dorsal surface temperature (as measured with infrared thermal imaging) and the dorsal s.c. temperature, the latter was affected to a smaller extent (Supplementary Fig. 7B).

Chickens respond to C-1 with a WDS-like behavior

The study of TRPM8-dependent WDS-like behaviors in birds has been limited by the insensitivity of avian TRPM8 to icilin. After identifying C-1 as a robust agonist for both recombinant and native cTRPM8 in vitro and demonstrating that rodents respond with vigorous WDS to C-1, we hypothesized that C-1 would elicit a putative TRPM8-dependent shaking behavior in chickens. Pharmacological preparations were s.c. (axillary) administered to chickens, as it was difficult to inject solutions i.p. while avoiding the abdominal air sacs.

C-1 (33 mg/kg) immediately evoked a range of behaviors: ample feather ruffling, preceding any vigorous body shaking, head and neck shaking while extending the neck forward, and finally, crowding in corners, often in a sleep-like behavior (Fig. 8A-B, Supplementary Movie 5 and 6). Head and neck shaking, with a maximum twist amplitude of about 180° was performed with closed eyelids (Fig. 8A i). Comparing head with body shakes in the six chickens treated with C-1, the elicited head shake bouts were more frequent and significantly shorter than body shake bouts (Fig. 8C). Feather ruffling was most visible around the neck, chest, and leg feathers, and could be seen from above by the large increase in feather volume (Fig. 8A ii). A few seconds after feathers were ruffled, a vigorous shaking of the body occurred, with the wings slightly spread and flapping (Fig. 8A iii, B, D). Escape-like jumping behavior was occasionally seen after C-1 administration. After the previously described behaviors ceased, the birds started pressing their chest against a corner or enclosure wall while having fluffed feathers (Fig. 8A iv, B, D, Supplementary Movie 6), and then entered a sleep-like state. This corner-crowding behavior occurred no later than 131 s (83.4 ± 13.7 s, mean ± SEM, n = 6) after the injection (Fig. 8B, D) and continued throughout the 15-min recording period. Two chickens were recorded for up to 30 min, a time they spent mostly in the same sleep-like state in a corner. C-1 elicited significantly more body, head, and total shake bouts than the vehicle, and a longer total shaking duration in the first 2 min, before chickens entered the sleep-like state (Fig. 8E, F). The few shaking movements induced by the vehicle can probably be explained by the temperature of the injected volume, which could not be precisely controlled and was therefore lower than the axillary s.c. temperature of the chickens. Corner-crowding and the sleep-like state were not present in vehicle-treated chickens, in contrast to the long duration of these behaviors in the same chickens after receiving C-1 (Fig. 8G).

Fig. 8. Subcutaneous C-1 elicits shaking behaviors in chickens.

A Four typical behaviors recorded in juvenile chickens after C-1 (33 mg/kg, s.c.). (i) Head shaking occurred with eyelids closed. (ii) Feather ruffling preceded all strong shakes. (iii) Vigorous body shakes were accompanied by moderate wing spreading and flapping. (iv) Corner-crowding in a sleep-like state while pressing the chest against the walls of the arena typically occurred within 2 min after C-1 administration. B A representative 2 min ethogram starting immediately after the C-1 injection. Typically, the body shakes were preceded by feather ruffling. C In C-1 injected chickens, body shake bouts were significantly longer than head shake bouts (***, p < 0.001, Mann-Whitney test, 62 head shakes and 12 body shakes, pooled from 6 chickens). The horizontal lines show the median. D Raster plots showing the behaviors of all 6 chickens in the study during the first 210 s from the s.c. C-1 injection. C-1 evoked feather ruffling, body and head shaking as well as feather preening and corner crowding. E The number of shake bouts counted over the first 2 min after injection. Body, head, and total number of shake bouts were significantly higher in chickens injected with C-1 compared with the same chickens receiving only the vehicle (***, p < 0.001; **, p < 0.01; *, p < 0.05, paired-sample t-test, n = 6). F The total shake time in the first 2 min after the injection (the same experimental set as in (D)), C-1 compared to vehicle (***, p < 0.001, paired-sample t-test, n = 6). G Total time spent during corner-crowding. This behavior characterized every C-1 injected chicken, while it was absent in vehicle-injected chickens (***, p < 0.001, paired- sample t-test, n = 6 chickens). All bar plots represent means ± SEM.

Chickens respond to water-spraying with a WDS-like behavior

We first examined the shaking behavior of domestic chickens by splashing adult farm chickens with water or sprinkling them with dust and found that the birds exhibited robust behaviors starting with feather ruffling, followed by vigorous body shaking and ending with neck twisting (Supplementary Movie 7, 8). Following these observations, to closely investigate shaking in response to naturalistic stimuli, similar to rain, we water-sprayed the chickens under controlled laboratory conditions. Chickens were sprayed with water at two temperatures (Fig. 9). A supplementary set of experiments was conducted on other chickens using room temperature water-spraying (Supplementary Fig. 8). Each chicken was sprayed 10 times in rapid succession (~0.9 ml per actuation), within a total spraying duration of ~4 s. The dorsal surface temperature of the chickens was measured using infrared thermal imaging (Fig. 9A). Chickens occasionally reacted with short head shakes during spraying (Fig. 9B, Supplementary Fig. 8A). Due to their startle-like nature, these events were not counted as proper head shake bouts. Chickens showed no feather ruffling or body shakes during the spraying interval, regardless of temperature. Feather ruffling, body and head shaking (Fig. 9C), as well as additional behaviors, such as feather preening and beak cleaning on the floor, followed soon after spraying (Fig. 9B, F, Supplementary Fig. 8A, Supplementary Movie 9). The corner-crowding in the sleep-like state observed in C-1 injected chickens was absent in all spraying experiments. Immediately after spraying, the dorsal surface temperature of chickens was 29.6 ± 0.7 °C (warm, mean ± SEM, n = 6) and 16.9 ± 0.6 °C (cold, mean ± SEM, n = 6). This temperature difference decreased to non-significant levels after one minute (Fig. 9D). As intended, there were no significant differences between the temperature measured before warm water-spraying and that recorded immediately after. Similarly to the shaking evoked by s.c. C-1, the average duration of body shake bouts was significantly longer than of head shake bouts (Fig. 9E). After cold spraying, most head and body shaking bouts occurred in the first 30 s. In contrast, the warm spraying evoked shaking bouts were fewer and more time-scattered (Fig. 9F). Warm spraying did not evoke any shaking in one chicken, and in another chicken, it only evoked one head shake in the first 5 min scored. Since the temperature difference between warm and cold spraying was significant at 30 s and not at 1 min, we counted shaking bouts in the first 30 s after spraying (Fig. 9G).

Fig. 9. Cold and warm water-spraying evokes shaking behaviors in chickens.

