Introduction

The cerebellum is important for motor coordination and learning through modulating motor commands and integrating sensory inputs; it is crucial for integration in the central nervous system and outputs to peripheral skeletal muscles. Neurons in the mature cerebellar cortex are organized into three layers: the granular, Purkinje cell, and molecular layers. Purkinje cells are the sole output cells of the cerebellar cortex and form inhibitory synapses with neurons in the deep cerebellar nuclei. The inhibitory control of Purkinje cells is mediated by γ-aminobutyric acid (GABA)ergic interneuron, basket cells, and stellate cells, which are distributed in the molecular layer of the cerebellum1. These inhibitory circuits regulate Purkinje cell activity and are the key to understanding the motor function of the cerebellar cortex. The descending branches of basket cell axons wrap around Purkinje cell bodies to make perisomatic synapses that are termed ‘pinceau.’ Interneurons provide major GABAergic inhibitory control and contain a variety of channels, including voltage-gated potassium channels and hyperpolarization-activated cyclic nucleotide-gated potassium channels2. These channels play an essential role in controlling GABA release from interneurons and thus the firing frequency of Purkinje neurons during motor behavior2,3. However, the channels on GABAergic inhibitory cells that are involved in the regulation of cerebellar motor functions are not yet fully understood.

Transient receptor potential (TRP) is a non-selective cation channel that is activated by a variety of chemical and physical stimuli, such as temperature, oxidative stress, and osmotic pressure. TRP channels are distributed in many tissues and are involved in a wide range of physiological functions as well as in disease development and progression4. In recent years, growing evidence has highlighted the presence and importance of TRP ion channels in the central nervous system. In the cerebellum, Purkinje cells contain many TRP canonical (TRPC) channels, and the genetic deletion of these channels in mice causes various symptoms including changes in motor function. These findings suggest an important role of TRPC in the normal functioning of the cerebellum, mediating processes such as balance and locomotion5,6. Although the functions of TRP channels that are expressed in Purkinje cells (such as TRPC, TRP melastatin 2, and TRP vanilloid 3) have been reported6,7,8,9, the roles of TRP channels that are expressed in cerebellar interneurons have not yet been explored. Cold-sensing TRP melastatin 8 (TRPM8) channels were observed in GABAergic inhibitory neurons by the precise mapping of the murine brain10,11. To investigate the role of TRP in interneurons, we selected TRPM8 from the various TRP channels because it contributes to inhibitory synaptic transmission in the central nervous system12. In the present study, we aimed to define the localization and involvement in motor functions of TRPM8 channels in the cerebellum using TRPM8-deficient (TRPM8KO) mice, pharmacological tools, and TRPM8-enhanced green fluorescent protein (EGFP) mice.

Results

Expression and characterization of TRPM8 immunoreactivity in the mouse cerebellum

To examine TRPM8 expression, we used a mouse transgenic line in which EGFP is expressed via TRPM813. Figure 1 showed TRPM8 expression in the TRPM8-EGFP mouse cerebellum. Neuronal-like TRPM8 immunoreactivity was detected in the molecular layer of the cerebellum (Fig. 1A). We then confirmed TRPM8 expression in the molecular layer using whole-cerebellum imaging with light sheet fluorescence microscopy (Fig. 1B and Supplementary Video S1). Next, we characterized TRPM8 immunoreactivity in the mouse cerebellum in double-labeling experiments (Fig. 1C). Double labeling revealed that TRPM8 immunoreactivity was present around calbindin (a Purkinje cell marker)-positive cell bodies but did not colocalize with them (Fig. 1C and Supplementary Video S2). TRPM8 immunoreactivity was colocalized with parvalbumin, a marker of GABAergic inhibitory interneurons. To confirm the presence of the neurotransmitter GABA in TRPM8-positive neurons, we conducted double-labeling experiments with vesicular GABA transporter (VGAT) and vesicular glutamate transporter 2 (VGLUT2). TRPM8-positive puncta colocalized with VGAT but not VGLUT2. To examine basket cell connectivity in more detail, we next conducted double-labeling experiments with GABAA receptor and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1), a pinceau marker. TRPM8 immunoreactivity around Purkinje cells was colocalized with HCN1 but not GABAA receptor. Furthermore, TRPM8 immunoreactivity did not colocalize with glial fibrillary acidic protein (GFAP), an astrocyte marker, or ionized calcium-binding adaptor molecule 1 (Iba-1), a glial cell marker, in the molecular layer of the cerebellum.

