Club-like receptors respond to light touch but not to whisking

Domain: physics
Published: 2025-12-24
Editorial outcome: minor_revision

Abstract
Rodents explore their environment by actively whisking and contacting objects with their whiskers. Each whisker follicle contains hundreds of mechanoreceptors, but how specific afferents distinguish between self-motion and touch remains unclear. Here, using artificial whisking in male rats, intra-axonal recordings, and morphological reconstruction, we identify a distinct mechanoreceptor subtype—club-like endings—that respond exclusively to touch and remain silent during whisking. In contrast, Merkel and lanceolate endings exhibit mixed selectivity. Club-like endings are arranged in a single-layer circular array near the center of mass of the whisker-follicle unit, embedded in a collagen-rich structure called the ring wulst. Using scanning electron microscopy, we show that these endings are tightly anchored to the glassy membrane and collagen fibers, forming a mechanically isolated zone. This configuration minimizes activation during whisking while preserving sensitivity to touch. We propose that these features evolved to enhance tactile precision in whisking species, as supported by the absence of such specializations in non-whisking animals such as cats.

Introduction
Natural perception is primarily active–animals control their sensory organs as part of perceiving their environments. While offering significant advantages over passive sensing1, 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 environment2. One of the active-sensing modes that has been extensively studied is that of active vibrissal touch3,4. Recent work has emphasized the dual modes of vibrissal information acquisition: generative, where the animal sweeps its whiskers forward and backwards, and receptive, where the animal keeps its whiskers still, and collects mechanical signals from the relative motion between the head and an object5.

Generative 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 10nm)6, 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)7,8. 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.

During generative sensing, the mechanical activation of the whisker follicle is shaped by a combination of kinematic and force-related variables—such as whisker angle, velocity, and bending moment—making the selective coding of specific stimulus features challenging9–11. Each whisker follicle contains hundreds of MRs, typically categorized into five types: Merkel, lanceolate, club-like, Ruffini-like, and free nerve endings12–15. Recently, genetic marking has enabled the recording from primary afferents of specific MR types16,17. Recordings from identified Merkel afferents during self-generated whisking in awake mice showed that Merkel receptors encode both whisking and touch, with some afferents responding predominantly to touch, some to whisking motion, and other to both17. In parallel, intracellular 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 follicle18,19. 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 minimizes sensitivity to whisking in air.

Results
Selectivity of the mechanoreceptors during artificial whisking
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 5Hz during trains of 2s (10 whisks), with inter-train intervals of 2s (Fig. 1b; 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. 1a).

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. 2a, Supplementary Fig. 1). In total, one reteridge 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).

In addition to identifying their type and location within the follicle (Fig. 2a), each primary afferent was classified as either a W, T or WT cell (Table 1) according to the difference between its response to whisking against an object and to whisking in air. W cells responded equally to both (Fig. 2b, cell No.14), T cells responded only upon touch (Fig. 2b, cell No. 3 and 8) and WT cells responded differently to both (Fig. 2b, cell No. 1 and 10). Note that (Fig. 2b) cell No. 3 responded to the onset of contact, “Contact T cell”7, whereas cell No.8 responded to detachment, “Detach T cell”7. Herein we do not distinguish between different subtypes of T cells. 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 cells20. With our current intra-axonal recordings, almost all T cells showed TI=1 (Table 1).

The three cell types recorded here, Merkel, lanceolate, and club-like endings, differed in both the distributions of their locations within the follicle (Fig. 2a) and the distributions of their response types (Figs. 2b, 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).

Morphological adaptations of the club-like endings
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 10nm)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 degrees18,21. Every whisker is innervated by ~60 club-like endings, each of them is attached to a particular shaft angle (Supplemntary Fig. 1b.). 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 air22,23. Thus, the location of club-like endings is the best for avoiding responding to whisking in air.