A Color-coded radiometric images recorded immediately after the same chicken was sprayed with warm (top) and cold water (bottom), in separate experiments. The white polygonal contours mark the areas used for measuring the average temperature after spraying. The color gradients on the right side of each image indicate the temperature range for each image. B A composite figure showing a representative ethogram of a chicken sprayed with cold water (first 45 s after spraying, upper part), synchronized with the thermal imaging measured temperature (“cold spray”, lower part) from the same experiment. The “warm spray” trace was added for comparison to illustrate the temperature variation of the same chicken when sprayed with warm water. Body shakes were typically preceded by feather ruffling. C All behaviors described above in Fig. 8A, except for corner-crowding, were present in water-sprayed chickens. D The average time-course of temperature, radiometrically measured on the back of the chickens, before and after warm and cold water-spraying. Comparisons between warm and cold spraying at the same time points (***, p < 0.001; n.s., p > 0.05) and between the initial temperature (30 s before spraying) and the temperature immediately after cold water-spraying (###, p < 0.001), two-way repeated measures ANOVA followed by Tukey’s post-hoc test, n = 6. E In cold water-sprayed chickens, body shake bouts were significantly longer than head shake bouts (***, p < 0.001, Mann-Whitney test, 20 head shakes and 11 body shakes, pooled from 6 chickens). The horizontal lines show the median. F Raster plots showing the behaviors displayed by all 6 chickens in the two-temperature water-spraying experiments. The behavior of each chicken in the first 60 s is shown on a single row. Warm (left) and cold (right) water-spraying evoked feather ruffling, body and head shaking as well as feather preening. Water-spraying-evoked behaviors were manifested earlier after cold than after warm spraying. G The number of shake bouts counted over the first 30 s after spraying with warm and cold water. Although cold water-spraying evoked more head, body and total shake bouts than warm water-spraying, all within group comparisons resulted in p > 0.05 (paired-sample t-test, n = 6). H The weighted score computed for body, head and total number of shakes in the first minute after spraying. The score = Σ(60 s − t₀), where t₀ is the onset measured from the end of the spraying, reveals that chickens shook more frequently and earlier after cold spraying than after warm spraying (**, p < 0.01; *, p < 0.05, paired-sample t-test, n = 6), as can seen in the raster plots in F. All bar plots represent means ± SEM.

To characterize the dispersion in time of the shaking events during the first minute after spraying, for each experiment, we computed a weighted score. This score calculates the sum of all shake bouts weighed with the remaining time until 60 s had elapsed: score = Σ(60 s − t₀), where t₀ is the onset measured from the end of the spraying. The more frequent the shaking events are towards the start, the larger the score is. The weighted score analysis revealed that chickens shook significantly more frequently earlier after cold than after warm water-spraying (Fig. 9H).

Altogether, the above suggest that shaking behaviors evoked in chicken by water-spraying are temperature-dependent and can be reproduced using the TRPM8 agonist C-1.

Discussion

Here, we have compared the chemically induced shaking behavior in mammals and birds with that induced by natural water-spray stimuli. We were able to show that chemical stimulation of TRPM8 elicits WDS-like behaviors in all tested species, and that TRPM8 contributes to the cold water evoked WDS in mice.

Since the cloning of the cold and menthol receptor TRPM8³⁵, a variety of synthetic small molecules have been validated as agonists of the channel, often with higher potencies than menthol²². However, of this large number of potent TRPM8 agonists, until recently only icilin was known to induce robust WDS^{3–7,10,12–15}. Our results demonstrate that a phosphoryl-alkane agonist of TRPM8, C-1, that we characterized in detail here, is capable of eliciting robust WDS-like responses in awake rodents and birds.

C-1 activated cTRPM8 but not the menthol-insensitive hTRPM8-Y745H mutant, confirming that it functions as a classical (menthol-like) TRPM8 agonist^24,36. The concentration-response dependence revealed a higher sensitivity of cTRPM8 to C-1, compared to mammalian TRPM8. Previously, cTRPM8 was shown to be more sensitive to menthol than mouse TRPM8^26,28. We show here, that for C-1 activation, cTRPM8 displays a lower EC₅₀ value than either rTRPM8 or hTRPM8.

During simultaneous stimulation with agonists and decreasing temperature, the whole-cell currents mediated by recombinant TRPM8 revealed a diffusion-limited conductivity. This suggests that the maximum open probability of the channel was reached and its temperature-dependent gating became saturated (Fig. 1F, Supplementary Fig. 3B). The property described here would allow testing of TRPM8 agonist efficacy: the higher the temperature at which the TRPM8 current starts to decrease during a cooling ramp in the presence of an agonist, the more efficacious the agonist is. Our results suggest that 10 μM C-1 can maximally activate cTRPM8, while 10 μM WS-12 can do the same for r/hTRPM8. A variety of ion channels have similarly low Q₁₀ values for open channel conductance³⁷ as those computed here for the current amplitudes of TRPM8 orthologs stimulated by C-1 or WS-12 (~1.3).

The experiments we performed in cultured DRG neurons showed that the sensitivities to WS-12 and C-1 overlap to a large extent in all tested species. The proportion of cultured rat DRG neurons sensitive to 10 μM C-1 (10.5%) is close to results reported by others for menthol (up to 10%^35,38). At 100 μM C-1, the proportion was larger (17.5%), but still in the range we reported previously for menthol (100 μM) sensitive neurons in the presence of cooling, when using the same culturing procedures^39,40.

Experiments in mouse DRG neurons revealed that C-1 is a more reliable pharmacological tool than WS-12 for testing the functional expression of TRPM8. Neurons from Trpm8-/- mice and C-1 insensitive neurons from WT mice displayed responses to WS-12, predominantly in a capsaicin-sensitive population (Supplementary Table 2). Moreover, we showed that heterologously expressed mTRPV1 was robustly activated by WS-12 (Supplementary Fig. 4A-B), indicating the nonselective nature of this molecule. This is in agreement with a report of small inward currents to WS-12 mediated by recombinant human TRPV1⁴¹, and our previous finding of WS-12 sensitivity in neurons from Trpm8-/- mice⁴².

In cultured chicken DRG neurons, C-1 (10 μM) activated 9.5% or 11.5% of the neurons (depending on experiment), which is higher than the 5.3% menthol sensitive neurons reported elsewhere²⁸, and lower than the 19.5% cold and menthol or 15.8% cold- and WS-12-sensitive neurons from a different study²⁶. Besides a large overlap with WS-12 sensitivity, C-1 responsiveness partially overlapped with a subpopulation of cold-sensitive neurons. The chicken cold-sensitive neurons we describe here (7.1% from total) represent a smaller proportion than reported elsewhere for the entire population of chicken DRG neurons presenting calcium transients evoked by cooling^26,28. We believe that factors contributing to these differences may encompass the different cooling stimulus used, the inclusion criteria for cold-sensitive neurons, as well as the breed and age of the chickens. Currently, the role of cold-evoked calcium transients independent of TRPM8 remains to be explored using electrophysiology to establish if these responses of chicken DRG neurons are associated with thermotransduction. Regarding the use of AMTB in chicken neurons, our results show that this TRPM8 antagonist evoked calcium transients by itself at its useful concentrations, adding to our initial observation coming from rat DRG neurons⁴².