Fig. 1
figure 1

Characterization of transient receptor potential melastatin 8 (TRPM8) immunoreactivity in the mouse cerebellum. (A) Green fluorescent protein (GFP) expression in the cerebellum of TRPM8-GFP mice. (B) TRPM8 expression in the whole cerebellum using light sheet fluorescent microscopy. (C) Double labeling of TRPM8 (green) with calbindin, parvalbumin, vesicular GABA transporter (VGAT), vesicular glutamate transporter 2 (VGLUT2), γ-aminobutyric acid (GABA)A receptor (GABAA-R), hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1), glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) (red) in the cerebellum of TRPM8-EGFP mice. Dotted squares indicate the magnified areas. Scale bars: 100 μm (A), 20 μm (C).

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TRPM8 involvement in motor function in mice

To explore the possible functional consequences of TRPM8 deficiency, we performed behavioral tests to assess motor function (Fig. 2). In the rotarod test, TRPM8KO mice exhibited deficits in the accelerating rotarod test compared with wild-type (WT) mice (Fig. 2A and Supplementary Video S3). Both the retention time in each trial and the average of the second and third trials were significantly lower in TRPM8KO mice than in WT mice. By contrast, in the open field test, there were no significant differences in the time spent in the central area, time spent in the peripheral area, distance traveled, or velocity between WT and TRPM8KO mice (Fig. 2B). Finally, we analyzed the footprint patterns of WT and TRPM8KO mice to examine gait characteristics (Fig. 2C). There were no differences between WT and TRPM8KO mice in terms of stride length, sway, or stance in the footprint test.

Fig. 2
figure 2

Effects of transient receptor potential melastatin 8 (TRPM8) deficiency on the rotarod, open field, and footprint tests. (A) Latency to fall off the rotarod in wild-type (WT) and TRPM8-deficient (TRPM8KO) mice. Data are presented as the mean ± standard error (SE) for 9–10 mice per group. *P < 0.05 compared with WT. (B) Distance traveled in the chamber, average movement speed (velocity), and percentage of time spent in the central and peripheral areas in WT and TRPM8KO mice. Data are presented as the mean ± SE for 10 mice per group. *P < 0.05 compared with WT. NS, not significant. (C) Quantification of footprints in terms of stride, sway, and stance in WT and TRPM8KO mice. Data are presented as the mean ± SE for 7 mice per group. NS, not significant.

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Effects of TRPM8 deficiency in cerebellar morphology and GABA-related factors

We next investigated cerebellar weight, cerebellar structure (using hematoxylin and eosin staining), and the structural features of cerebellar neurons (using Nissl staining) in WT and TRPM8KO mice. There were no differences between WT and TRPM8KO mice in terms of cerebellar weight, structure, or neurons (Fig. 3A, B). We then investigated whether TRPM8 deficiency affects the organization of inhibitory terminals in the cerebellar cortex (Fig. 3C). TRPM8 deficiency did not affect the number or size of Purkinje cells (calbindin-positive cells). Moreover, the numbers of parvalbumin-positive cells and VGAT-immunoreactive puncta in the molecular layer of the cerebellar cortex did not differ between WT and TRPM8KO mice. GABA is synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD6714. We therefore quantified the protein amounts of GAD67 and GAD65 in the cerebral cortex using western blot analyses (Fig. 3D and Supplementary Fig. 1S). There were no significant differences between WT and TRPM8KO mice in GAD65 or GAD67 protein expression. We also measured tissue GABA levels using liquid chromatography–tandem mass spectrometry (LC-MS/MS) (Fig. 3E). TRPM8 deficiency did not affect basal GABA levels in the molecular layer of the cerebellum.

