Club-like Receptors Respond to Light Touch but not to Whisking

Club-like Receptors Respond to Light Touch but not to Whisking | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Club-like Receptors Respond to Light Touch but not to Whisking Taiga Muramoto, Takahiro Furuta, Taro Koike, Knarik Bagdasarian, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6369001/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Rodents acquire environmental information through their whiskers by employing active whisking and touch. Each whisker extends from a highly complex follicle containing hundreds of mechanoreceptors (MRs) of various types, positioned in different locations and surrounded by diverse tissue structures. Previous studies have shown that individual primary sensory afferents from these follicles respond selectively either to whisking (W) or touch (T), or non-selectively to both (WT). Given that MRs are sensitive to tiny, sub-micron deformations, the mechanisms enabling such selectivity were enigmatic. In this study, using artificial whisking, intra-axonal recordings and single-cell morphological analysis, we identify one class of MRs, the club-like endings, as T cells - all responding selectively to active touch and not to active whisking. The other MR types examined here, Merkel and lanceolate endings, exhibited various selectivity patterns. Club-like endings are arranged as a one-layer circular array around the whisker shaft near its center of mass, within the neck of a specialized structure called ringwulst (Rw). Using scanning electron microscopy (SEM), we found that individual club-like endings were directly attached to the glassy membrane covering the epithelial sheath attached to the whisker shaft, while being anchored to a specialized network of collagen fibers. This morphology minimizes deformations during whisking in air while allowing high sensitivity to whisker deformations induced by touch. We hypothesize that these specific morphological adaptations evolved to enable the detection of light touch, which is crucial for precise object localization during active whisking and show that cats, which do not use active whisking, lack several of these adaptations. Biological sciences/Neuroscience/Somatosensory system/Whisker system Biological sciences/Neuroscience/Sensory processing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Natural perception is primarily active – animals move and move their sensory organs as part of perceiving their environments. While offering significant advantages over passive sensing 1 , active sensing has computational costs. One of these costs is the need to differentiate changes occurring due to self-motion from those resulting from the interactions of the moving sensors with the environment 2 . One of the active-sensing modes that has been extensively studied is that of active vibrissal touch 3,4 . Active vibrissal sensing introduces a major mechanical challenge – allowing selective sensitivity to light touch. As the whiskers constantly move, such selectivity necessitates differentiating mechanical deformation induced by whisker-object touch from that induced by vigorous whisking in air. Since mechanoreceptors (MRs) have evolved to remarkable sensitivity (down to deformations of 10 nm) 5 , the major challenge lies in preventing MR activation during whisking in air. Nevertheless, evolution has risen to this challenge. Using artificial whisking (achieved by electrical stimulation of the facial nerve in anesthetized rats), it was found that individual primary afferents from these follicles respond selectively either to whisking (W) or touch (T), or non-selectively to both (WT) 6,7 . Thus, W cells are sensitive to whisking and insensitive to light touch (i.e., touch that does not block the movement of the follicle); T cells are insensitive to whisking in air and respond upon contact or detachment and WT cells are sensitive to both. Each whisker follicle contains hundreds of MRs, typically categorized into five types: Merkel, lanceolate, club-like, Ruffini-like and free nerve endings 8-11 . Recently, genetic marking has enabled the recording from primary afferents of specific MR types 12,13 . Additionally, intra-cellular or intra-axonal recording and labeling from MR neurons have allowed the associations of spatial selectivities of responses of single MR neurons with their precise receptor morphology and location within the follicle 14,15 . However, these methods have not yet been applied to study receptor selectivities during active sensing. In this study, we combined artificial whisking with intra-axonal recording and labeling methods to identify the type and location of individual MRs along with their functional selectivity. We found that club-like endings respond selectively to touch. Furthermore, we explored the morphological adaptations that enable this remarkable selectivity and found that the rat’s follicle possesses a unique mechanical design that maximizes sensitivity to whisker touch while minimizing sensitivity to whisking in air. Results We recorded 201 primary afferents from 104 anesthetized rats (males, ages 10 to 15 weeks old, weighting 250 to 350g, SLC, Hamamatsu, Japan) during artificial whisking in air and against objects. Artificial whisking was applied at 5 Hz during trains of 2 s (10 whisks), with inter-train intervals of 2 s (Fig. 1; see Methods). Intra-axonal recordings were done using glass electrodes filled with biotinylated dextran amine (5% BDA) or neurobiotin (20% NB) for labeling (see Methods). The neuronal tracers were injected intra-axonally to each of the recorded axons (Fig. 1). Stable recordings were difficult to obtain during artificial whisking and varied in duration. Of the 201 afferents, only 18 met the criteria of being recorded for at least one train of artificial whisking in air (active whisking), three trains against objects (active touch), and having fully reconstructed morphology (Fig. 2, Extended Data Fig.1). In total, one rete ridge collar (RRC)-Merkel ending, five club-like endings, six ring sinus (RS)-Merkel endings, and six lanceolate endings met these criteria and are reported here (Table 1). Each primary afferent was classified as either W, T or WT cell (Table 1, Extended Data Table 1) according to the difference between their responses to whisking against the object and to whisking in air. W cells responded equally to both (Fig. 2, cell No.14), T cells responded only upon touch (cell No.3 and 8) and WT cells responded differently to both (cell No.1 and 10). Quantitatively, their touch index (TI = (St-Sw)/(St+Sw), where St = spike count upon touch and Sw = spike count during whisking in air, see Methods) was previously found to be between [-0.2, 0.2] for W, larger than 0.8 for T, and otherwise for WT cells 16 . With our current intra-axonal recordings, all T cells showed TI = 1 (Table 1). The three cell types recorded here, Merkel, lanceolate and club-like endings, differed in the distributions of response types (Fig. 2, 3). Whereas Merkel and lanceolate endings were sensitive to both whisking and touch, club-like endings were sensitive only to touch (Table 1, Fig. 3). The stereotypic T response of the club-like endings suggests that they were selectively adapted through evolution to avoid responding to whisking in air. Given the remarkable sensitivity of mechanosensitive molecules to minute mechanical deformations (on the scale of 10 nm) 5 , resisting responses to whisking in air requires precise adaptation. Therefore, we examined the morphological adaptations of the club-like endings. The club-like endings are arranged as a one-layer ring around, and very close to, the whisker shaft, covering all shaft angles except for the dorso-caudal 45 degrees 14,17 . Every whisker is innervated by ~60 club-like endings, each of them is attached to a particular shaft angel (Table 1). The one-layer ring of the club-like endings is located near the center of mass of the whisker-follicle unit, which is moved during whisking in air by the moment generated by a pair of intrinsic muscles, one attached to the follicle near its bottom and one near its top; the center of mass is very close to the floating pivot of whisker rotation, a point that moves the least during whisking in air 18,19 . Thus, the location of club-like endings is the best for avoiding responding to whisking in air. Using semi-thin array tomography 20 , we show that, consistently with previous studies 14,15 , club-like endings possess short axon terminals. Each axon terminal is covered by terminal Schwann cell (TS) sheaths (Fig. 4, 5). Thin processes of the TS sheath extended upwardly as a long longitudinal process (Fig. 5g-j). The TS sheath covering the club-like endings were sandwiched between dense thin fibers connecting with the glassy membrane and another set of thin fibers connecting with thick collagen bundles ascending from the marginal zone of the Rw (Fig. 5e-f). This tight TS-collagen connection was observed only with club-like endings (Fig. 4), adding another factor isolating mechanosensitive channels, such as piezo II 12,13 , on these endings from movements that are common to the follicle and the whisker shaft encompassed by it. The third factor found to contribute to the decoupling of club-like endings from deformations during whisking in air was the structure of the Rw in the rat and its attachment to the glassy membrane surrounding the epithelial follicle. The Rw is hanging on a curved edge of the glassy membrane (Fig. 6). The mass of the Rw determines the amount of