In our experiments, both icilin and C-1 elicited robust WDS after i.p. injections in rats, consistent with a previous report of WDS in anesthetized rats induced by phosphoryl-alkane TRPM8 agonists²⁰. We have also tested in rats the potent agonist WS-12^41,43, without any success in inducing WDS. The uncommon property of certain TRPM8 agonists of inducing WDS is likely due to a favorable relationship between potency and in vivo bioavailability, as indicated by a LogD at physiological pH between 2 and 3. This would allow quick access to cutaneous nerve terminals expressing TRPM8 at sufficiently high concentrations.

We demonstrated the TRPM8 dependence of C-1-triggered WDS by showing that Trpm8-/- mice do not shake in response to i.p.-administered C-1. This is consistent with previous findings of icilin-induced WDS and jumping in mice^10,11. Besides WDS, another striking difference between WT and Trpm8-/- mice, when injected with C-1, was the grooming duration, which was significantly longer in WT mice and frequently accompanied by a crouched posture (Fig. 6). We also observed grooming evoked by C-1 and icilin in rats, in line with previous reports on the response to i.p. icilin in rats and other mammals²⁹.

We also approached the study of TRPM8-dependent shaking in a more naturalistic manner, by looking at how cold and TRPM8 expression (in mice), affects the shaking of wet rodents. In rats, the two temperatures of the water used for spraying the animals revealed a dramatic difference in behavior, with only cold spraying evoking WDS in the first minute after spraying. In mice, we found that the latency of the first WDS after spraying was significantly shortened in a cooling-dependent manner in WT but not in TRPM8 null mutant animals, indicating that TRPM8 controls the urgency of shaking (Fig. 7H). Cutaneous TRPM8 activation likely acts as a warning signal of potentially critical body heat loss due to wetness, reducing the latency of shaking compared to mechanical stimulation alone (Supplementary Fig. 9). In agreement with our findings, applying a mechanical stimulus (oil droplets) on the back of the neck was reported to evoke WDS in mice, with an average latency of ~10 s ², similar to that measured in our neutral-warm water-spraying assay (~10.1 s).

Bird shaking behaviors have been described in some bird species^8,16–19 but their underlying mechanisms have not been investigated. Here, we report the elicitation of WDS-like behaviors in birds by pharmacological means. It is noteworthy that body shaking was a much less frequent event in chickens compared to rats or mice. Also different from rodents, in chickens the head and body shaking appeared to be separate events. In chickens, the duration of individual shake bouts was longer for body shakes than for head shakes, in both C-1 injected and sprayed chickens. The gradual propagation of the twisting movement from the head to the tail, as seen in mammals, was not as evident in chickens, owing perhaps to vertebral fusion, common in birds. In chickens, vertebral fusion is age-dependent and occurs at several levels, particularly the thoracic (forming the notarium) and thoracic to caudal levels (forming the synsacrum)⁴⁴. Our experiments were conducted with young birds, which most likely still had free vertebrae, but the same shaking behavior was also observed in older chickens (Supplementary Movie 7).

Interestingly, C-1 injections also elicited feather ruffling and, after about 2 min, a long-lasting crowding against the enclosure corners in a sleep-like state with fluffed feathers. These are known heat-loss limiting behaviors encountered in chickens during cold exposure^45,46 that might also be relevant for bird huddling in these adverse conditions. The extended duration of the crowding behavior is consistent with a prolonged in vivo effect of C-1 on chicken TRPM8.

Spraying chickens with water at two temperatures evoked similar shaking behaviors as those elicited by C-1, and these experiments revealed that the frequency and timing of the shaking behaviors is temperature-dependent (Fig. 9F-H). Here, the absence of the corner-crowding behavior, seen in C-1 injected chickens, suggests that cold water-spraying did not decrease body temperature enough to evoke all behavior types associated with cold exposure^45,46. The involvement of TRPM8 in the chicken shaking behaviors evoked by cold water-spraying is partially supported by the data obtained in recombinant cTRPM8 and chicken DRG neurons. However, a definitive answer on this will only be obtained once cTRPM8 antagonists with suitable potency, selectivity and solubility properties become available for in vivo use, or TRPM8 knock out chickens are generated⁴⁷.

Understanding how shaking behaviors evolved, and their partial dependence on TRPM8 as a peripheral trigger, requires knowledge about the evolution of hairs and feathers, spine twisting capacity, and TRPM8 itself. While mammals and birds share similar TRPM8-dependent shaking behavior, it’s likely that these behaviors evolved independently, as the most recent common ancestor of mammals and birds - an early amniote – had no capacity for axial twisting of the thoracic spine^48,49 and probably no need to shake, as it likely had a self-cleaning hydrophobic integument, as some modern lizards^50,51. The convergent evolution of a strikingly similar shaking behavior in furred and feathered animals reveals the importance of keeping body coverings dry and clean. These behaviors could already have taken advantage of early TRPM8’s sensitivity to cold, which was likely acquired as the first tetrapods emerged, before hairs and feathers evolved⁵². As we show here, the cold sensitivity of TRPM8, as well as neuronal cold-sensitivity that relies on TRPM8, is species-dependent, which may partly explain the different magnitude of shaking responses to cold and TRPM8 agonists we have observed when comparing rodents and chickens.

While flying birds may also benefit from removing water from their feathers to become lighter (including during flight⁸), all birds appear to have similar shaking behaviors. Skeletal and muscular adaptations for flight are not a prerequisite for shaking in birds, as Palaeognathae (ratites) can shake to remove water from their plumage (e.g. emu^53,54 and cassowary⁵⁵), despite a nearly absent furcula, non-keeled sternum, and reduced pectoral girdle musculature⁵⁶. It can be speculated that extensive coating of the body with fur or feathers evolved in parallel with skeletons allowing axial rotation, enabling the shaking and grooming behaviors important for cleaning and thermoregulation⁴⁸.

Since proper hygroreceptors have only been found in invertebrates⁵⁷, wetness sensing in furred or feathered animals is thought to be synthetic, arising from mechanical and cold sensing, by analogy with human perception⁵⁸. Mechanical stimulation alone (e.g., coat touching, dust pressure following dust bathing) can evoke WDS in mammals and birds⁵⁹, Supplementary Movie 3, 8). Recently, it was shown that C-LMTR neurons and Piezo2 channels partially mediate mechanically-evoked WDS via the spinoparabrachial pathway². Although not investigated here, it is likely that TRPM8-expressing afferents transmit the cold information via a population of calcitonin-receptor like (CALCRL)-positive spinal projection neurons, as well as spinal interneurons expressing the thyrotropin-releasing hormone receptor (TRH), to the lateral parabrachial nucleus, to mediate WDS behaviors^2,60,61.