Fig. 3
figure 3

Effects of transient receptor potential melastatin 8 (TRPM8) deficiency on cerebellar weight, structure, and γ-aminobutyric acid (GABA)-related factors. (A) Cerebellar weights in wild-type (WT) and TRPM8-deficient (TRPM8KO) mice. (B) Representative images of hematoxylin and eosin staining (left) and Nissl staining (right) in WT and TRPM8KO mice. (C) Quantification of the number and soma size of Purkinje cells (calbindin-positive cells), number of parvalbumin-positive cells, and number of vesicular GABA transporter (VGAT)-positive puncta in WT and TRPM8KO mice. (D) Representative western blotting images of glutamate decarboxylase (GAD)65 and GAD67 proteins from WT and TRPM8KO mice, and quantitative western blotting results. (E) Comparison of GABA levels in WT and TRPM8KO mice. Data are presented as the mean ± standard error for 5–7 mice. NS, not significant. Scale bars: 500 μm (B), 20 μm (C).

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Effects of RQ-00203078, a TRPM8 antagonist, on motor coordination

To confirm the role of TRPM8 in motor coordination, we investigated the effects of a selective TRPM8 antagonist, RQ-00203078, using the rotarod test (Fig. 4). Both the intraperitoneal (Fig. 4A) and intracerebellar (Fig. 4B) administration of RQ-00203078 significantly decreased retention time compared with vehicle treatment. After the third trial of the rotarod test, we assessed the double labeling of TRPM8 with c-Fos or GABA in the molecular layer of the cerebellum (Fig. 4C). In vehicle-treated mice, TRPM8-positive cell bodies colocalized with c-Fos immunoreactivity, and GABA immunoreactivity was detected around TRPM8. By contrast, after intracerebellar RQ-00203078 administration, both c-Fos and GABA immunoreactivities were markedly reduced in the molecular layer.

Fig. 4
figure 4

Effects of transient receptor potential melastatin 8 (TRPM8) antagonist (RQ-00203078) administration on motor coordination. (A) Latency to fall off the rotarod in mice that were intraperitoneally treated with vehicle (control) or RQ-00203078. Data are presented as the mean ± standard error (SE) for 8–10 mice per group. *P < 0.05 compared with vehicle. (B) Latency to fall off the rotarod in mice that were treated with vehicle or RQ-00203078 via intracerebellar administration. Data are presented as the mean ± SE for 8 mice per group. *P < 0.05 compared with vehicle. (C) Double labeling of TRPM8 with c-Fos and γ-aminobutyric acid (GABA) in the cerebellum of a TRPM8-EGFP mouse. Scale bars: 20 μm.

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Discussion

The present study provides the first evidence to suggest that TRPM8 channels expressed in inhibitory neurons of the cerebellar molecular layer contribute to motor coordination. We identified both a GABAergic neuronal marker and an interneuronal marker, but not a Purkinje cell marker, on TRPM8-expressing neurons. We then conducted behavioral tests to investigate the functional connectivity between TRPM8-positive interneurons and motor activity in the cerebellar cortex using TRPM8-deficient mice and pharmacological tools. Both TRPM8 deficiency and the intracerebellar administration of a TRPM8 antagonist significantly affected motor coordination activity compared with the corresponding control groups. Together, these results suggest that TRPM8 channels expressed on inhibitory GABAergic neurons contribute to motor coordination by modulating synaptic transmission between Purkinje cells and interneurons.

Previous studies have detected cold-sensitive TRPM8 expression in the mouse hypothalamus, septum, thalamic reticular nucleus, and other limbic structures, as well as in specific nuclei of the brainstem10,15. Furthermore, TRPM8 reportedly colocalizes with GABA or VGAT on neurons in the mouse hypothalamus and reticular thalamic nucleus10,11. In the present study, we observed that TRPM8-immunopositive cell bodies were present throughout the molecular layer and colocalized with the interneuronal marker parvalbumin. Cerebellar stellate and basket cells are the predominant interneuronal cell types of the molecular layer; these different classes of interneurons play essential roles in controlling cerebellar cortical output during motor behavior16,17. The descending branches of basket cell axons wrap around Purkinje cell bodies, making pinceau18. Our double-staining data of TRPM8 with the basket cell marker HCN1 suggest that TRPM8 is expressed in the region of basket cell pinceau. Basket cell axons surround the cell bodies of Purkinje cells and form a characteristic plexus around the axonal initial segment, whereas stellate cells make synapses exclusively on the dendritic arbor19. Brown et al.17 used a tamoxifen-inducible Cre method to demonstrate that parvalbumin-positive neurons in the apical and basal molecular layers specifically reflect stellate and basket cells, respectively. However, it was difficult to assign parvalbumin- and TRPM8-positive neurons to specific stellate or basket cell identities, although the distribution patterns of TRPM8-positive cell bodies suggested that TRPM8 are expressed on both cell types.