inertial damping of the movements during whisking in air – the larger the mass the smaller the movement at the neck of the Rw, where the club-like endings are located. To test whether the mass and specific morphology of the Rw and its attachment to the epithelial follicle are unique to whisking animals, we compared the anatomy of the Rw in rats and cats (Fig. 6). The differences are striking: in the cat, (i) the Rw lacks a dense array of thick collagen fiber bundles, (ii) the glassy membrane is not curved, (iii) there is no lumen between the glassy membrane and the Rw body, (iv) the neck of the Rw is also innervated by Merkel endings, which intermingle with club-like endings (Fig.6, Extended Data Fig.2) 21 (v) the club-like endings are unevenly arranged at the middle part of the Rw, not forming a one-layer ring. As a result, the Rw in cats provides less effective inertial resistance to whisking than in rats due to its smaller mass and different attachment to the glassy membrane. The absence of Merkel ending innervation in the rat is analogous to the absence of interfering structures—such as additional innervations and blood vessels—in the anatomical fovea of the primate retina. In both cases, this lack of interference suggests an adaptation optimized for high precision and sensitivity. Discussion Club-like endings form a highly specialized class of receptors in the whisker follicle. They are arranged in a unique structure – a single-layer ring around the whisker shaft, protected in a unique manner by Schwann cells, embraced by a unique collagen structure – the Rw 22 , innervated in a 1:1 manner – one axon per one ending – and exhibit the smallest sizes of receptive fields among all follicle receptors 10,11,14,15,17,23 . Here we revealed their unique role in the encoding of active touch - club-like receptors respond selectively to active contacts and ignore whisking in air. Selectivity to active contacts during whisking requires an extremely delicate mechanical selectivity. Piezo channels, responsible for detecting contacts in most mechanoreceptors, are sensitive to deformations as small as several nanometers 12,24 . Active whisking is a vigorous movement involving extrinsic and intrinsic muscles and rotating the follicles at high speeds and high accelerations 25 . We show here that evolutionary adaptation selected the best position for minimizing deformations during whisking in air – the whisker’s center of mass – for localizing the club-like endings. This may explain the single-layer ring structure, keeping all ~60 endings near the center of mass. Adding extra care of Schwann protection and inertial damping by the Rw likely complete the supreme isolation of club-like ending from whisking in air deformations. The circular arrangement of club-like endings raises an intriguing hypothesis: these endings may not only detect contact but also encode its azimuth relative to the snout. This potential azimuth coding is facilitated by the circular organization of club-like endings, which form a single-layer ring around the whisker shaft of about 60, nearly identical, endings. Consequently, the first ending to respond upon contact is likely the one experiencing the strongest deformation. Upon contact, the deformation induced by the bending moment is expected to be non-uniform around the shaft due to the uneven distribution of contact stress. This leads to a key coding principle: because whisker torsion (rotation around its own axis) 25,26 correlates with azimuthal protraction, the location of maximal deformation will vary with the azimuthal position of the contact. Thus, the identity of the first responding club-like ending encodes the azimuth of contact, with a resolution on the order of protraction amplitude divided by 60. Whether rodents utilize this form of coding remains to be tested. The precise 1:1 innervation pattern and the central localization of the club-like endings along the follicle resemble the specialized adaptation of the primate retina. The distinct sensitivities of peripheral and foveal cells in the primate retina optimize their ability to detect scene-related and object-related features, respectively. Peripheral vision is crucial for efficient search, guiding eye movements toward areas of interest. In turn, foveal vision provides high-resolution analysis of the area of interest 27 . Similarly, the unique characteristics of receptors distributed along the whisker follicle may be adapted to optimize sensing during whisking in air by non-club-like endings located far from the center of mass, and sensing precise contact timings by club-like endings that are concentrated near the center of mass. High-resolution selective sensitivity to touch necessitates evolutionary specializations that eliminate, or strongly dampen, mechanical deformations that can be induced during whisking in air. By comparing follicle morphology between non-whisking (cat) and whisking (rat) animals we identified five major differences that might represent such evolutional adaptation. Three of the differences increase the inertial resistance of the Rw, which contains the club-like endings ring in its neck, in the rat compared to the cat: increased mass, curving the glassy membrane and creating a lumen between the Rw body and the glassy membrane. The fourth difference, indicating the cleaning the Rw’s neck area from interfering structures in the rat, might be needed for coping with the ultimate challenge of high sensitivity together with high accuracy, similar to the cleaning of fovea centralis in the primate retina from interfering structures. The fifth difference, the formation of a one-layer ring in the rat, suggests an accurate metric coding by club-like endings. The specialization of club-like endings was substantially different from those of the other receptor types studied here: Merkel and lanceolate endings. Individual Merkel and lanceolate endings are typically 2 and 3-fold larger than club-like endings, respectively 11,17 . Their distribution in the follicle is broad and their surrounding tissues do not exhibit any highly specific morphological adaptations. Merkel endings are located within the basal layer of the epithelial sheath, an area free of connective tissue. This positioning enables them to detect deformations in their vicinity, whether these are caused by whisking or touch. Their large variability in receptor size, number of endings per cell, location relative to the whisker shaft and morphological relationships with their surrounding tissue 10,15 , allows substantial variability in response selectivity, as observed here. Lanceolate endings are like lancets suspended in the loose space between the mesenchyme and the glassy membrane, called the intermediary zone, supported by Schwann cell processes 10,15,23 . Their vertical hanging morphology suggests different sensitivities than those of Merkel endings. Yet, nothing in their surrounding tissue, or location, prevents them from responding to mechanical deformations induced by either whisking or object contacts. These comparisons suggest that while individual Merkel and lanceolate endings may exhibit selective sensitivities to whisking and touch, they were not selected as a group to address a specific sensory selectivity. In contrast, it seems that the special morphology of club-like endings and their surrounding tissue, including their one-layer ring arrangement around the center of mass of the whisker-follicle unit, was selected through evolutionary and developmental processes to address a specific need. This need is likely the detection of contact with external objects at a high confidence, i.e., at a high signal-to-noise ratio, and at a high temporal reliability. High temporal reliability is required for accurate object localization 3,25 , thus an evolutionary postdiction of this study is that the special arrangement of club-like endings in whisking rodents evolved along with their ability to localize external objects at high accuracy. Conclusive remarks This study shows, for the first time, morphological specialization that enables a specific functional selectivity in active touch. The challenge that this specialization solves is that of elimination of responses to whisking in air while allowing high sensitivity to contacts made during such whisking. The solution is localization of tiny receptor endings near the center of mass of the whisker-follicle unit, arranging them in a one-layer ring close to and around the shaft, protecting the receptor endings by a thin Schwann layer that is completely embedded within a dense network of thin collagen fibers and generating an inertial resistance by a specially-adapted collagen weight. Whether similar adaptations accompany the refinement of active sensing in other modalities remained to be discovered. Declarations Acknowledgements We thankfully appreciate to Dr. Sebastian Haidaliu, Dr. Eldad Assa, and Dr. Guy Nelinger (Weizmann Institute of Science, Israel), Dr. Mari Hirose, Dr. Tomokazu Murase (Meiji University of Integrative Medicine, Japan), Dr. Masaaki Kitada (Kansai Medical University, Japan), Dr. Yumi Tsutsumi (Hyogo Medical University, Japan), Dr. Fumihiko Sato (Osaka University, Japan) for scholar and professional discussions and excellent technical support. We also thank Keiko Okamoto-Furuta (Kyoto University, Japan) for helpful discussion in electron microscopic analysis, Biomedical Central Research Center (Kansai Medical University, Japan) for the use of an ultramicrotome, and Dr. Edward L. White (Ben-Gurion university, Israel) for