To our knowledge, there is yet no evidence that natural cold stimuli alone (i.e., exposure to cold still air) can trigger WDS by themselves. One explanation may lie in the relatively slow variation of air temperature, in contrast to raindrops and bathing, which produce a much faster surface temperature change, while being also paired with mechanical stimuli. Regardless of the conceivable dissociation of strong cold and mechanical sensing, we believe that shaking in response to wet-cold conditions is an important thermoregulatory behavior in mammals and birds. Without the ability to shake, it is likely that a non-aquatic endothermic tetrapod would require high energy costs for thermoregulation after cold water soaking of its coat, especially in a small animal⁶². Our research shows that the cold sensor TRPM8 controls the urgency to shake, providing a rapid regulatory response required for avoiding excessive heat-loss.

Materials and methods

Animal models

Rats: the animals included in the study for the C-1 and icilin experiments were in total 12 Wistar rats (6 males and 6 females) purchased from the “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 5–8 weeks old at the time of experiments. The weight of the males was 189.2 ± 62.2 g (mean ± SD, n = 6), and that of females 167.2 ± 47.0 g (mean ± SD, n = 6 for each sex). For the two temperature water-spraying assay, further 5 male rats were used, purchased from the “Cantacuzino” National Medical-Military Research-Development Institute. These were 9 weeks old and weighed 283 ± 23.3 (mean ± SD, n = 5).

Rats were housed in cages of 2 animals and kept on a 12-h light-dark cycle with the experiments being conducted during the light phase. Rats were supplied with water and food ad libitum.

Mice: the mice used for the C-1 experiments were WT C567BL/6 J (purchased from Jackson Laboratory, https://www.jax.org/strain/000664), called WT, and TRPM8 knock-out mice (Trpm8^-/- mice on C56BL/6 background⁶³, also from Jackson Laboratory, https://www.jax.org/strain/008198), called further Trpm8-/-, in total 20 mice of both sexes, aged 12–22 weeks (118.2 ± 31.3 days, n = 20, mean ± SD). The weight (mean ± SD) of the WT mice was 27.8 ± 3.0 g (males, n = 6) and 22.0 ± 2.0 g (females, n = 4), while that of Trpm8-/- mice was 27.2 ± 1.2 g (males, n = 6) and 19.8 ± 1.0 g (females, n = 4). Mice were housed up to 5 per cage, kept on a 12-h light–dark cycle, and were supplied with water and food ad libitum.

The mice used for the two temperature water-spraying assay were WT C57BL/6 J (purchased from Jackson Laboratory, RRID: IMSR_JAX:008198) and TRPM8 KO mice (Trpm8^EGFPf/EGFPf on C57BL/6 background¹⁰, a generous gift from Prof. Ardem Patapoutian, The Scripps Research Institute), called further Trpm8-/-, in total 26 mice of both sexes, 8–16 weeks old (83.0 ± 12.2 days, mean ± SD, n = 26). The weight (mean ± SD) of the WT mice was 26.6 ± 1.7 g (males, n = 7) and 22.4 ± 1.2 g (females, n = 6), while that of Trpm8-/- mice was 25.1 ± 1.7 g (males, n = 7) and 22.0 ± 0.8 g (females, n = 6). Mice were housed in group cages inside a ventilated cabinet (Scantainer, Scanbur Technology A/S, Karlslunde, Denmark), kept at constant temperature (22 °C) and humidity (55%) on a 12-h light–dark cycle and were supplied with water and food ad libitum.

Chickens: for the C-1 experiments and spraying with water at room temperature, 6 Plymouth Rock barred chickens of both sexes were purchased from Agromar Balotești (Ilfov, Romania). These were determined to be 3 males and 3 females, and were all of approximately the same age, 30–38 days old during the study, weighing 644.2 ± 98.2 g (mean ± SD, n = 6).

For the two temperature water-spraying assay, further 6 Plymouth Rock barred chickens were used, determined as 3 males and 3 females, aged 43–45 days, and weighing 645.2 ± 71.5 g (mean ± SD, n = 6). Chickens were housed together in a large bird cage, at about 23 °C and 40% relative humidity, on a 12-h light–dark cycle and were supplied with water and food ad libitum. The sex of the chickens was determined based on head plumage color⁶⁴, and later by the development of the comb and wattles in males.

For all used species, we have complied with all relevant ethical regulations for animal use.

Behavioral experiments in rats

The procedures for investigating the effects of i.p. C-1 and water spraying in rats were approved by the Research Ethics Committee of the University of Bucharest (No. 75/20.04.2019) and complied with EU Directive 2010/63/EU and the Romanian law 43/11.04.2014 on laboratory animal protection. The recording arenas consisted of bottomless transparent acrylic boxes (length = 21 cm; depth = 16 cm; height = 31 cm) with polycarbonate plastic floors. All rats in the spraying experiments were habituated with the enclosures during 3 sessions of 15 min each over 2 days before experiments. Icilin (1 mg/kg, in 100 μl solution/rat) was administered i.p. dissolved in polyethylene glycol 400 (PEG, Kollisolv 06855, Sigma-Aldrich), while C-1 (33 mg/kg, in 100 μl solution/rat) was dissolved in Ringer solution. This contained (in mM): NaCl, 140; KCl, 4; CaCl₂, 2; MgCl₂, 1; HEPES, 10; NaOH, 4.54 (pH 7.4 at 25 °C adjusted with NaOH). In control experiments, the animals received only the vehicle of each active compound. WS-12 (1, 2, 20 or 33 mg/kg) was dissolved in either PEG or mixtures of two or three solvents (from ethanol, DMSO, PEG, Tween 20 and sunflower oil). The boxes and floors were completely dry and clean before each experiment. The room temperature in the icilin and C-1 experiments was kept between 24 and 26 °C. In the spraying experiments, the average room temperature and relative humidity were 22 °C and 44%, respectively. Each rat was sprayed with water at room temperature 12 times in quick succession, with 0.9 ml of water per spray, from a distance of ca. 25–30 cm, targeting the rostral back. The water temperature in the spraying bottle was measured using a K-type thermocouple wire connected to a digital thermometer. The average temperature inside the bottle was 56.1 ± 0.2 °C for warm and 3.6 ± 0.1 °C for cold water-spraying (mean ± SEM, n = 5). Each spray lasted less than 0.5 s and the next one was delivered immediately after. The total spraying duration was 5.1 ± 0.2 s (mean ± SEM, n = 10), measured by analyzing the peak spectral density of the sound between the first and last spray, using the spectrogram feature in the Solomon Coder program (version: beta 19.08.02, RRID: SCR_016041).