Next, we speculated on the relationship between neurotransmitters released from TRPM8-positive interneurons by targeting their receptors in double-staining experiments. TRPM8-positive puncta colocalized with VGAT, and TRPM8-positive nerve endings densely covered GABAA receptor-positive cells and Purkinje cells (Fig. 5 and Supplementary Video S2). Glutamate is the major excitatory neurotransmitter in the cerebellum20, and TRPM8-positive puncta did not colocalize with VGLUT2-positive puncta in the molecular layer. Taken together, these immunohistochemical data suggest that TRPM8 channels are expressed in GABAergic interneurons in the mouse cerebellar molecular layers.

Fig. 5
figure 5

Schematic diagram of how transient receptor potential melastatin 8 (TRPM8) channels on inhibitory γ-aminobutyric acid (GABA)ergic neurons may contribute to motor coordination by modulating synaptic transmission at Purkinje cell–interneuron synapses. This image was generated using the Motifolio illustration toolkit (Motifolio Inc., Ellicott City, MD, USA).

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To investigate the role of TRPM8 in motor functions, we subjected TRPM8KO and WT mice to the rotarod, footprint, and open field tests. In mice, the rotarod, open field, and footprint tests are used to assess motor coordination, general locomotor function, and body balance, respectively21. Compared with WT mice, TRPM8KO mice had significantly lower latency to fall off in the accelerating rotarod test. Furthermore, both the systemic and cerebellar administration of a selective TRPM8 antagonist led to impaired motor coordination in this test. By contrast, in the open field and footprint tests, TRPM8 deficiency did not affect locomotor activities and footprint patterns, respectively. Consistent with previous data, TRPM8KO and WT mice traveled similar distances and spent similar amounts of time in the central/peripheral areas in the open field test at room temperature22. The rotarod test is reportedly a sensitive method for measuring sensorimotor function and motor coordination23. Mice deficient in systemic VGAT and GABA transporter 1 display motor dysfunction in the rotarod test and abnormal footprint patterns, suggesting that GABA transporter-positive neurons are critical for motor coordination and body balance24,25. Furthermore, GAD67 conditional knockout mice (in which GAD67 is deleted in parvalbumin-expressing GABAergic interneurons only) are reported to exhibit abnormal motor coordination in the rotarod test but normal gait patterns in the footprint test26. Furthermore, Brawn et al.17 reported that the loss of VGAT in basket and satellite cells alters the rate of Purkinje cell spike firing, which suggests that molecular layer interneurons cooperate to establish Purkinje cell function in vivo. We therefore speculate that TRPM8 may indirectly regulate Purkinje cells by modulating the activation of GABAergic interneurons under accelerating rotarod conditions. Taken together, the data from behavioral and immunohistochemical experiments suggest that TRPM8 expressed in VGAT-/parvalbumin-positive GABAergic interneurons especially contributes to motor coordination.

Although the molecular mechanisms of TRPM8 activation and regulation by chemical and physical stimuli are largely unknown, TRPM8 can be activated by cool temperatures (15–28 °C), cooling compounds, and a number of cellular signaling pathways,27,28,29. The expression and activity of TRPM8 is regulated by cellular signaling pathways, notably phosphatidylinositol 4,5-bisphosphate (PIP2), Ca2+-sensitive phospholipase C, and protein kinase C30. PIP2 contributes to the activity of cerebellar granule neurons, which is involved in neurotransmitter regulation31. TRPM8 sensitivity requires PIP2 which regulates the lifetime of channel opening and closing29. In the present study, we observed that the cerebellar administration of a TRPM8 antagonist reduced the number of c-Fos-positive cells and GABA immunoreactivity after the third rotarod trial. However, the stimuli that activated TRPM8 and the downstream molecular mechanisms contributing to motor coordination in this experimental condition remain unclear. The cerebellum produces corrections that allow for strength regulation and appropriate muscular activation after receiving, analyzing, and recognizing a sensorial or motor pattern32,33. It has been reported that TRPM8-positive neurons are present in the dorsal root ganglion, hypothalamus, septum, thalamic reticular nucleus, and brainstem in mice10,13. It is therefore possible that other TRPM8-positive neurons in the central nervous system contribute to motor processes, in addition to TRPM8-positive interneurons in the cerebellum. Further detailed investigations are necessary to clarify the activation mechanism of TRPM8 in motor coordination.