a continuous generous support. This work was supported by KAKENHI (JP21H03529, JP23K21711 and JP22K19403 to T.F., and JP23K06311 and JP22H04926 “Advanced Bioimaging Support (ABiS)” to S.E.) from the Japan Society for the Promotion of Science (JSPS). This study was also supported by the Japan Agency for Medical Research and Development (AMED) (JP23dm0207112 to T.F.). Author contributions T. M. Designed the study, conducted the experiments, analyzed the data, generated the figures and wrote the paper. T. F. and A.T. developed the intra-axonal recording and labeling and contributed to writing. S. T. and K. B. established the intra-cellular recording & labeling with artificial whisking and contributed to writing. T. K., Y. K., M. M., A. E. and K. K. pursued electron microscopic studies and contributed to writing. E. A. Designed the study and wrote the paper. S. E. led and supervised the study, designed the study and wrote the paper. Competing interests All authors declare no competing interests. Author information 1 Department of Anatomy, Meiji University of Integrative Medicine, Nanatan, Kyoto, 629-0392, Japan 2 Department of Systematic Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, Suita, Osaka, 565-0871, Japan 3 Department of Anatomy, Kansai Medical University, Hirakata, Osaka, 573-1010, Japan 4 Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel 5 Department of Anatomy, Kawasaki Medical University, Kurashiki, Okayama, 701-0192, Japan 6 Graduate school of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, 650-0047, Japan 7 Laboratory for Chemical Biology, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, 650-0047, Japan 8 Japan Electron Optics Laboratory (JEOL) Ltd., Akishima, Tokyo, 196-8558, Japan References Gibson, J. Observations on active touch. Psychol Rev. 69 , 477-91 (1962). Ahissar, E. & Assa, E. Perception as a closed-loop convergence process. Elife. 5 , e12830 (2016). 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J Vis . 9 , 1-16 (2009) Methods Fourteen (14 of 104 rats were used this experiments) adults male Wistar rats (10 to 15 weeks old, weighting 250 to 350g, SLC, Hamamatsu, Japan) were used in this study. This study was carried out accordance with the Meiji university of Integrative Medicine Animal Care and Use Committee (#2016-004, 2019-002, 2022-001, 2023-006,009). Intra-axonal recording and labeling Under deep anesthesia (Isoflurane 1.5-2.0%, 0.5ml/sec), rats were fixed in a stereotaxic apparatus while breathing freely. Body temperature was maintained at 37-38℃ by a heating pad. Intra-axonal recording and labeling of primary afferents were performed using the methods described in previous studies 14,15 , here alongside facial nerve stimulation 6, 7 . In brief, a small opening on the skull was made to expose the cerebellar surface, then a glass pipet filled with neuronal tracer was inserted to explore a proper axon in the trigeminal tract. The skull opening was located around 3.5 mm posterior and 3.2 mm laterally from Lambda, and the electrode was lowered 5-7 mm from the cerebellar surface. Polished grass pipette (Kwik-fill, World precision Instruments, Florida, USA) filled with biotinylated dextran amine (5% BDA MW:3,000; Invitrogen, Eugene, OR), or neurobiotin (20% NB Vector Lab. Inc., Burlingame, CA USA) dissolved in 1M potassium acetate, were used (electrode resistance: 50-150 MΩ, tip diameter: 1-2 µm). Resting potential immediately decreased when the pipette was successfully entered to a single axon. Then the receptive field of the vibrissa was characterized using a bamboo stick. After that, artificial whisking was applied. Recording signals were amplified (IR-183, Cygnus Technology, USA) and A/D converter (Power Lab 8/30, AD Instruments, New Zealand, sampling rate: 2500 per second,) and an audio speaker, digital display, and computer with recording software (Chart 5, AD instruments) were used to monitor the responses. Recording data were analyzed using dedicated software (Spike 2, Cambridge Electronic Design). After completing electrophysiological recordings, neuronal tracer was electrophoretically injected into the single axon via the glass pipette (25nA-50nA, 2Hz, 15-20 min). Finally, the skin was closed by silk thread. Artificial whisking Artificial whisking was induced by stimulating the facial nerves, as described in detail previously 28 . Briefly, bipolar, rectangular electrical pulses (7 pulses, 2.0 V, 2 ms duration) were applied to the distal part of the facial nerve through an isolated pulse stimulator (PG4000A, CYGNUS, Dorset, UK, BSI-950, Dagan MN, USA) at 83 Hz. Trains of 4 s were employed, each composed of 2 s of facial nerve stimulation (at 5 Hz) followed by a quiescent interval of 2 s. Artificial whisking were performed in air and against an object. The object (vertical pole of 3 mm diameter) was placed rostrally to the whisker, distanced from the skin by the equivalent of 70-90% of the whisker’s length. Analysis of physiological data Physiological data were analyzed using raster plots, post stimulus time histograms (PSTHs) and computations of the Touch Index. Following Yu et al. (2006) 16 , we quantified the responses during their steady states, which were typically stable during the last 6 cycles in each train. To standardize the quantification across neurons, each having a different recording length, we quantified the last 6 cycles of the last 5 trains in each condition – total of 30 cycles per condition. For two cells (No.8 and 14), shorter stable recording times allowed only 18 cycles of the last 3 trains per condition. Touch Index (TI) was calculated as TI = (St-Sw)/(St+Sw), where St = spike count upon touch and Sw = spike count during whisking in air. St and Sw were quantified by calculating the firing rate during protraction, i.e., during the first 100 ms in each cycle. TI = 0 means that the neurons responded the same during whisking in air and whisking against object. TI = 1 means that the neurons only responded during whisking against object. TI = -1 means that the neuron was completely inhibited by the object. Following Yu et al 16 we classified Whisking cells as showing -0.2 <= TI < 0.2, Whisking / Touch cells as showing 0.2<= TI < 0.8 and Touch cells as showing 0.8<= TI <=1; none of our cells showed TI < -0.2. Histology Visualization of neuronal markers. After sufficient survival time, up to 20 hours for neurobiotin or 10 days for BDA, the animals were deeply anesthetized (Isoflurane, 7% Chloral Hydrate) and then perfused transcardially with saline (0.9%NaCl, room temperature) followed by a fixative solution of formalin (10-20%) with 0.1M sodium phosphate buffer (PB). Whisker pads were removed and then immersed in 30% sucrose in 0.1M PB. Specimens were made into frozen serial sections (100-140µm) by cryostat (Leica CM3050S). The maxillary nerve bundles were separated with forceps into tiny bundles. All serial sections were rinsed in 0.1M phosphate buffered saline containing 0.3% Triton-X100 (PBS-T) and then incubated in 3% hydrogen peroxide (H 2 O 2 ) overnight. The tissues were rinsed several times in PBS-T, and then they were immersed in a horseradish peroxidase conjugated avidin-biotin complex (ABC, Elite, 1:300, Vector Labs, USA) in PBS-T at 4℃ for 1 day. After that the tissues were rinsed several times in PBS-T and reacted in a solution of diaminobenzidine (0.02%, DAB) including 0.01% cobalt-nickel ammonium sulfate added 0.3% H 2 O 2 in 0.05M tris-HCl buffer (pH6.8). All serial sections were mounted on gelatin coated glass slides. Finally, tissues were enhanced by 0.05% osmium (OsO4), dehydrated by ethanol and coverslipped with mounting medium (Entellan New, Merch-Aldrich Japan, Tokyo, Japan). These samples were observed using a light microscope (i80, Nikon, Tokyo, Japan). High resolution digital photomicrographs were obtained with a DXM1200 camera (Nikon) using image analyzing software (NIS-Elements, BR, Nikon). (Extended Data Fig.1) Immunohistochemistry Whisker pads were horizontally processed into serial frozen thick sections as mentioned above. The follicles were processed immunohistochemically using rabbit collagen type I antibodies (1:1,000, Vector, CA, USA), Alexa Fluor 488 conjugated anti-rabbit IgG (1:300, Vector, USA) and pyridinium iodide for nuclei. The most perfectly stained samples were selected and submitted for observation by confocal scanning microscopy (Nikon-C1, Japan) (Fig.5 c, d). Scanning electron microscopic semi-thin array tomography Rats were deeply anesthetized and perfused with 0.1M PB followed by 4% formaldehyde (FA) and 0.05% glutaraldehyde (GA) in 0.1M PB. Whisker follicle was removed and immersed in 4% FA in 0.1M PB for 12 hours at 4℃. Whisker follicle was embedded in 3% agarose in 0.1M PB and sliced with a linear slicer (45 µm-thick, Neo-Linear Slicer AT, Dosaka EM, Kyoto, Japan). Agarose was removed and the sections were fixed with 2% FA and 2% GA in 0.1M PB for 10 minutes at room temperature followed by OsO 4 in 0.1M PB at 4℃, and then dehydrated with ascending concentrations of ethanol solution and embedded in epoxy resin. Serial semi-thin sections (250 nm thickness, Leica, Wetzlar Germany) were mounted on a piece of silicon wafer 29 . The sections were stained with 1% uranyl acetate for 15 minutes followed by Sato’s lead staining solution for 5 minutes 30 . The sections were observed by field-emission SEM using JSM-IT800 (JEOL, Tokyo, Japan) or SU8600 (HITACHI, Tokyo, Japan), were aligned the XY axes and segmented manually using dedicated software (Photoshop 2024, Adobe System Co., Ltd., Tokyo Japan). 