Rat behavior was recorded using a color camera from the side or above the arenas at a resolution up to 2224 ×1080 pixels at 25 or 30 frames per second (fps). In the water-spraying experiments, an infrared thermal imaging camera (TiX580, Fluke Thermography, Everett, Washington, USA) was added, to record the animals from above. The recording duration was 30 min after the injection of C-1, and 15 min from water-spraying. After recordings, the animals were returned to their home cages. At the end of the spraying experiments, the excess water was gently swept from the rat’s fur with paper towels.

Behavioral experiments in mice treated with C-1

The procedures for investigating the effects of i.p. C-1 on mice were approved by the UC Davis Institutional Animal Care and Use Committee (IACUC), Protocol for Animal Care and Use #22313. All animals were habituated (1 h/day) to the recording arenas (open cylindrical restrainers, diameter = 12 cm; height = 25 cm) for 3 days before testing. The enclosures sat on a plastic floor and during the recordings were covered with a transparent acrylic plate. The laboratory temperature was controlled and maintained in the range of 20–22 °C, while the relative humidity was in the 30–40% range. C-1 (33 mg/kg in 10 ml/kg of Ringer solution, contents described above) was injected i.p. In control experiments, the same animals received only the vehicle. The behavior was recorded from above the arenas for 30 min after the injection, at a resolution of 1920 × 1080 pixels, at 25 fps. After 30 min elapsed, the animals were returned to their home cages.

Behavioral experiments in water-sprayed mice

The procedures investigating the behavioral responses in mice following warm and cold water-spraying, were performed according to regulations of animal care and welfare and approved by the Animal Protection Authority of the District Government of Lower Franconia (Würzburg, Germany) and the ethics committee of Friedrich-Alexander University Erlangen-Nürnberg under the file number 55.2.2-2532-2-1014. Mice were habituated in a bottomless transparent polycarbonate box (length = 15 cm; depth = 14 cm; height = 20 cm) during three sessions of 15 min each, over 2–3 days. The box sat on a polypropylene floor. The box and the floor were completely dry and clean before each experiment. On the day of the experiment, the mice were left in their home cages in the room where the experiments were performed, for at least 20 min, to acclimatize to room conditions. The average room temperature and relative humidity during the experiments were 24.8 ± 0.1 °C and 40.2 ± 0.5%, respectively (mean ± SEM, n = 52 experiments).

The mice were sprayed with a pump bottle with the nozzle adjusted to deliver ~0.85 ml per each actuation. Each spray lasted less than 0.5 s and the next one was delivered as soon as possible. Each mouse was sprayed 4 times in quick succession. The spraying duration was 1.61 ± 0.03 s (mean ± SEM, n = 52 experiments), measured by analyzing the peak spectral density of the sound between the first and last spray, using the spectrogram feature in Solomon Coder. The warm and cold spraying duration averages were comparable: 1.57 ± 0.04 s and 1.65 ± 0.05 s, respectively (mean ± SEM, n = 26 experiments).

Spraying targeted the rostral back area of each mouse. On the ground (from the height of ca. 25 cm), the shape of the sprayed area was an oval with extreme diameters of ca. 18 and 11 cm. Each mouse was sprayed with water at two temperatures, measured inside the bottle with a thermocouple, and on the surface of the mouse, using a forward-looking infrared (FLIR) thermal imaging camera (T420bx, FLIR Systems Inc, FLIR(R) Systems GmbH, Frankfurt, Germany). Distilled water was warmed and cooled with two liquid baths programmed at 62 and 0.5 °C, respectively, then transferred in the pump bottle, which was equipped with a thermocouple wire connected to a digital thermometer. At 58 °C in the bottle, the measured dorsal surface temperature reached a value between 27 and 38 °C, while at 4 °C in the bottle, the dorsal surface temperature was between 11 and 20 °C.

Each mouse was sprayed inside the box where it was video-recorded. The recordings started with spraying and ended 15 min afterwards. Two cameras were used simultaneously: a high-definition color camera recording through the front panel of the box, and the FLIR camera, recording from above. The front camera was used to record video files of 2560 ×1440 pixels at 30 fps, while the FLIR camera was used to record radiometrically at 320 × 240 pixels and 30 fps.

Each mouse underwent a single spray experiment per day and most mice were sprayed during two consecutive days. The order of exposure to warm and cold spray experiments was alternated in all animal groups, so that approximately half of the mice were sprayed first with warm water. If the temperature measured immediately after the end of last spray was not in the interval desired for neutral-warm (27–37 °C) or cold (12–19 °C) temperatures, the experiment was repeated the following day. The average number of spray trials to which a mouse was exposed was 1.23 ± 0.10 (minimum 1, maximum 3) for warm spray and 1.38 ± 0.13 (minimum 1, maximum 3) for cold spray (mean ± SEM, n = 26). The average temperature difference between warm and cold spray per mouse was 17.4 ± 0.5 °C (mean ± SEM, n = 26). After 15 min elapsed, the mouse was removed from the box, the excess water from its fur was gently swept with a paper towel, and the mouse was returned to its home cage.

A different group of mice, 3 WT and 3 Trpm8-/-, all males, 13-14 weeks old (95.3 ± 2.5 days, weighing 25.8 ± 2.2 g, mean ± SD) was implanted s.c. with miniature transponders (temperature microchip UCT-2112, Unified Information Devices, Lake Villa, IL, USA) under the back skin, above the middle thoracic spine, using the manufacturer supplied microchip injector and hypodermic needles. The temperature transducers were pre-calibrated by the manufacturer. The measured s.c. temperature was read with a receptor unit (URH 1HP reader, Unified Information Devices) placed under the floor of the enclosure and connected to a computer. Temperature acquisition started at least two minutes before cold spraying. The interval between consecutive data points was typically one second but was dependent on the position of the mouse inside the enclosure, relative to the receptor. Simultaneously, the dorsal surface temperature of the mice was recorded using a FLIR camera. One Trpm8-/- mouse was excluded from the FLIR data acquisition due to a technical problem.

Behavioral experiments in chickens

The procedures for investigating the effects of s.c. C-1 and water-spraying in chickens were approved by the Research Ethics Committee of the University of Bucharest (No. 75/20.04.2019) and complied with the EU Directive 2010/63/EU and the Romanian law 43/11.04.2014 on laboratory animal protection. Chickens were habituated to the arena, a bottomless transparent polycarbonate box (length = depth = height = 30 cm), during 2 sessions of 15 min each, over 2 days. The floor of the box was made from polycarbonate plastic covered with paper. The box and the floor were completely dry and clean before each experiment. Glass covers, or adding another box for a total height of 60 cm, were used for covering the enclosures during recordings, to discourage escape attempts.

C-1 (33 mg/kg) was dissolved in Ringer solution (see above) in a total volume of 1 ml/kg and s.c. injected under an axillary fold of skin. In control experiments the same animals received only the vehicle. The average room temperature in the C-1 experiments was 25.1 ± 0.3 °C (mean ± SEM, n = 6). The behavior was recorded using a color camera from above the arena for up to 30 min after the injection, at a resolution of 2560 ×1440 pixels, at 30 fps.