There are multiple limitations that must be considered when interpreting the results of the present study. First, we did not detect alterations in the synaptic properties of interneuron terminals. In vitro electrophysiological analysis using TRPM8-selective pharmacological tools and/or TRPM8-deficient mice is therefore required to detect the role of TRPM8-mediated GABA signaling on Purkinje cell activation. Second, the physiological importance of TRPM8 in the cerebellum remains unclear, although we detected abundant TRPM8-positive interneurons in the molecular layer of the mouse cerebellum. TRPM8 serve as the primary sensors for cold temperature fluctuations. As previously reported, the expression of TRPM8 in mouse hypothalamic neurons is activated and upregulated by cold temperatures34. It is thus possible that TRPM8 play some physiological role in motor coordination under the cold environments. In cerebellar molecular layer interneurons, NMDA increases spontaneous GABA release by activating presynaptic NMDA receptors which regulate motor learning in the cerebellum35. In sensory neurons, TRPM8 may interact with TRPV1, modifying the firing of mechanosensitive colonic afferents36. However, interaction of TRPM8 with NMDA receptors and other TRP channels have not been explored in cerebellar interneurons. A further characterization of TRPM8 expression would provide additional insights, such as the molecular interactions between channels and receptors in motor coordination and the environment-related alterations of TRPM8 in the molecular layer of the cerebellum. Third, the contribution of TRPM8 to cerebellar disorders such as cerebellar ataxia remains unclear. We revealed that TRPM8 knockout or antagonism decreased rotarod retention time in exercise load conditions but did not affect general spontaneous activity, body balance, or GABA-related factors in the cerebellum under daily exercise conditions. These effects of TRPM8 on motor activity may be useful for investigating novel therapeutic targets in various cerebellar diseases.

The present findings indicate that TRPM8 is expressed on HCN1-labeled inhibitory plexuses that wrap around GABAA receptor-expressing Purkinje cells. A lack of TRPM8 did not affect cerebellar structure, interneurons, Purkinje cells, enzymes for GABA synthesis, or GABA levels. Under accelerating rotarod conditions, it is likely that TRPM8 activation leads to Ca2+ influx in interneurons and GABA release, which then regulates GABAA receptors in Purkinje cells (Fig. 5). Moreover, our data suggest that TRPM8 channels on inhibitory GABAergic neurons contribute to motor coordination by modulating synaptic transmission at Purkinje cell–interneuron synapses.

Materials and methods

All experiments were performed in accordance with relevant guidelines and regulations. All animal studies are reported in compliance with the ARRIVE guidelines37. The protocols were approved by the committee of the Ethics of Animal Research of Kyoto Pharmaceutical University and Doshisha Women’s College of Liberal Arts.

Mice

Male C57BL/6 mice (8–10 weeks) were purchased from Japan SLC Inc. (Shizuoka, Japan) and were used as control. Male TRPM8KO mice (8–10 weeks) were originally established by Dr. A. Patapoutian from a C57BL/6 background38 and then provided by Dr. M. Tominaga. This strain is maintained by breeding TRPM8KO mice together. Male TRPM8-EGFP transgenic mice (8–10 weeks), which express EGFP under the direction of a TRPM8 promoter, were established from a C57BL/6 background as described previously13, and were purchased from Jackson Laboratories (Bar Harbor, ME, USA). In the present study, we used male mice to avoid the metabolic, hormonal, and behavioral changes that accompany the female estrous cycle. All mice were maintained in plastic cages with free access to food and water and were housed at 22 ± 1 °C with a 12-h light/dark cycle. The number of animals used was kept to the minimum necessary for the meaningful interpretation of the data, and animal discomfort was kept to a minimum.