3D reconstructions were made using a dedicated software (Dragonfly, Comet Technology Canada Inc, Canada) (Fig.5, Extended Data Fig. 2, Supplementary Video 1). References Semba, K., Egger, M.D. The facial "motor" nerve of the rat: control of vibrissal movement and examination of motor and sensory components. J Comp Neurol. 1986 May 8;247(2):144-58. doi: 10.1002/cne.902470203. PMID: 3722437. Koike, T., Kataoka, Y., Maeda, M., Hasebe, Y., Yamaguchi, Y., Suga, M., Saito, A., & Yamada, H. A device for ribbon collection for array tomography with scanning electron microscopy. Acta Histochem Cytochem . 50 , 135-140 (2017) Sato, T. A modified method for lead staining of thin sections. J Electron Microsc (Tokyo) . 17 , 158-159 (1968) Table Table 1 is available in the Supplementary Files section Additional Declarations There is NO Competing Interest. Supplementary Files Table1.docx SupplementaryVideo1Muramotoetal.mp4 Supplementary Video 1. | Reconstruction of adjacent two club-like endings and surrounding tissues. file name: Supplementary Video 1 Muramoto et al.mp4 ExtendedData.docx Cite Share Download PDF Status: Published Journal Publication published 24 Dec, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6369001","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":438540922,"identity":"44581442-7b85-4309-8c74-ff7cbc220815","order_by":0,"name":"Taiga Muramoto","email":"","orcid":"","institution":"Meiji University of Integrative Medicine","correspondingAuthor":false,"prefix":"","firstName":"Taiga","middleName":"","lastName":"Muramoto","suffix":""},{"id":438540923,"identity":"737277c3-0de0-4602-8347-7d2f99c1b517","order_by":1,"name":"Takahiro Furuta","email":"","orcid":"","institution":"Osaka 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11:40:26","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6369001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6369001/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-67514-w","type":"published","date":"2025-12-24T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80198032,"identity":"5fb9bdc4-967f-480b-9e3c-594dbefcfc47","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174361,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntra-axonal recording during artificial whisking.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eIntra-axonal recording and labeling. \u003cstrong\u003eb. \u003c/strong\u003eInduction of artificial whisking at 5 Hz. Each whisking cycle lasted 200 ms, where protraction was actively induced by facial nerve stimulation (7 pulses at 83 Hz) and retraction was passive. Each stimulation train lasted 4.0 s and consisted of 2.0 s of artificial whisking (10 cycles at 5 Hz) and 2.0 s pause.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/21cbaf4923cb5e9bf43ce4c0.png"},{"id":80198528,"identity":"5a25d450-4a6b-4db1-9344-eff3634d1597","added_by":"auto","created_at":"2025-04-09 06:17:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":783432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReceptor identification and intra-axonal responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eMorphological visualization mechanoreceptors, 5 examples shown: RRC-Merkel (No.1), club-like (No.3), RS-Merkel (No.8 and 10), and lanceolate (No.14) (see Table 1). Scale bar, 50 µm. Black dots, location of the ending(s) along the longitudinal axis of the follicle. Color cylinders or ring, the area where other endings of the same type are distributed. Black dot or elongated shape, azimuthal location of the mechanoreceptor ending(s). The object (gray square) was placed rostrally to the whisker. \u003cstrong\u003eb. \u003c/strong\u003eResponses of the neurons shown in \u003cstrong\u003ea \u003c/strong\u003eduring 10 whisking cycles. Right, their inferred response types.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/688503cc40b3ae867afa4813.png"},{"id":80198035,"identity":"1fc35621-56a6-40ae-93d7-85fcba531964","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":202406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResponse selectivities.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll neurons were identified morphologically. Line graphs show the single whisk PSTHs of all recorded neurons (n=18). Blue, Touch (T); yellow, Whisking/Touch (WT); red, Whisking (W). Cell No 1. innervated RRC-Merkel endings and was a WT cell (shading on the pie chart).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/801fda9ae058219226fda364.png"},{"id":80198037,"identity":"6b8b8bb8-4692-46d4-b2bf-414ee23e595a","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":191007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative morphological diagram of mechanoreceptors in the rat.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIndividual club-like endings are composed of a small flat axon terminal (green) sandwiched between the two terminal Schwann cell sheaths. The processes extend upward. Each club-like ending is enclosed completely by the collagen mass of the ringwulst (Rw), which in turn “floats” in the ring sinus (RS). Lanceolate endings are consisting thickened lancet-like axon terminals holed between sides by the terminal Schwann cell sheaths. They are distributed within the intermediary zone (IMZ), where collagen fibers are scarce. Several terminal Schwann cell processes (arrows) are bridging between the glassy membrane (GM) and the inner connective tissue sheath (ICS) supporting the endings. Merkel terminal disks are embedded in the basal layer of the epithelial sheath (ES).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/2c213b61394cd3982c4af683.png"},{"id":80198039,"identity":"c473cef4-5266-4ee6-bc68-1abe0de05f57","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1556561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEmbedding of club-like endings within surrounding tissue.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, b, e–j. \u003c/strong\u003eScanning electron microscopy (SEM) of serial semi-thin sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eThe top surface, outlined by a dashed gray square, represents a cut plane at the level of the inner conical body (ICB), a connective tissue structure forming the ceiling of the ring sinus (RS) (see corresponding dashed gray line in c). *, indicates the thick collagenous capsule. CS, cavernous sinus; VS, vibrissal shaft. \u0026nbsp;\u003cstrong\u003eb.\u003c/strong\u003e The follicle is sectioned along a plane indicated by the black dashed line in a (see also c). The C-shape of the ringwulst (Rw) is clearly visible. The C-shape opening is located dorsocaudally (arrowheads). Club-like endings are distributed along the medial edge of the Rw (highlighted by a blue dotted line).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec, d\u003c/strong\u003e. Immunohistochemical staining of collagen I (yellow) on a longitudinal frozen section. The Rw is filled with collagen I. Nuclei are counterstained in magenta with Pyridinium iodide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee.\u003c/strong\u003e A club-like ending (white arrow) abuts the terminal curve (open arrow) on the basal face of the GM. The ending is embedded within a dense network of fine collagen bundles (enclosed by the dashed line). Thick collagen bundles radiate from the core of the Rw outward, toward the RS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef, g. \u003c/strong\u003eAfter removal of the myelin sheath (S) at the terminal point (TP), the axon terminal (AT) of the club-like ending is enveloped by a terminal Schwann cell (TS). The TS process extends upward and is embedded in fine collagen fibers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eh. \u003c/strong\u003eSchematic diagram of the three-dimensional organization of two club-like endings (see Supplementary Video 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ei, j. \u003c/strong\u003e2D visualization of two cross sections marked in h.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/ae0b7da157e9894a2afd60e5.png"},{"id":80198036,"identity":"9b81c75c-3068-4096-b9e3-b5d65853fed5","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":579990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparative organization of club-like endings and the ringwulst (Rw) in rats and cats.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003ea,\u003c/strong\u003e In rats, the open ring sinus extends deep into the Rw (arrows), whereas in cats it does not. Club-like endings in rats are distributed along the terminal curve (open arrow) of the GM. In contrast, cats lack a terminal curve, and their club-like endings are irregularly positioned within the mid-region of the Rw. Thick collagen bundles are regularly arranged in rats, while finer, less organized fibers are observed in cats. VS, vibrissal shaft. ICS continues to *. \u003cbr\u003e\n \u003cstrong\u003eb,\u003c/strong\u003e Simplified comparative schematics. A star indicates the basal border of the GM. I, terminal curve (open arrow), present only in rats; II, core region of the Rw containing embedded endings; III, outer region of the Rw; IV, lumen allowing continuity between the Rw and the ring sinus in rats.