In the spraying experiments, the average room temperature and relative humidity were 22.7 ± 0.1 °C and 45.7 ± 1.5% (mean ± SEM, n = 9), respectively. Each chicken was sprayed 10 times in quick succession, with ~0.9 ml of water per spray, from a distance of ca. 30 cm. Water temperature in the spraying bottle, measured as above (see rat spraying), was 55.4 ± 0.4 °C for warm and 3.3 ± 0.2 °C for cold water-spraying (mean ± SEM, n = 6). Each spray lasted less than 0.5 s and the next one was delivered immediately after. The spraying interval was measured using the spectrogram available in the Solomon Coder program (as described above) and by visually following the stream of droplets in the recorded videos. The mean spraying duration was 4.0 ± 0.1 s (mean ± SEM, n = 12) and the behavior was recorded for 15 min from water-spraying. At the end of the experiments, the excess water on the plumage was gently swept with paper towels. After recordings, the chickens were returned to their home cages.

Rat DRG cell culture

Rat DRG neurons were obtained from all spinal levels of adult male Wistar rats (purchased from the “Nicolae Simionescu” Institute of Cellular Biology and Pathology, Bucharest, Romania). Male rats (150–200 g; 6–8 weeks, n = 3) were killed in a gradually increasing CO₂ concentration followed by decapitation. The procedures were approved by the Research Ethics Committee of the University of Bucharest and complied with EU Directive 2010/63/EU and the Romanian law 43/11.04.2014 on laboratory animal protection. Upon excision from all spinal levels, DRGs were incubated with a mixture of 1.5 mg/ml collagenase type XI (C 7657, Sigma-Aldrich) and 3 mg/ml dispase (Gibco 17105-041, Thermo Fisher Scientific, Waltham, Massachusetts, USA) in the IncMix solution for 1 h at 37 °C and 5% CO₂. Neurons were dissociated by gentle trituration with fire-polished Pasteur pipettes, plated on poly-D-lysine treated (0.1 mg/ml for 30 min) 25-mm borosilicate coverslips (0.17 mm thick) and maintained at 37 °C with 5% CO₂ in a 1:1 mixture of DMEM and Ham’s F-12 medium (D 8900, Sigma-Aldrich), supplemented with 50 ng/ml mouse Nerve Growth Factor-7S (NGF, N0513, Sigma-Aldrich), 10% horse serum and 50 μg/ml gentamicin. The cultures were used for experiments within 36 h after plating.

Mouse DRG cell culture

Mouse DRG neurons were excised from all spinal levels of adult male C57BL/6 J mice (8 weeks, n = 2, in-house breeding colony; originally from Jackson Laboratory, Bar Harbor, ME, USA, RRID: IMSR_JAX:000664), and Trpm8^EGFPf/EGFPf mice¹⁰ (8 weeks, n = 2, in-house breeding colony; a generous gift from Prof. Ardem Patapoutian, The Scripps Research Institute). All animal procedures were performed according to regulations of animal care and welfare (EU Directive 2010/63/EU) and approved by the Animal Protection Authority of the District Government (Ansbach, Germany). Mice were euthanized by breathing an increasing CO₂ concentration. DRG were dissected out, transferred to DMEM containing 50 μg/ml gentamicin, treated with 1 mg/ml collagenase type XI (C 7657, Sigma-Aldrich) and 0.1 mg/ml protease (P5147, Sigma-Aldrich) for 30 min, mechanically dissociated with a fire-polished silicone-coated Pasteur pipette, and plated on poly-D-lysine treated (0.2 mg/ml) 10 mm diameter glass coverslips. DRG neurons were cultured in serum-free TNB 100 cell culture medium (F8023 Biochrom, Berlin, Germany) supplemented with TNB 100 lipid-protein complex (F8820, Biochrom) and streptomycin/penicillin (100 μg/ml). Mouse NGF 2.5S (N-240, Alomone Labs, Tel Aviv, Israel) was added at 100 ng/ml and the cells were kept at 37 °C and 5% CO₂. Cells were used for experiments within 24 h.

Chicken DRG cell culture

For obtaining chicken DRG cultures, the procedures closely followed those used for culturing rat DRG neurons. Plymouth Rock barred chickens (purchased from Agromar Balotești, Ilfov, Romania, 320–420 g; 3–5 weeks, n = 7) were killed in a gradually increasing CO₂ concentration followed by decapitation. The procedures were approved by the Research Ethics Committee of the University of Bucharest and complied with EU Directive 2010/63/EU and the Romanian law 43/11.04.2014 on laboratory animal protection. DRG neurons were obtained from all spinal levels. See above (rat sensory neuron culture) for the neuron dissociation and culturing procedures.

Heterologous expression of TRP ion channels

Recombinant DNA was transiently transfected into HEK293T cells (RRID: CVCL_0063) using the jetPEI reagent (Polyplus-transfection S.A., Illkirch, France). Human TRPM8 (hTRPM8, RefSeq NM_024080.5), rat TRPM8 (rTRPM8, RefSeq NM_134371.3), and chicken TRPM8 (cTRPM8, GenBank OQ657222.1) were gifts from Dr. Gordon Reid (University College Cork, Ireland). Plasmids containing the hTRPM8 mutant Y745H were a kind gift from the laboratory of Dr. Viktorie Vlachova (Institute of Physiology, Academy of Sciences of the Czech Republic, Prague). All of the above constructs were inserted in pcDNA3 or pcDNA3.1 vectors. Mouse TRPV1 (mTRPV1) was inside a pIRES2-EGFP vector and was a kind gift from Prof. Joris Vriens (KU Leuven), while rat TRPV1 (rTRPV1) was inserted in a pcDNA3 vector. Approximately 24 h after transfection, the cells were detached with trypsin-EDTA, washed with complete medium, centrifuged at 1000 g for 5 min, resuspended in medium and plated onto 25-mm borosilicate glass coverslips (0.17 mm thick), which had been treated with poly-D-lysine (0.1 mg/ml for 30 min, and left to dry for another 30 min). The culture medium was a 1:1 mixture of DMEM and Ham’s F-12 medium (D 8900, Sigma-Aldrich). For the concentration-response experiments, cells transfected with hTRPM8 or rTRPM8 were co-cultured at a 1:1 ratio with cells transfected with cTRPM8 to control for inter-dish variability in the calcium imaging experiments. The cultures were used for experiments within 36 h after plating.