Immunohistochemistry

TRPM8-EGFP, WT, and TRPM8KO mice were anesthetized with a combination of hydrochloric acid medetomidine (0.3 mg/kg, Domitor, ZENOAQ, Koriyama, Japan), butorphanol (5 mg/kg, Vetorphale, Meiji Seika Pharma, Tokyo, Japan), and midazolam (4 mg/kg, Dormicum, Maruishi Pharmaceutical Co., Osaka, Japan), and perfused transcardially with heparinized phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA; Fujifilm Wako Pure Chemical, Osaka, Japan) in 0.1 M phosphate buffer (pH 7.4). Segments of the mouse cerebellum were then removed, fixed by immersion in fresh 4% PFA in 0.1 M phosphate buffer for 2 h at 4 °C, and washed three times with PBS. They were then cryoprotected overnight in 0.1 M phosphate buffer containing 20% sucrose. Next, the tissues were frozen in optimal cutting temperature compound (Sakura Finetek, Toyko, Japan) and sectioned on a cryostat (Leica Instruments, Nussloch, Germany) at a thickness of 30 μm. The sections were thaw-mounted onto Superfrost Plus slides (Matsunami Glass Ind., Osaka, Japan). Prior to staining, the slide-mounted sections were incubated in 10% normal donkey serum containing 0.2% Triton X-100, and then in 0.1% sodium azide in PBS for 1 h. Next, they were washed three times for 10 min each with PBS. Immunohistochemical procedures were performed as previously described39. To detect TRPM8 immunoreactivity, sections were incubated in chicken anti-GFP antibody (1:10000, GFP-1010, Aves Labs, Davis, CA, USA) for 18 h at room temperature. After washing in PBS, sections were incubated for 3 h with donkey anti-chicken secondary antibody linked to Alexa Fluor® 488 (1:800, 703-545-155, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The specificity of the GFP antibody could be confirmed by detecting no staining in the negative control (Supplementary Fig. 2SA). Furthermore, we confirmed TRPM8 immunoreactivities using TRPM8KO mice because the mice were genetically engineered by knocking in EGFP38. Neuronal-like TRPM8 immunoreactivity was detected in the molecular layer of the cerebellum (Supplementary Fig. 2SB). TRPM8-positive axons formed basket-shaped pinceau like structure in the basal part of the cerebellar molecular layer (Supplementary Fig. 2SB). A strong expression of GFP was detected more frequently in TRPM8-EGFP mice cerebellum than in TRPM8KO mice. Therefore, we used TRPM8-EGFP transgenic mice for immunohistochemical analysis.

Next, double labeling was conducted using specific markers to characterize TRPM8 immunoreactivity. Sections were incubated with rabbit anti-calbindin (1:1000, ab108404, Abcam, Cambridge, UK), rabbit anti-parvalbumin (1:2000, ab181086, Abcam), rabbit anti-VGAT (1:4000, 131-002, Synaptic Systems, Göttingen, Germany), guinea-pig anti-VGLUT (1:1000, Af810, Frontier Institute, Tokyo, Japan), rabbit anti-GABAA receptor (1:4000, AGA-025, Alomone Labs, Jerusalem, Israel), rabbit anti-HCN1 (1:4000, APC-056, Alomone Labs), rabbit anti-Iba-1 (1:1000, 019-19741, Fujifilm), rabbit anti-GFAP (1:1000, ab4674, Abcam), rabbit anti-c-Fos (1:10000, 226 008, Synaptic Systems), and rabbit anti-GABA (1:40000, A2052, Millipore, Burlington, MA, USA) for 18 h at room temperature. After washing in PBS, sections were incubated for 3 h at room temperature with the corresponding secondary antibodies: anti-rabbit secondary antibody linked to Alexa Fluor 594 (1:800, A-21207, Invitrogen) and anti-guinea pig secondary antibody linked to Alexa Fluor 594 (1:800, 706-585-148, Jackson ImmunoResearch Laboratories). The stained tissues were then observed using a confocal microscope (A1R+, Nikon, Tokyo, Japan).