Each axon terminal is covered by terminal Schwann cell (TS) sheaths (Fig. 4). Using semi-thin array tomography24, we show that, consistently with previous studies18,19, club-like endings possess short axon terminals embedded within a dense network of fine collagen fibers occupying core region of the C-shaped Rw (Figs. 5a–e, f, g, i, 6, Supplementary Movie 1). Thin processes of the TS sheath extended upwardly as a long longitudinal process (Fig. 5f, g, i–k). 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–h). The bundles were collagen I-immunopositive and showed typical banding pattern (Fig. 5d, h, i). This tight TS-collagen connection was observed only with club-like endings (Fig. 4), adding another factor isolating mechanosensitive channels, such as piezo II16,17, on these endings from movements that are common to the follicle and the whisker shaft encompassed by it.

The third factor found to potentially contribute to the decoupling of club-like endings from tissue 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 suspended from a curved edge of the glassy membrane (Fig. 6). The mass of the Rw determines the extent 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, should be. This damping effect is analogous to that achieved using pendulum-type mass dampers in engineering applications, such as those used to suppress vibrations in tall buildings25. The operating principle involves suspending a mass (forming a pendulum) within the structure, allowing it to swing freely. When the structure undergoes lateral motion, the pendulum oscillates out of phase with the movement, thereby exerting an inertial counter force that reduces the amplitude of the structural vibrations (see Supplementary Movie 2).

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, Supplementary Fig. 2)26 (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.

Methods
Animals
Adult male Wistar rats (10 to 15 weeks old, weighting 250 to 350g, SLC, Hamamatsu, Japan) were used in this study. Cat samples obtained in previous studies14,26, using the same fixation methods as those described herein, were reprocessed and analyzed. The study was carried out in 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%, 0.5 L/min), rats were fixed in a stereotaxic apparatus while breathing freely. Body temperature was maintained at 37–38°C by a heating pad. Intra-axonal recording and labeling of primary afferents were performed using the methods described in previous studies18,19, here alongside facial nerve stimulation7,8. 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 3.5mm posterior and 3.2mm laterally from Lambda, and the electrode was lowered 5–7mm from the cerebellar surface. Polished glass pipette (Kwik-fill, World precision Instruments, Florida, USA) filled with biotinylated dextran amine (5% BDA MW: 3000; Invitrogen, Eugene, OR), or neurobiotin (20% NB Vector Lab. Inc., Burlingame, CA USA) dissolved in 1M potassium acetate, were used (electrode resistance: 50–150MΩ, tip diameter: 1–2µm). 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. Recorded signals included low-frequency components (<20Hz), likely reflecting physiological activity such as cardiac and respiratory rhythms, and high-frequency components, primarily attributed to neural electrical activity. Recorded data were analyzed using dedicated software (Spike2, Cambridge Electronic Design).

After completing electrophysiological recordings, neuronal tracer was electrophoretically injected into the single axon via the glass pipette (25–50nA, 2Hz, 15–20min). Finally, the skin was closed by silk thread.

Artificial whisking
Artificial whisking was induced by stimulating the facial nerves, as described in detail previously37. Briefly, bipolar, rectangular electrical pulses (7 pulses, 2.0 V, 2ms 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 83Hz. Trains of 4s were employed, each composed of 2s of facial nerve stimulation (at 5Hz) followed by a quiescent interval of 2s. Artificial whisking were performed in air and against an object. The object (vertical pole of 3mm 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.20, we quantified the responses during their steady states, which were typically stable during the last 6 cycles in each train. 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 100ms 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.20, 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.

Classification reliability was evaluated by repeating the classification process 10 times, each time with only one train in air and one train against an object, randomly selected. Classification reliability (Table 1) was defined as the fraction of identical classifications among the 10 repetitions.