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/5b4fb641d915bec201655bf9.png"},{"id":98984079,"identity":"3e398a96-df88-42fb-a0e8-8e78c0c663d9","added_by":"auto","created_at":"2025-12-25 08:05:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4907954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/6c1010d7-bc53-4e5c-8597-15bcc43d3c32.pdf"},{"id":80198034,"identity":"b679d15f-2ab8-4ba4-8ea7-1359b18316ad","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":115008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/97031d03faf7c227420b2bb5.docx"},{"id":80198041,"identity":"f133b494-de30-4040-a307-668642a6d72c","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10372714,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Video 1. | Reconstruction of adjacent two club-like endings and surrounding tissues.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003efile name: Supplementary Video 1 Muramoto et al.mp4\u003c/p\u003e","description":"","filename":"SupplementaryVideo1Muramotoetal.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/6a9233a8b72a9a687fb05102.mp4"},{"id":80198040,"identity":"9e5f49de-c7db-4ca0-9388-9fcb0d4ceb1f","added_by":"auto","created_at":"2025-04-09 06:09:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3720146,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedData.docx","url":"https://assets-eu.researchsquare.com/files/rs-6369001/v1/a01968c4e22cf63eba868b58.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Club-like Receptors Respond to Light Touch but not to Whisking","fulltext":[{"header":"Introduction","content":"\u003cp\u003eNatural perception is primarily active \u0026ndash; animals move and move their sensory organs as part of perceiving their environments. While offering significant advantages over passive sensing\u003csup\u003e1\u003c/sup\u003e, active sensing has computational costs. One of these costs is the need to differentiate changes occurring due to self-motion from those resulting from the interactions of the moving sensors with the environment\u003csup\u003e2\u003c/sup\u003e. One of the active-sensing modes that has been extensively studied is that of active vibrissal touch\u003csup\u003e3,4\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eActive vibrissal sensing introduces a major mechanical challenge \u0026ndash; allowing selective sensitivity to light touch. As the whiskers constantly move, such selectivity necessitates differentiating mechanical deformation induced by whisker-object touch from that induced by vigorous whisking in air. Since mechanoreceptors (MRs) have evolved to remarkable sensitivity (down to deformations of 10 nm)\u003csup\u003e5\u003c/sup\u003e, the major challenge lies in preventing MR activation during whisking in air. Nevertheless, evolution has risen to this challenge. Using artificial whisking (achieved by electrical stimulation of the facial nerve in anesthetized rats), it was found that individual primary afferents from these follicles respond selectively either to whisking (W) or touch (T), or non-selectively to both (WT)\u003csup\u003e6,7\u003c/sup\u003e. Thus, W cells are sensitive to whisking and insensitive to light touch (i.e., touch that does not block the movement of the follicle); T cells are insensitive to whisking in air and respond upon contact or detachment and WT cells are sensitive to both.\u003c/p\u003e\n\u003cp\u003eEach whisker follicle contains hundreds of MRs, typically categorized into five types: Merkel, lanceolate, club-like, Ruffini-like and free nerve endings\u003csup\u003e8-11\u003c/sup\u003e. \u0026nbsp;Recently, genetic marking has enabled the recording from primary afferents of specific MR types\u003csup\u003e12,13\u003c/sup\u003e. Additionally, intra-cellular or intra-axonal recording and labeling from MR neurons have allowed the associations of spatial selectivities of responses of single MR neurons with their precise receptor morphology and location within the follicle\u003csup\u003e14,15\u003c/sup\u003e. However, these methods have not yet been applied to study receptor selectivities during active sensing.\u003c/p\u003e\n\u003cp\u003eIn this study, we combined artificial whisking with intra-axonal recording and labeling methods to identify the type and location of individual MRs along with their functional selectivity. We found that club-like endings respond selectively to touch. Furthermore, we explored the morphological adaptations that enable this remarkable selectivity and found that the rat\u0026rsquo;s follicle possesses a unique mechanical design that maximizes sensitivity to whisker touch while minimizing sensitivity to whisking in air.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe recorded\u0026nbsp;201 primary afferents from 104 anesthetized rats (males, ages 10 to 15 weeks old, weighting 250 to 350g, SLC, Hamamatsu, Japan) during artificial whisking in air and against objects. Artificial whisking was applied at 5 Hz during trains of 2 s (10 whisks), with inter-train intervals of 2 s (Fig. 1; see Methods).\u0026nbsp;Intra-axonal recordings were done using\u0026nbsp;glass electrodes\u0026nbsp;filled with\u0026nbsp;biotinylated dextran amine\u0026nbsp;(5% BDA) or\u0026nbsp;neurobiotin\u0026nbsp;(20% NB) for labeling (see Methods).\u0026nbsp;The neuronal tracers\u0026nbsp;were\u0026nbsp;injected intra-axonally\u0026nbsp;to each of the recorded axons (Fig. 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStable recordings were difficult to obtain during artificial whisking and varied in duration. Of the 201 afferents, only 18 met the criteria of being recorded for at least one train of artificial whisking in air (active whisking), three trains against objects (active touch), and having fully reconstructed morphology (Fig. 2, Extended Data Fig.1). In total, one rete ridge collar (RRC)-Merkel ending, five club-like endings, six ring sinus (RS)-Merkel endings, and six lanceolate endings met these criteria and are reported here (Table 1).\u003c/p\u003e\n\u003cp\u003eEach primary afferent was classified as either W, T or WT cell (Table 1, Extended Data Table 1) according to the difference between their responses to whisking against the object and to whisking in air. W cells responded equally to both (Fig. 2, cell No.14), T cells responded only upon touch (cell No.3 and 8) and WT cells responded differently to both (cell No.1 and 10). Quantitatively, their touch index (TI = (St-Sw)/(St+Sw), where St = spike count upon touch and Sw = spike count during whisking in air, see Methods) was previously found to be between [-0.2, 0.2] for W, larger than 0.8 for T, and otherwise for WT cells\u003csup\u003e16\u003c/sup\u003e. With our current intra-axonal recordings, all T cells showed TI = 1 (Table 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe three cell types recorded here, Merkel, lanceolate and club-like endings, differed in the distributions of response types (Fig. 2, 3). Whereas Merkel and lanceolate endings were sensitive to both whisking and touch, club-like endings were sensitive only to touch (Table 1, Fig. 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe stereotypic T response of the club-like endings suggests that they were selectively adapted through evolution to avoid responding to whisking in air. Given the remarkable sensitivity of mechanosensitive molecules to minute mechanical deformations (on the scale of 10 nm)\u003csup\u003e5\u003c/sup\u003e, resisting responses to whisking in air requires precise adaptation. Therefore, we examined the morphological adaptations of the club-like endings.\u003c/p\u003e\n\u003cp\u003eThe club-like endings are arranged as a one-layer ring around, and very close to, the whisker shaft,\u0026nbsp;covering all shaft angles except for the dorso-caudal 45 degrees\u003csup\u003e14,17\u003c/sup\u003e. Every whisker is innervated by ~60 club-like endings, each of them is attached to a particular shaft angel\u0026nbsp;(Table 1). The one-layer ring of the club-like endings is located near the center of mass of the whisker-follicle unit, which is moved during whisking in air by the moment generated by a pair of intrinsic muscles, one attached to the follicle near its bottom and one near its top; the center of mass is very close to the floating pivot of whisker rotation, a point that moves the least during whisking in air\u003csup\u003e18,19\u003c/sup\u003e. Thus, the location of club-like endings is the best for avoiding responding to whisking in air.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing semi-thin array tomography\u003csup\u003e20\u003c/sup\u003e, we show that, consistently with previous studies\u003csup\u003e14,15\u003c/sup\u003e, club-like endings possess short axon terminals. Each axon terminal is covered by terminal Schwann cell (TS) sheaths (Fig. 4, 5). Thin processes of the TS sheath extended upwardly as a long longitudinal process (Fig. 5g-j). The TS sheath covering the club-like endings were sandwiched between dense thin fibers connecting with the glassy membrane and another set of thin fibers connecting with thick collagen bundles ascending from the marginal zone of the Rw (Fig. 5e-f). This tight TS-collagen connection was observed only with club-like endings (Fig. 4), adding another factor isolating mechanosensitive channels, such as piezo II\u003csup\u003e12,13\u003c/sup\u003e, on these endings from movements that are common to the follicle and the whisker shaft encompassed by it.\u003c/p\u003e\n\u003cp\u003eThe third factor found to contribute to the decoupling of club-like endings from deformations during whisking in air was the structure of the Rw in the rat and its attachment to the glassy membrane surrounding the epithelial follicle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Rw is hanging on a curved edge of the glassy membrane (Fig. 6). The mass of the Rw determines the amount of inertial damping of the movements during whisking in air \u0026ndash; the larger the mass the smaller the movement at the neck of the Rw, where the club-like endings are located.