Image analysis

Quantitative determinations were made from three random locations of each mouse cerebellum. For the quantitative analysis, all images were captured from the cerebellar molecular layer with an All-In-One Fluorescence Microscope (BZ-X800, Keyence, Osaka, Japan) using the same settings. For Purkinje cell analysis, the number of calbindin-positive cells/length of the basal part of the cerebellar molecular layer (mm) was counted and the area of calbindin-positive cell bodies (radius × radius × π) of each cell was measured. For the other analyses, the numbers of parvalbumin-immunopositive cells and VGAT-immunopositive puncta were counted per 104 µm2 area of tissue.

Laser confocal microscopic analysis with super-resolution Airyscan detector

Super-resolution fluorescence images were captured using a confocal laser scanning microscope (LSM800) equipped with an Airyscan detector (Carl Zeiss, Oberkochen, Germany). Images were acquired as Z-stack images (settings: size = 3016 × 3016 pixels, digital zoom = 1.3×, scan speed = 4, Z-step size 0.22 μm, numerical aperture = 1.2, detector gains = approximately 800) and were adjusted for the signal-to-noise ratio using ZEN 3.6 software (Carl Zeiss). Images were further reconstructed to three-dimensional pictures and movies (13.00 frames/s) using IMARIS imaging software (Oxford Instruments, Abingdon, UK).

Light sheet fluorescence microscopy

Mice were perfused and fixed with PBS and 4% PFA, and whole cerebellar blocks were then immediately removed and post-fixed in 4% PFA for 24 h. The fixed mouse cerebellum was delipidated in 50% CUBIC-L buffer (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) overnight followed by 100% CUBIC-L buffer for 9 days with gentle shaking at room temperature. The cerebellum was then simultaneously incubated with primary rabbit polyclonal anti-GFP antibody (10 µg/mL, ab13970, Abcam) and secondary Alexa Fluor 647-conjugated anti-chicken antibody (7.5 µg/mL, 103-607-008, Jackson ImmunoResearch Laboratories) using a CUBIC-HVTM1 kit (Tokyo Chemical Industry Co., Ltd.) to a total volume of 250 µL, according to the manufacturer’s instructions. Next, the whole cerebellum block was serially immersed in 50% CUBIC-R buffer (Tokyo Chemical Industry Co., Ltd.) for 1 day and 100% CUBIC-R buffer for 2 days at room temperature in the dark. Finally, the fluorescent signal in the whole cerebellum block was detected using a light sheet fluorescence microscope (UltraMicroscope II, Miltenyi Biotec, Bergisch Gladbach, Germany) with a 2× lens. Images were acquired using the following parameters: laser power = 10%, thickness = 3.97 μm, sheet width = 70%, S-step size = 3.97 μm, and numerical aperture = 0.156. The three-dimensional images and movies (50.00 frames/s) were then reconstructed using IMARIS imaging software.

Behavioral tests

For the rotarod (47600 Rota-Rod, Ugo Basile, Gemonio, Italy) test, mice were pre-trained in two independent sessions at a constant rate of 5 rpm on the first training day and 8 rpm on the second training day. On the test day (the day after the second training day), the mice were placed on a rod and evaluated for three trials, each of which had an acceleration from 5 to 40 rpm in 300 s. There was a resting time of 180 s between each trial. The end of a trial was considered to occur when a mouse fell off the rod or reached 300 s, and the latency to fall was recorded for each trial.

For the open field test, each mouse was placed in the center of a circular open field chamber. Activity in the open field chamber was video recorded for 10 min and the time spent in the central area, time spent in the peripheral area, distance traveled, and velocity were analyzed using EthoVision XT Software (Noldus, Wageningen, Netherlands).

For the footprint test, the hind paws of the mice were painted with blue (WT) or red (TRPM8KO) acrylic paint, and the mice were allowed to walk along a runway (length, 32 cm; width, 10 cm). For each run, a fresh sheet of paper was placed on the runway floor. Each mouse was tested twice, and the footprint patterns of the hind paws were evaluated in terms of three parameters: (i) stride length, measured as the average distance in forward movement between each stride; (ii) stance length, measured as the average distance between the left and right hind footprints; and (iii) sway length, taken as the average perpendicular distance between each footprint, as reported previously27. The footprint parameters were determined by drawing lines through the center of each footprint and measuring the distance (in centimeters) between the appropriate lines.

Hematoxylin and Eosin and Nissl staining

Mouse cerebellums were fixed in 10% buffered formalin, embedded in paraffin, and cut into 4-µm-thick sections for routine hematoxylin and eosin and Nissl staining as reported previously40,41.