Visualization of neuronal markers
After sufficient survival time, up to 20h 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 (H2O2) 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°C 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% H2O2 in 0.05M tris-HCl buffer (pH 6.8). 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). (Supplementary Fig. 1)

Immunohistochemistry
Whisker pads were horizontally processed into serial frozen thick sections as mentioned above. The follicles were processed immunohistochemically using goat collagen type II IgG antibodies (1:200, Arigo, Taiwan, ROC), Alexa Fluor 488 conjugated anti-goat 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. 5c, 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 12h at 4°C. Whisker follicle was embedded in 3% agarose in 0.1M PB and sliced with a linear slicer (45µm-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 10min at room temperature followed by OsO4 in 0.1M PB at 4°C, and then dehydrated with ascending concentrations of ethanol solution and embedded in epoxy resin. Serial semi-thin sections (250nm thickness, Leica, Wetzlar Germany) were mounted on a piece of silicon wafer24. The sections were stained with 1% uranyl acetate for 15min followed by Sato’s lead staining solution for 5min38.

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, Supplementary Fig. 2, Supplementary Movie 1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In their paper ‘Club-like Receptors Respond to Light Touch but not to Whisking‘ Muranoto et al. investigate structure function relationships of vibrissa afferents. Specifically, the authors apply intra-axonal recordings combined with neurobiotin-labeling, electron-microscopy and electric whisking paradigms, an astounding range of analysis technologies.\nThe authors identify a range of afferents including significant numbers of vibrissal Merkel-receptors, laneolate-endings and club-like endings. The highlight of the study is the author’s conclusion that club-like endings are so-called touch cells, i.e. cells that do not respond to whisking alone but are responsive to object contact.\nThe authors also do beautiful electron-microscopy work and reveal novel details about the anchoring of club-like endings, which they show to be attached to the glassy membrane and also being anchored by collagen fibers. On grounds of these morphological reconstructions and by based on additional comparative evidence (comparisons to non-whisking cats) the authors argue that the ringwulst might actually contribute to dampening whisking related responses in club-like afferents. I think the whisking-response-dampening by the ringwulst is an interesting hypothesis, but the authors present it as a fact, which I think is putting this idea too strongly.\nMajor Points\n1. This is an important paper. The authors are pioneers in understanding the vibrissa follicle and whisker response properties. The intra-axonal recordings performed here in whisking animals are nontrivial, but absolutely decisive for understanding the vibrissal system. Classifying club-like endings as touch-cells is an important advance that fully justifies publication.\n2. What do the authors think is origin of the prominent subthreshold activity seen in many afferents (Figure 2)? I am asking, because the authors penetrate cells a centimeter or more away from the mechanosensory tip of the afferents and I expected that we should see little subthreshold signal as a result of axonal filtering.\n3. I think the ms will benefit if the results followed a bit more tightly the Figures and individual panels. In general, the paper is well written, but the detailed link to the Figures would often be advantageous.\n4. As I said earlier the whisking-response-dampening by the ringwulst is an interesting hypothesis. It would be great is the authors could explain thinking in more detail, perhaps adding a schematic as to how the ringwulst is supposed to do this trick. Also, I am mot sure the idea is correct and the authors need to present this more cautiously.\n5. I enjoyed the careful electron-microscopy.\nMinor\n1. Make sure all abbreviations are explained in all legends; I could not find IMZ.\n2. Figure 5 C, D please show higher magnification of the immunofluorescence.", "reviewer_2": "This study is commendable for tackling a fundamental and unresolved question in sensory neuroscience: the structure-function relationships in low-threshold mechanoreceptors (LTMRs). The authors employ a technically demanding approach — intra-axonal recordings — to address this issue, and they present valuable data on whisker LTMR responses during artificial whisking, along with anatomical insights.\nHowever, I have concerns regarding the sample size and the generalizability of the findings to natural whisking behavior.