\u003c/p\u003e\n\u003cp\u003eTo test whether the mass and specific morphology of the Rw and its attachment to the epithelial follicle are unique to whisking animals, we compared the anatomy of the Rw in rats and cats (Fig. 6). The differences are striking: in the cat, (i) the Rw lacks a dense array of thick collagen fiber bundles, (ii) the glassy membrane is not curved, (iii) there is no lumen between the glassy membrane and the Rw body, (iv) the neck of the Rw is also innervated by Merkel endings, which intermingle with club-like endings (Fig.6, Extended Data Fig.2)\u003csup\u003e21\u003c/sup\u003e (v) the club-like endings are unevenly arranged at the middle part of the Rw, not forming a one-layer ring.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a result, the Rw in cats provides less effective inertial resistance to whisking than in rats due to its smaller mass and different attachment to the glassy membrane. The absence of Merkel ending innervation in the rat is analogous to the absence of interfering structures\u0026mdash;such as additional innervations and blood vessels\u0026mdash;in the anatomical fovea of the primate retina. In both cases, this lack of interference suggests an adaptation optimized for high precision and sensitivity.\u003c/p\u003e"},{"header":"Discussion ","content":"\u003cp\u003eClub-like endings form a highly specialized class of receptors in the whisker follicle. They are arranged in a unique structure – a single-layer ring around the whisker shaft, protected in a unique manner by Schwann cells, embraced by a unique collagen structure – the Rw\u003csup\u003e22\u003c/sup\u003e, innervated in a 1:1 manner – one axon per one ending – and exhibit the smallest sizes\u0026nbsp;of receptive fields\u0026nbsp;among all follicle receptors\u003csup\u003e10,11,14,15,17,23\u003c/sup\u003e. Here we revealed their unique role in the encoding of active touch - club-like receptors respond selectively to active contacts and ignore whisking in air.\u003c/p\u003e\n\u003cp\u003eSelectivity to active contacts during whisking requires an extremely delicate mechanical selectivity. Piezo channels, responsible for detecting contacts in most mechanoreceptors, are sensitive to deformations as small as several nanometers\u003csup\u003e12,24\u003c/sup\u003e. Active whisking is a vigorous movement involving extrinsic and intrinsic muscles and rotating the follicles at high speeds and high accelerations\u003csup\u003e25\u003c/sup\u003e. We show here that evolutionary adaptation selected the best position for minimizing deformations during whisking in air – the whisker’s center of mass – for localizing the club-like endings. This may explain the single-layer ring structure, keeping all ~60 endings near the center of mass. Adding extra care of Schwann protection and inertial damping by the Rw likely complete the supreme isolation of club-like ending from whisking in air deformations.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe circular arrangement of club-like endings raises an intriguing hypothesis: these endings may not only detect contact but also encode its azimuth relative to the snout. This potential azimuth coding is facilitated by the circular organization of club-like endings, which form a single-layer ring around the whisker shaft of about 60, nearly identical, endings. Consequently, the first ending to respond upon contact is likely the one experiencing the strongest deformation.\u003c/p\u003e\n\u003cp\u003eUpon contact, the deformation induced by the bending moment is expected to be non-uniform around the shaft due to the uneven distribution of contact stress. This leads to a key coding principle: because whisker torsion (rotation around its own axis)\u003csup\u003e25,26\u003c/sup\u003e correlates with azimuthal protraction, the location of maximal deformation will vary with the azimuthal position of the contact. Thus, the identity of the first responding club-like ending encodes the azimuth of contact, with a resolution on the order of protraction amplitude divided by 60. Whether rodents utilize this form of coding remains to be tested.\u003c/p\u003e\n\u003cp\u003eThe precise 1:1 innervation pattern and the central localization of the club-like endings along the follicle resemble the specialized adaptation of the primate retina. The distinct sensitivities of peripheral and foveal cells in the primate retina optimize their ability to detect scene-related and object-related features, respectively. Peripheral vision is crucial for efficient search, guiding eye movements toward areas of interest. In turn, foveal vision provides high-resolution analysis of the area of interest\u003csup\u003e27\u003c/sup\u003e. Similarly, the unique characteristics of receptors distributed along the whisker follicle may be adapted to optimize sensing during whisking in air by non-club-like endings located far from the center of mass, and sensing precise contact timings by club-like endings that are concentrated near the center of mass.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHigh-resolution selective sensitivity to touch necessitates evolutionary specializations that eliminate, or strongly dampen, mechanical deformations that can be induced during whisking in air. By comparing follicle morphology between non-whisking (cat) and whisking (rat) animals we identified five major differences that might represent such evolutional adaptation. Three of the differences increase the inertial resistance of the Rw, which contains the club-like endings ring in its neck, in the rat compared to the cat: increased mass, curving the glassy membrane and creating a lumen between the Rw body and the glassy membrane. The fourth difference, indicating the cleaning the Rw’s neck area from interfering structures in the rat, might be needed for coping with the ultimate challenge of high sensitivity together with high accuracy, similar to the cleaning of fovea centralis in the primate retina from interfering structures. The fifth difference, the formation of a one-layer ring in the rat, suggests an accurate metric coding by club-like endings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe specialization of club-like endings was substantially different from those of the other receptor types studied here: Merkel and lanceolate endings. Individual Merkel and lanceolate endings are typically 2 and 3-fold larger than club-like endings, respectively\u003csup\u003e11,17\u003c/sup\u003e.\u0026nbsp;Their distribution in the follicle is broad and their surrounding tissues do not exhibit any highly specific morphological adaptations.\u003c/p\u003e\n\u003cp\u003eMerkel endings are located within the basal layer of the epithelial sheath, an area free of connective tissue. This positioning enables them to detect deformations in their vicinity, whether these are caused by whisking or touch. Their large variability in receptor size, number of endings per cell, location relative to the whisker shaft and morphological relationships with their surrounding tissue\u003csup\u003e10,15\u003c/sup\u003e, allows substantial variability in response selectivity, as observed here. \u0026nbsp;Lanceolate endings are like lancets suspended in the loose space between the mesenchyme and the glassy membrane, called the intermediary zone, supported by Schwann cell processes\u003csup\u003e10,15,23\u003c/sup\u003e. Their vertical hanging morphology suggests different sensitivities than those of Merkel endings. Yet, nothing in their surrounding tissue, or location, prevents them from responding to mechanical deformations induced by either whisking or object contacts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese comparisons suggest that while individual Merkel and lanceolate endings may exhibit selective sensitivities to whisking and touch, they were not selected as a group to address a specific sensory selectivity. In contrast, it seems that the special morphology of club-like endings and their surrounding tissue, including their one-layer ring arrangement around the center of mass of the whisker-follicle unit, was selected through evolutionary and developmental processes to address a specific need. This need is likely the detection of contact with external objects at a high confidence, i.e., at a high signal-to-noise ratio, and at a high temporal reliability. High temporal reliability is required for accurate object localization\u003csup\u003e3,25\u003c/sup\u003e, thus an evolutionary postdiction of this study is that the special arrangement of club-like endings in whisking rodents evolved along with their ability to localize external objects at high accuracy.