Single-wavelength calcium imaging

Intracellular non-ratiometric calcium imaging was used in HEK293T cells and rat and chicken DRG neurons. Coverslips with attached cells were incubated for 30 min at 37 °C in extracellular solution (ES) containing 2 μM Calcium Green-1 AM (Invitrogen C3011MP, Thermo Fisher Scientific) and 0.02% Pluronic F-127 (Invitrogen P6867). Cells were washed and left to recover for 30 min before use. Coverslips were then mounted in a Teflon chamber (MSC TD, Harvard Apparatus, Holliston, MA, USA) on the stage of an Olympus IX70 inverted microscope and imaged with a 20× NA 0.75 objective. All solutions continuously superfused the cultured cells with a gravitational-driven 6-channel perfusion system through a miniature manifold (MM-6; Harvard Apparatus) at a rate of ~0.75 ml/min. Temperature was controlled locally using a Peltier-based system^65,66. To select microscopic fields rich in cold-sensitive neurons, the cells were tested first with a mild cooling step. In order to measure the temperature experienced by the cells during the experiment, a miniature thermocouple (1T-1E, Physitemp, Clifton, NJ, USA) was placed very close to the imaged cells and the temperature was measured in real time with a digital thermometer (AZ 8851, TME, Łódź, Poland) connected to the imaging computer (Supplementary Fig. 1A). Cells were illuminated with a 470 nm LED powered by a Dual OptoLED unit (Cairn Research, Faversham, UK) controlled by the Axon Imaging Workbench 2.2 software (AIW, Axon Instruments, Union City, CA, USA), which was also used for image acquisition and analysis. Fluorescence images were recorded with a CCD camera (Cohu 4910, Pieper GmbH, Schwerte, Germany) at a rate of ~0.3–1 Hz and digitized to 8 bits. Regions of interest were used in AIW to mark individual cells. Background intensity was subtracted before computing the relative change of fluorescence (ΔF/F₀). The average area under the ΔF/F₀ curve (AUC), measured in seconds, during and before each stimulus, was used to discriminate cell responses. The threshold set to identify a cell response to chemical stimuli was set at the average baseline AUC plus its 2 standard deviations, for an interval equal to the ensuing stimulus duration. The difference values were rounded by truncation to 2 decimal places. To identify neurons responding to cooling or inhibited by warming, by excluding the small intrinsic temperature sensitivity of Calcium Green-1 fluorescence²⁷, we set a threshold at 4 standard deviations from the center of the normal distribution fitting the small amplitude responses. Neurons with spontaneous activity, equal in amplitude with the activity recorded during chemical stimulation, were excluded from further analysis. Cells responding to 50 mM KCl were considered viable neurons. To analyze the data from the concentration-response experiments performed on coverslips with co-cultured HEK293T cells expressing hTRPM8, rTRPM8, or cTRPM8, icilin (10 μM) was applied at the end of experiments. The average amplitude (ΔF/F₀) of the response of hTRPM8 and rTRPM8 was normalized to the response of cTRPM8 from the same culture dish. Unless otherwise stated, the experiments were performed at a constant temperature of 25 °C.

Ratiometric calcium imaging

Mouse DRG neuron cultures were loaded for 30 min at 37 °C with 3 μM Fura-2 AM (Invitrogen F1221) in ES containing also 0.02% Pluronic F-127 and left to recover for about 10 min in ES before recording. Coverslips were then mounted on the stage of an Olympus IX71 inverted microscope and imaged using a 10× objective. Cells were continuously superfused with ES using a software-controlled 7-channel gravity-driven common-outlet system⁶⁷. Fura-2 was excited at 340 and 380 nm using a Polychrome V monochromator (Till Photonics, Gräfelfing, Germany), with a full width half-maximum wavelength range of 10 nm. Fluorescence emission was long-passed at 495 nm and pairs of images were acquired at a rate of 0.5–1 Hz with an exposure time of 10 ms, using a 12-bit digital CCD camera and processed off-line (Imago Sensicam QE and TILLvisION software, Till Photonics, Gräfelfing, Germany). After background intensity was subtracted, the ratio between fluorescence emitted when Fura-2 was excited at 340 nm and at 380 nm (R = F₃₄₀ /F₃₈₀) was computed. Responsive cells were identified using the method described above (single-wavelength calcium imaging). Neurons with spontaneous activity, equal in amplitude with the activity recorded during chemical stimulation, were excluded from further analysis. Cells responding to 1 μM capsaicin, or 50 mM KCl, were considered viable neurons. For the HEK-293T recordings using Fura-2 AM (Supplementary Fig. 4), imaging was performed at ~23 °C using an IX-73 Olympus inverted microscope with an UAPO 340 20× objective and an Orca R2 CCD camera (Hamamatsu Photonics, Japan), a real time controller (U-RTC, Olympus) and xCellence RT software (Olympus).

Patch clamp electrophysiology

For patch-clamp recordings, HEK293T cells transiently transfected with TRPM8 orthologs and EGFP (DNA ratio 3:1), were plated on 25 mm borosilicate glass coverslips coated with poly-D-lysine and used within 24 h. Whole-cell patch clamp currents were recorded using a WPC-100 patch clamp amplifier (E.S.F. Electronic, Göttingen, Germany), filtered at 2 kHz and digitized at 5 kHz using an Axon Instruments DigiData 1322 A interface driven by pCLAMP 8.2 (RRID:SCR_011323, Molecular Devices, Sunnyvale, CA, USA). Capacitive transients were compensated using the R-series, C-slow and C-fast adjustments of the amplifier. The extracellular solution was nominally calcium-free, the same as the solution for calcium imaging, except it contained no CaCl₂ and was supplemented with 1 mM EGTA. The intracellular solution was K⁺-free with low Cl^- and contained (in mM): 134 Cs gluconate, 6 NaCl, 1 MgCl₂, 2 Na₂ATP, 5 EGTA, 10 HEPES; pH 7.22 at 25 °C adjusted with CsOH. Borosilicate capillaries with filament (GC-150F-10, Harvard Apparatus, Holliston, MA, USA) were pulled using a horizontal micropipette puller (P-1000, Sutter Instruments, Novato, CA, USA) and the tip polished for resistances of 2–4 MΩ. The reference electrode was placed in the same intracellular solution as the recording electrode and connected to the bath through an agar bridge. Currents were recorded either at −80 mV or during voltage ramps, from −100 mV to 100 mV (400 ms every 2 s, holding at −80 mV). During the experiments, the temperature inside the bath was controlled via a custom-made Peltier-driven perfusion system^65,66 and cooling ramps were triggered using pCLAMP or a custom-written software. The temperature was measured as above (see calcium imaging) before and after the recording, at the place where the cell had been located. Unless otherwise stated, the recordings were performed with the temperature fixed at 25 °C. The representative current traces shown in the figures were decimated with a factor of 1000 in Clampfit (pCLAMP).