\nMajor Comments\nLine 94: Were the recordings of sufficient duration to yield robust estimates of the response index for each afferent? Depending on the number of spikes in each recording, the classification of an afferent could potentially shift based on the presence or absence of few spikes. Please provide more specific information on the duration of each recording (in seconds) and the number of spikes recorded. Including this information in Table 1 for each afferent would be helpful.\nLine 143: The sample sizes are quite limited (N = 5–6 for each of the three main LTMR types). The conclusion that club endings differ from Merkel and lanceolate endings in their touch versus whisking responsivity hinges on a small number of cells with W/WT properties. This makes the reliability of the classification (as noted above) particularly critical.\nThe whisking responses in this study are elicited via electrical stimulation of the whisking muscles. While this method effectively mimics the bending forces associated with whisker-object contact, it only partially replicates the forces present during natural whisking. Thus, while the evidence for touch responses in club-like endings is convincing, the conclusion that these endings do not respond to self-motion during natural whisking is less so. Prior work (e.g., Severson et al., 2017) has shown that most afferents respond to self-motion during natural whisking. If club-like endings also respond to self-motion, this would challenge the rationale for seeking an anatomical basis for selective touch responses in the latter half of the manuscript. While intra-axonal recordings in awake, head-fixed animals may not be feasible, a paragraph acknowledging this limitation should be added to the Discussion.\nMinor Comments\nThe Introduction focuses exclusively on artificial whisking. Studies examining mechanoreceptor function during natural whisking should be explicitly discussed in both the Introduction and Discussion.\nLine 457: If a W cell is defined as having abs(TI) < 0.2, should the range for a WT cell not be –0.2 to 0.8?\nFigure 3: The PSTHs are missing time scales.\nLine 137: The latency difference (~100 ms) between the two example T cells is surprisingly large. Could the authors clarify how this is possible?", "reviewer_3": "This is an exciting study which brings to bear the most elegant methodologies of morphological anatomy and electrophysiology into a sensory paradigm that is, as much as can be achieved given the goals, a naturalistic and behaviorally relevant one – whisking and touching. The authors have identified the termination structure of tactile afferents from the rat whisker follicle, confirming the functional properties of previously identified classes and, beyond that, working out a class of receptor until now seen anatomically but never functionally documented. This latter class are Club-like Receptors and, as stated straight off in the title, these respond to light touch but not to whisking. Though the number that could be studied was limited to 5 due to the delicateness of the experimental setup, those sampled receptors appear to stand out qualitatively as having a distinct functional repertoire. Finally, the authors examine the physical substrate in which the receptors are embedded and provide a convincing account for how this substrate might isolate them from the mechanical inputs of whisking while leaving them exquisitely sensitive to the mechanical inputs that occur when the moving whisker collides with an object.\nThe text is clearly written and illustrated.\nOverall, this work will be a significant contribution to sensory neuroscience.\nWhile this reviewer has no criticisms of methodologies or results, I believe that the findings would be strengthened by including somewhat broader perspectives on some points.\nFirst, with regard to the behavioral role of whisking, the authors have broken down behavior into active versus passive: “Natural perception is primarily active – animals move and move their sensory organs as part of perceiving their environments.” This is somewhat limiting and perhaps not entirely accurate. Just as humans carry out many tactile judgments by holding the fingertips immobile – thus for instance the potter lightly touches their hands against the rotating clay to assess its consistency, moisture, and symmetry, using tactile feedback from the moving surface. Rubbing ones hands against the clay is not the optimal sensorimotor strategy under some conditions. Likewise, among freely moving rodents the whisking mode of perception is observed more frequently, but this is because experimentalists less often create conditions in which tactile information can be acquired by the immobile whiskers. Recent work has emphasized the dual modes of vibrissal information acquisition: generative, where the animal sweeps its whiskers forward and backwards to palpate objects, and receptive, where the animal keeps its whiskers still, and collects mechanical signals from the motion of an object such as a vibrating surface (Diamond & Arabzadeh, 2013; Diamond & Toso, 2023; Fassihi et al., 2014). The dichotomy generative vs receptive is far preferable to active vs passive because (1) holding receptor surfaces stable (finger tips or whiskers) is hardly a passive task from the neuromuscular point of view, but is actually quite complex, and (2) the sensory nervous system is not “passive” during receptive sensing, but rather is highly engaged in optimizing information acquisition and likely is actively forming predictive models. In my view, the present study would be better presented in the generative/receptive framework, not the active/passive framework, also discussing the relevant work.