Western blotting

Protein concentrations of the samples were quantified using a Pierce bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A polyvinylidene fluoride membrane was blocked with 5% skim milk and washed with PBS containing Tween 20. The membrane was then incubated 18 h at 4 °C with the following primary antibodies: anti-GAD65/67 antibody (1:5000 diluted in skim milk, ab11070, Abcam) and anti-β-actin antibody (1:2000 diluted in skim milk, 4970, Cell Signaling, Danvers, MA, USA). Next, the membrane was incubated in secondary antibodies (anti-rabbit IgG horseradish peroxidase-linked antibody; 1:5000 diluted in skim milk, Cell Signaling). Results were quantified using Evolution Capt. (Garvan Institute of Medical Research, Darlinghurst, Australia) andβ-actin protein was used as a reference to the data.

Chemicals and administration protocol

Animals received an intraperitoneal or intracerebellar administration of the TRPM8 selective antagonist RQ-00203078 (MedChemExpress, Monmouth Junction, NJ, USA). The rotarod test was conducted 13 min after the intraperitoneal administration of RQ-00203078 (3 mg/kg) or vehicle (2% dimethyl sulfoxide/saline), which was administered using a volume of 0.1 mL/10 g mouse body weight. The RQ-00203078 dose was selected based on a previously described protocol42. Intracerebellar administration was performed as follows. First, mice were anesthetized by the intraperitoneal injection of a combination of hydrochloric acid medetomidine, butorphanol, and midazolam. Mice were then placed on a stereotaxic instrument (Stoelting, Wood Dale, IL, USA) and a small hole was made in the occipital bone using a 23-gauge needle (Terumo, Tokyo, Japan). Next, a syringe (Hamilton, Reno, NV, USA) with a 22-gauge blunt needle was set on an automated syringe pump (KD Scientific, Holliston, MA, USA), and the needle was inserted into the cerebellar vermis (coordinates from bregma: 0.0 mm lateral, 7.0 mm posterior, and 2.0 mm ventral). The RQ-00203078 was dissolved in PBS containing 5.0% dimethyl sulfoxide, and the concentration was adjusted to 2.5 µg/µL (4.5 mM). The solutions (4.0 µL of RQ-00203078 or vehicle) were administered at an injection rate of 1 µL/min.

LC-MS/MS analysis of GABA

The cerebellar cortex was isolated, weighed, and homogenized in PBS. The homogenates of cerebellar cortex were stored at − 80 °C until analysis. The GABA concentrations in tissue homogenates were determined using an API 3200 triple-quadrupole mass spectrometer (SCIEX, Foster City, CA, USA), as previously reported with minor modifications43. Briefly, 50 µL tissue homogenate was spiked with 10 µL acetamidophenol solution (10 µg/mL in water/methanol/formic acid [20/80/0.2, volume (v)/v/v]) as an internal standard. To precipitate the protein, 200 µL acetonitrile was added to the mixture. After vigorous mixing for at least 30 s, the mixture was centrifuged for 15 min at 14,000 × g. Next, 10 µL supernatant was diluted with 90 µL mobile phase (acetonitrile/0.1% formic acid [50:50, v/v]). This diluted solution was injected (10 µL) into the LC–MS/MS system, which was equipped with a turbo ion spray sample inlet as an interface for the electrospray ionization. The flow rate of the mobile phase was 0.2 mL/min, and chromatographic separation was conducted using a COSMOSIL HILIC Packed Column (2.0 × 150 mm, 5 μm; Nacalai Tesque, Inc., Kyoto, Japan) maintained at 50 °C. The mass spectrometer used a selected reaction monitoring method in positive ion mode, with 104.0→87.0 for GABA and 152.0→110.0 for acetamidophenol. The lower limit of quantification for GABA was < 0.01 µg/mL in the tissue homogenate samples.

Data and statistical analyses

Data are presented as the mean ± standard error of the mean. Statistical analyses were performed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). For comparisons, Student’s t-test or two-way analysis of variance (genotype versus rotarod trial or TRPM8 antagonist versus rotarod trial) with Bonferroni’s multiple comparison test were performed. P-values < 0.05 were considered significant.