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusive remarks","content":"\u003cp\u003eThis study shows, for the first time, morphological specialization that enables a specific functional selectivity in active touch. The challenge that this specialization solves is that of elimination of responses to whisking in air while allowing high sensitivity to contacts made during such whisking. The solution is localization of tiny receptor endings near the center of mass of the whisker-follicle unit, arranging them in a one-layer ring close to and around the shaft, protecting the receptor endings by a thin Schwann layer that is completely embedded within a dense network of thin collagen fibers and generating an inertial resistance by a specially-adapted collagen weight. Whether similar adaptations accompany the refinement of active sensing in other modalities remained to be discovered. \u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thankfully appreciate to Dr. Sebastian Haidaliu, Dr. Eldad Assa, and Dr. Guy Nelinger (Weizmann Institute of Science, Israel), Dr. Mari Hirose, Dr. Tomokazu Murase (Meiji University of Integrative Medicine, Japan), Dr. Masaaki Kitada (Kansai Medical University, Japan),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDr. Yumi Tsutsumi (Hyogo Medical University, Japan), Dr. Fumihiko Sato (Osaka University, Japan) for scholar and professional discussions and excellent technical support. \u0026nbsp;We also thank \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKeiko Okamoto-Furuta (Kyoto University, Japan) for helpful discussion in electron microscopic analysis, Biomedical Central Research Center (Kansai Medical University, Japan) for the use of an ultramicrotome, and Dr. Edward L. White (Ben-Gurion university, Israel) for a continuous generous support. \u0026nbsp;This work was supported by KAKENHI (JP21H03529, JP23K21711 and JP22K19403 to T.F., and JP23K06311 and JP22H04926 “Advanced Bioimaging Support (ABiS)” to S.E.) from the Japan Society for the Promotion of Science (JSPS). This study was also supported by the Japan Agency for Medical Research and Development (AMED) (JP23dm0207112 to T.F.).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT. M. Designed the study, conducted the experiments, analyzed the data, generated the figures and wrote the paper.\u003c/p\u003e\n\u003cp\u003eT. F. and A.T. developed the intra-axonal recording and labeling and contributed to writing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS. T. and K. B. established the intra-cellular recording \u0026amp; labeling with artificial whisking and contributed to writing.\u003c/p\u003e\n\u003cp\u003eT. K., Y. K., M. M., A. E. and K. K. pursued electron microscopic studies and contributed to writing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eE. A. Designed the study and wrote the paper. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eS. E. led and supervised the study, designed the study and wrote the paper. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Department of Anatomy, Meiji University of Integrative Medicine, Nanatan, Kyoto, 629-0392, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003e Department of Systematic Anatomy and Neurobiology, Graduate School of Dentistry, Osaka University, Suita, Osaka, 565-0871, Japan\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Department of Anatomy, Kansai Medical University, Hirakata, Osaka, 573-1010, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e Department of Brain Sciences, Weizmann Institute of Science, Rehovot, 7610001, Israel\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003e Department of Anatomy, Kawasaki Medical University, Kurashiki, Okayama, 701-0192, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eGraduate school of Science, Technology and Innovation, Kobe University, Kobe, Hyogo, 650-0047, Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e7\u0026nbsp;\u003c/sup\u003eLaboratory for Chemical Biology, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Hyogo, 650-0047,\u0026nbsp;Japan\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eJapan Electron Optics Laboratory (JEOL) Ltd., Akishima, Tokyo, 196-8558,\u0026nbsp;Japan\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n \u003cli\u003eGibson, J. 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A Device for Ribbon Collection for Array Tomography with Scanning Electron Microscopy. \u003cem\u003eActa Histochem Cytochem\u003c/em\u003e. \u003cstrong\u003e50\u003c/strong\u003e, 135-140 (2017). \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eEbara, S., Kumamoto, K. \u0026amp; Rice, F.L. Confocal microscopic analysis of Merkel innervation in the cat mystacial vibrissa follicles. \u003cem\u003eThe Merkel Cell\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e Eds, Baumann, K.I., Halata, Z. \u0026amp; Moll, I. Springer, 137-141\u0026nbsp;(2003)\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eVincent, S.B. The tactile hair of the white rat. J. Comp. Neurol. \u003cstrong\u003e23\u003c/strong\u003e, 1-34 (1913).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTakahashi-Iwanaga, H. Three-dimensional microanatomy of longitudinal lanceolate endings in rat vibrissae. \u003cem\u003eJ. Comp. Neurol\u003c/em\u003e. \u003cstrong\u003e426\u003c/strong\u003e, 259-269 (2000).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHandler, A., Zhang, Q., Pang, S., Nguyen, T.M., Iskols, M., Nolan-Tamariz, M., Cattel, S., Plumb, R., Sanchez, B., Ashjian, K., Shotland, A., Brown, B., Kabeer, M., Turecek, J., DeLisle, M.M., Rankin, G., Xiang, W., Pavarino, EC., Africawala, N., Santiago, C., Lee, W.A., Xu, C.S. \u0026amp; Ginty, D.D. Three-dimensional reconstructions of mechanosensory end organs suggest a unifying mechanism underlying dynamic, light touch. \u003cem\u003eNeuron\u003c/em\u003e. \u003cstrong\u003e111\u003c/strong\u003e, 3211-3229 (2023).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eKnutsen, P.M. \u0026amp; Ahissar E. Orthogonal coding of object location. \u003cem\u003eTrends Neurosci.\u003c/em\u003e\u003cstrong\u003e32\u003c/strong\u003e, 101-109 (2009). \u0026nbsp;Erratum in: \u003cem\u003eTrends Neurosci\u003c/em\u003e. \u003cstrong\u003e32\u003c/strong\u003e, 187-188 (2009). \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eHaidarliu, S., Bagdasarian, K., Shinde, N. \u0026amp; Ahissar, E. Muscular basis of whisker torsion in mice and rats. \u003cem\u003eAnat Rec (Hoboken)\u003c/em\u003e.\u003cstrong\u003e\u0026nbsp;300\u003c/strong\u003e, 1643-1653 (2017).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLarson, A.M. \u0026amp; Loschky, L.C. The contributions of central versus peripheral vision to scene gist recognition. \u003cem\u003eJ Vis\u003c/em\u003e.\u003cstrong\u003e\u0026nbsp;9\u003c/strong\u003e, 1-16 (2009)\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003eFourteen (14 of 104 rats were used this experiments) adults male Wistar rats (10 to 15 weeks old, weighting 250 to 350g, SLC, Hamamatsu, Japan) were used in this study. This study was carried out accordance with the Meiji university of Integrative Medicine Animal Care and Use Committee (#2016-004, 2019-002, 2022-001, 2023-006,009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIntra-axonal recording and labeling\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder deep anesthesia (Isoflurane 1.5-2.0%, 0.5ml/sec), rats were fixed in a stereotaxic apparatus while breathing freely. Body temperature was maintained at 37-38℃ by a heating pad. Intra-axonal recording and labeling of primary afferents were performed using the methods described in previous studies\u003csup\u003e14,15\u003c/sup\u003e,\u0026nbsp;here alongside facial nerve stimulation\u003csup\u003e6, 7\u003c/sup\u003e. In brief, a small opening on the skull was made to expose the cerebellar surface, then a glass pipet filled with neuronal tracer was inserted to explore a proper axon in the trigeminal tract. The skull opening was located around 3.5 mm posterior and 3.2 mm laterally from Lambda, and the electrode was lowered 5-7 mm from the cerebellar surface. Polished grass pipette (Kwik-fill, World precision Instruments, Florida, USA) filled with biotinylated dextran amine (5% BDA MW:3,000; Invitrogen, Eugene, OR), or neurobiotin (20% NB Vector Lab. Inc., Burlingame, CA USA) dissolved in 1M potassium acetate, were used (electrode resistance: 50-150 M\u0026Omega;, tip diameter: 1-2 \u0026micro;m). Resting potential immediately decreased when the pipette was successfully entered to a single axon. Then the receptive field of the vibrissa was characterized using a bamboo stick. After that, artificial whisking was applied. \u0026nbsp;Recording signals were amplified (IR-183, Cygnus Technology, USA) and A/D converter (Power Lab 8/30, AD Instruments, New Zealand, sampling rate: 2500 per second,) and an audio speaker, digital display, and computer with recording software (Chart 5, AD instruments) were used to monitor the responses. Recording data were analyzed using dedicated software (Spike 2, Cambridge Electronic Design).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter completing electrophysiological recordings, neuronal tracer was electrophoretically injected into the single axon via the glass pipette (25nA-50nA, 2Hz, 15-20 min). Finally, the skin was closed by silk thread.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eArtificial whisking\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArtificial whisking was induced by stimulating the facial nerves, as described in detail previously\u003csup\u003e28\u003c/sup\u003e. Briefly, bipolar, rectangular electrical pulses (7 pulses, 2.0 V, 2 ms duration) were applied to the distal part of the facial nerve through an isolated pulse stimulator (PG4000A, CYGNUS, Dorset, UK, BSI-950, Dagan MN, USA) at 83 Hz. Trains of 4 s were employed, each composed of 2 s of facial nerve stimulation (at 5 Hz) followed by a quiescent interval of 2 s. Artificial whisking were performed in air and against an object. The object (vertical pole of 3 mm diameter) was placed rostrally to the whisker, distanced from the skin by the equivalent of 70-90% of the whisker\u0026rsquo;s length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis of physiological data\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysiological data were analyzed using raster plots, post stimulus time histograms (PSTHs) and computations of the Touch Index. Following\u0026nbsp;Yu et al. (2006)\u003csup\u003e\u0026nbsp;16\u003c/sup\u003e, we quantified the responses during their steady states, which were typically stable during the last 6 cycles\u0026nbsp;in each train.