\nSecond, as the authors well know, the whisking cycle in freely moving rats relies on the coordinated action of intrinsic and extrinsic muscles within the mystacial pad (Berg & Kleinfeld, 2003; Hill et al., 2011). Intrinsic muscles, located entirely within the pad, insert on individual vibrissae and are primarily responsible for generating protraction movements of the whiskers. Extrinsic muscles, such as the nasolabialis and maxillolabialis, originate outside the pad and provide broader support, contributing to retraction of whisker positioning. The interplay between these two muscle groups ensures precise control of vibrissa motion, enabling rats to generatively sample their tactile environment during exploration. For reasons that are not entirely clear (at least to me), electrical whisking produces only protraction (Arabzadeh et al., 2005). As noted in the present manuscript, “retraction was passive.” The implication is that, because the electrical whisking protocol did not produce a full, natural protraction AND RETRACTION, authors should be more circumspect in designating the Club-like Receptors as being receptive only to touch. It is possible, even if not likely, that the club receptors could fire during whisker retraction if that retraction were active rather than passive. The receptors might be sensitive to extrinsic muscle contraction.", "reviewer_4": "Muramoto and colleagues describe a specific class of mechanoreceptors that responds to touch, but not whisking in air, by recording from their axons, characterizing their responses, and reconstructing their morphology.\nThe study is important for the field of vibrissa biophysics and has implications for understanding the low-level coding of whisker touch coding in rodents. The findings seem to be well supported by the data, and the presentation is clear.\nMy only requests concern slight clarifications and discussion points and do no warrant another round of review.\nThe argument that the club-lice receptors avoid activation during whisking in air due to their location is convincing, but this logic does not immediately explain how they still respond to whisker touch, which should, at least in one major component, contain torque along the whisker shaft that acts on a similar point of rotation in the follicle as the momentum and air resistance during whisking in air. Are the club-like receptor expected to predominantly activate to axial whisker deflections, lateral non-bending loads, or high-frequency vibrations or stick-clip events maybe? It would be interesting to at least add a sentence or two discussing this.\nRelated to this, a bit more information about the nature of the object contacts in the main text would be useful to readers to make it easier to interpret the findings. For example, what kind of whisker deflections were observed, at what speeds, etc.\nAll in all, this is a solid contribution to the literature and is improving our understanding of the vibrissa system."}, "author_response": "Dear Reviewers,\nWe are deeply grateful for the fair and constructive comments and suggestions that have helped us improve our manuscript.\nBelow are the Reviewer’s comments, in black, and our replies, in blue, starting with “>>>Reply:”. Please note that all line numbers refer to the revised manuscript when viewed in “All Markup” mode (PDF).\n[... Author responses to all reviewers ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed my concerns. This is an outstanding paper. I support publication.", "reviewer_2": "Concerns about sample size and reliability addressed through an elegant and convincing analysis.", "reviewer_3": "In my first-round review I pointed out the merits fo this study so I omit that form of summary in the second-round review.\nAll of the queries raised by me and by the other reviewers (who I congratulate for the preciseness of their observations) have been addressed to satisfaction.", "reviewer_4": "The revised manuscript addresses my comments about clarifying the degree to which the results an be used to infer a mechanism for how touch but not whisking selective responses could arise and how far the findings can be expected to generalize. The revised version also addresses related comments made by other reviewers. In my opinion this can be published as is."}, "author_response": ">>>Reply: Thank you very much. We greatly appreciate this constructive review."}]

Decision letter