Solutions and chemicals

The standard ES used in all experiments contained (in mM): 140 NaCl, 4 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, 4.54 NaOH, and 5 glucose   (pH 7.4 at 25 °C). The solution for DRG incubation (IncMix) contained (in mM): 155 NaCl, 1.5 K₂HPO₄, 5.6 HEPES, 4.8 NaHEPES, 5 glucose, and 50 mg/ml gentamicin. Drugs were added from the following stock solutions: (2S,5 R)-2-isopropyl-N-(4-methoxyphenyl)-5-methylcyclohexanecarboximide (WS-12, Tocris #3040, Bio-Techne Ltd, Abingdon, UK) 5 mM in ethanol; 1-diisopropylphosphorylheptane (Cryosim-1, C-1, CAS Registry Number 1487170-15-9, supplied by E.T. Wei) 1, 10 and 100 mM in H₂O; 1-(2-hydroxyphenyl)-4-(V3-nitrophenyl)-l,2,3,6-tetrahydropyrimidine-2-one (icilin, Tocris #1531) 10 mM in DMSO; 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC, Tocris #3875) 10 mM in DMSO; N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB, Tocris #3989) 10 mM in H₂O; (1 R, 2S, 5 R) - (-) menthol (M278-0, Sigma-Aldrich, Saint Louis, MO, USA) 200 mM in ethanol; capsaicin (M2028, Sigma-Aldrich) 5 mM in ethanol; allyl isothiocyanate (AITC, #377430, Sigma Aldrich) 100 mM in DMSO. Fresh stock solutions of AITC and for all working drug dilutions were prepared on the day of the experiment. All unspecified chemicals were from Sigma-Aldrich.

Statistical analysis and reproducibility

Power analysis based on data from pilot studies with water sprayed mice and data from previous experiments with icilin in rats were used to determine the animal sample sizes for C-1 and water-spray evoked shaking experiments in all species. Data describe biological replicates (animals or cells).

Data analysis and statistical tests were performed with OriginPro 8 (RRID: SCR_014212, OriginLab Corporation, Northampton, MA, USA) and GraphPad Prism 9 (RRID: SCR_002798, Dotmatics, Boston, MS, USA). All data sets were analyzed with the Kolmogorov-Smirnov normality test. Normally distributed data were compared using the two-tailed Student’s t-test (paired or unpaired, as indicated), or ANOVA with Tukey’s HSD post-hoc test to compare all possible group pairings. Ion current densities elicited in the same cells by one agonist at several temperatures were compared using one-way repeated measures ANOVA, while current densities elicited by two agonists and temperature values were compared using two-way repeated measures ANOVA. Behavioral parameters and surface temperature after spraying, measured at multiple time points, were compared with either two-way mixed ANOVA (with factors set as genotype and time), or two-way repeated measures ANOVA (with factors set as treatment and time). The Geisser-Greenhouse correction was applied to the repeated measures ANOVA each time the Mauchly’s test of sphericity did not indicate that sphericity can be assumed. Non-parametric statistical testing for data found not normally distributed was performed using the Mann-Whitney test or the Kruskal-Wallis test with Dunn’s post-hoc test for multiple comparisons. Responsiveness to TRPM8 agonists in DRG neurons from WT and Trpm8-/- mice was compared with the chi-square test for homogeneity. A value of p < 0.05 was considered statistically significant. Data are presented as mean ± SEM, except the age and weight of the animals which are expressed as mean ± SD, and for non-normally distributed data, where the median is indicated. In graphs where individual data points were connected with lines, the points were arranged on the horizontal axis so that all slopes (of the connecting lines) are represented proportional to the variation. For illustration purposes, the averaged ΔF/F₀ and F₃₄₀/F₃₈₀ were smoothed using a Savitzky-Golay filter set at a window length of 5–7.

The dose-response curves for non-ratiometric imaging were fitted using the Hill equation (without weighting), $y=v_{\max }{x}^n/\left(k^n+x^n\right)$, where v_max is equivalent to the maximal response amplitude, k to EC₅₀, and n is the Hill coefficient. Data points that showed obvious desensitization (smaller response at 100 μM than at 10 μM C-1) were excluded from the fitting data sets.

The Q₁₀ coefficients, defined as $Q_{10}={\left(I_2/I_1\right)}^{10^{\circ }\mathrm{C}/\left(T_2-T_1\right)}$ were computed for linear regions of maximal slope spanning at least 2 °C when analyzing log(-current) against temperature. Linear fitting was used to find the slope and Q₁₀ was calculated as $Q_{10}={10}^{10slope}$.

Predicted logP values were retrieved from ACD/Labs Percepta Platform (PhysChem Module) computed properties, available in the ChemSpider database.

Behavioral scoring was performed by investigators blind to all experimental conditions, using the Solomon Coder or BORIS (RRID: SCR_021509⁶⁸) programs. A shaking event in rats and mice was considered any visible head, neck, or body rotation. Shaking behavior was similarly scored in chickens, whereas, to avoid scoring spontaneous head twitches, we set a head shake bout duration threshold of 0.1 s. The thermal infrared recording was analyzed with the FLIR Tools Professional and FLIR Thermal Studio programs (Teledyne FLIR LLC, Wilsonville, OR, USA, RRID:SCR_016330) to measure the average temperature on the back of each mouse, using the polygon selection tool available in FLIR Thermal Studio. Rat and chicken thermal imaging data was analyzed using the Smartview Classic 4.3 program (Fluke Thermovision) to measure the average dorsal temperature using a polygonal selection. A standard emissivity of 0.98 was used for all temperature determinations. Vector graphs were exported and compiled together with illustrations in InkScape 1.0 (RRID: SCR_014479).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Author contributions

T.S. designed the study and established the methodology, performed and analyzed calcium imaging experiments, the patch clamp recordings, the behavioral experiments in rats and chickens, the spraying experiments in mice, contributed with resources, and wrote the manuscript with feedback from all authors. R.A.B. performed and analyzed calcium imaging experiments and behavioral experiments in rats and chickens. V.M.C. performed and analyzed behavioral experiments in rats, mice and chickens, and performed additional data analysis. M.I.C. performed and analyzed the C-1 experiments in mice. A.M. performed and analyzed calcium imaging experiments. D.E.H. performed and analyzed calcium imaging experiments and performed additional data analysis. R.M. recorded and analyzed the s.c. temperature in sprayed mice. E.T.W. contributed with resources (chemical compounds). E.C. performed and analyzed the C-1 experiments in mice, and contributed with resources. K.Z. contributed with resources and methodology for the mouse spraying experiments. A.B. performed and analyzed calcium imaging experiments in mouse DRG neurons and contributed with resources.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Joao Valente.

Data availability

All data analyzed during this study are included in the manuscript and supporting files. Numerical source data for all graphs in the manuscript can be found in the supplementary data file (Supplementary Data 1). All other data can be obtained upon request from the corresponding author.

Competing interests

E.T.W. is an inventor and has patent rights to use di-alkyl-phosphinoyl-alkanes in skin disorders (US Patent 10,195,217). All other authors declare they have no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s42003-025-09370-4.