\u0026nbsp;To standardize the quantification across neurons, each having a different recording length, we quantified the last 6 cycles of the last 5 trains in each condition \u0026ndash; total of 30 cycles per condition. For two cells (No.8 and 14), shorter stable recording times allowed only 18 cycles of the last 3 trains per condition.\u003c/p\u003e\n\u003cp\u003eTouch Index (TI) was calculated as TI = (St-Sw)/(St+Sw), where St = spike count upon touch and Sw = spike count during whisking in air. St and Sw were quantified by calculating the firing rate during protraction, i.e., during the first 100 ms in each cycle. TI = 0 means that the neurons responded the same during whisking in air and whisking against object. TI = 1 means that the neurons only responded during whisking against object. TI = -1 means that the neuron was completely inhibited by the object. Following Yu et al\u003csup\u003e16\u003c/sup\u003e we classified Whisking cells as showing -0.2 \u0026lt;= TI \u0026lt; 0.2, Whisking / Touch cells as showing 0.2\u0026lt;= TI \u0026lt; 0.8 and Touch cells as showing 0.8\u0026lt;= TI \u0026lt;=1; none of our cells showed TI \u0026lt; -0.2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistology\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eVisualization of neuronal markers.\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter sufficient survival time, up to 20 hours for neurobiotin or 10 days for BDA, the animals were deeply anesthetized (Isoflurane, 7% Chloral Hydrate) and then perfused transcardially with saline (0.9%NaCl, room temperature) followed by a fixative solution of formalin (10-20%) with 0.1M sodium phosphate buffer (PB). Whisker pads were removed and then immersed in 30% sucrose in 0.1M PB. Specimens were made into frozen serial sections (100-140\u0026micro;m) by cryostat (Leica CM3050S). The maxillary nerve bundles were separated with forceps into tiny bundles.\u003c/p\u003e\n\u003cp\u003eAll serial sections were rinsed in 0.1M phosphate buffered saline containing 0.3% Triton-X100 (PBS-T) and then incubated in 3% hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) overnight. The tissues were rinsed several times in PBS-T, and then they were immersed in a horseradish peroxidase conjugated avidin-biotin complex (ABC, Elite, 1:300, Vector Labs, USA) in PBS-T at 4℃ for 1 day. After that the tissues were rinsed several times in PBS-T and reacted in a solution of diaminobenzidine (0.02%, DAB) including 0.01% cobalt-nickel ammonium sulfate added 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in 0.05M tris-HCl buffer (pH6.8). All serial sections were mounted on gelatin coated glass slides. Finally, tissues were enhanced by 0.05% osmium (OsO4), dehydrated by ethanol and coverslipped with mounting medium (Entellan New, Merch-Aldrich Japan, Tokyo, Japan).\u003c/p\u003e\n\u003cp\u003eThese samples were observed using a light microscope (i80, Nikon, Tokyo, Japan). High resolution digital photomicrographs were obtained with a DXM1200 camera (Nikon) using image analyzing software (NIS-Elements, BR, Nikon). (Extended Data Fig.1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Whisker pads were horizontally processed into serial frozen thick sections as mentioned above. \u0026nbsp;The follicles were processed immunohistochemically using rabbit collagen type I antibodies (1:1,000, Vector, CA, USA), Alexa Fluor 488 conjugated anti-rabbit IgG (1:300, Vector, USA) and pyridinium iodide for nuclei. The most perfectly stained samples were selected and submitted for observation by confocal scanning microscopy (Nikon-C1, Japan) (Fig.5 c, d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eScanning electron microscopic semi-thin array tomography\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRats were deeply anesthetized and perfused with 0.1M PB followed by 4% formaldehyde (FA) and 0.05% glutaraldehyde (GA) in 0.1M PB. Whisker follicle was removed and immersed in 4% FA in 0.1M PB for 12 hours at 4℃. Whisker follicle was embedded in 3% agarose in 0.1M PB and sliced with a linear slicer (45 \u0026micro;m-thick, Neo-Linear Slicer AT, Dosaka EM, Kyoto, Japan). Agarose was removed and the sections were fixed with 2% FA and 2% GA in 0.1M PB for 10 minutes at room temperature followed by OsO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003ein 0.1M PB at 4℃, and then dehydrated with ascending concentrations of ethanol solution and embedded in epoxy resin. Serial semi-thin sections (250 nm thickness, Leica, Wetzlar Germany) were mounted on a piece of silicon wafer\u003csup\u003e29\u003c/sup\u003e. The sections were stained with 1% uranyl acetate for 15 minutes followed by Sato\u0026rsquo;s lead staining solution for 5 minutes\u003csup\u003e30\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe sections were observed by field-emission SEM using JSM-IT800 (JEOL, Tokyo, Japan) or SU8600 (HITACHI, Tokyo, Japan), were aligned the XY axes and segmented manually using dedicated software (Photoshop 2024, Adobe System Co., Ltd., Tokyo Japan). 3D reconstructions were made using a dedicated software (Dragonfly, Comet Technology Canada Inc, Canada) (Fig.5, Extended Data Fig. 2, Supplementary Video 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReferences\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"28\"\u003e\n \u003cli\u003eSemba, K., Egger, M.D. The facial \u0026quot;motor\u0026quot; nerve of the rat: control of vibrissal movement and examination of motor and sensory components. J Comp Neurol. 1986 May 8;247(2):144-58. doi: 10.1002/cne.902470203. PMID: 3722437.\u003c/li\u003e\n \u003cli\u003eKoike, T., Kataoka, Y., Maeda, M., Hasebe, Y., Yamaguchi, Y., Suga, M., Saito, A.,\u003cbr\u003e\u0026amp; Yamada, H. A device for ribbon collection for array tomography with scanning electron microscopy. \u003cem\u003eActa Histochem Cytochem\u003c/em\u003e. \u003cstrong\u003e50\u003c/strong\u003e, 135-140 (2017)\u003c/li\u003e\n \u003cli\u003eSato, T. A modified method for lead staining of thin sections. \u003cem\u003eJ Electron Microsc (Tokyo)\u003c/em\u003e.\u003cstrong\u003e\u0026nbsp;17\u003c/strong\u003e, 158-159 (1968)\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6369001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6369001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRodents acquire environmental information through their whiskers by employing active whisking and touch. Each whisker extends from a highly complex follicle containing hundreds of mechanoreceptors (MRs) of various types, positioned in different locations and surrounded by diverse tissue structures. Previous studies have shown that individual primary sensory afferents from these follicles respond selectively either to whisking (W) or touch (T), or non-selectively to both (WT). Given that MRs are sensitive to tiny, sub-micron deformations, the mechanisms enabling such selectivity were enigmatic. In this study, using artificial whisking, intra-axonal recordings and single-cell morphological analysis, we identify one class of MRs, the club-like endings, as T cells - all responding selectively to active touch and not to active whisking. The other MR types examined here, Merkel and lanceolate endings, exhibited various selectivity patterns. \u0026nbsp;Club-like endings are arranged as a one-layer circular array around the whisker shaft near its center of mass, within the neck of a specialized structure called ringwulst (Rw). Using scanning electron microscopy (SEM), we found that individual club-like endings were directly attached to the glassy membrane covering the epithelial sheath attached to the whisker shaft, while being anchored to a specialized network of collagen fibers. This morphology minimizes deformations during whisking in air while allowing high sensitivity to whisker deformations induced by touch. We hypothesize that these specific morphological adaptations evolved to enable the detection of light touch, which is crucial for precise object localization during active whisking and show that cats, which do not use active whisking, lack several of these adaptations.\u003c/p\u003e","manuscriptTitle":"Club-like Receptors Respond to Light Touch but not to Whisking","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-09 06:09:23","doi":"10.21203/rs.3.rs-6369001/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a3b9ff21-0956-4772-ae46-77793c7f8a4a","owner":[],"postedDate":"April 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46700863,"name":"Biological sciences/Neuroscience/Somatosensory system/Whisker system"},{"id":46700864,"name":"Biological sciences/Neuroscience/Sensory processing"}],"tags":[],"updatedAt":"2025-12-25T08:05:21+00:00","versionOfRecord":{"articleIdentity":"rs-6369001","link":"https://doi.org/10.1038/s41467-025-67514-w","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-12-24 05:00:00","publishedOnDateReadable":"December 24th, 2025"},"versionCreatedAt":"2025-04-09 06:09:23","video":"","vorDoi":"10.1038/s41467-025-67514-w","vorDoiUrl":"https://doi.org/10.1038/s41467-025-67514-w","workflowStages":[]},"version":"v1","identity":"rs-6369001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6369001","identity":"rs-6369001","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}