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Nav1.8-positive afferent fibers are mostly nociceptors playing a role in mediating thermal and mechanical pain, but mechanoreceptors within these afferents have not been fully investigated. In this study, we generated mice ChR2 expressing channel rhodopsin 2 (ChR2) in Nav1.8-positive afferents (Nav1.8 ), which showed avoidance responses to mechanical stimulation and nocifensive responses to blue light stimulation applied to hindpaws. Using ex vivo hindpaw skin-tibial nerve preparations made from these mice, we characterized properties of mechanoreceptors on ChR2 ChR2 Nav1.8 -positive and Nav1.8 -negative afferent fibers that innervate the hindpaw glabrous skin. Of all Aβ-fiber ChR2 mechanoreceptors, small portion was Nav1.8 -positive. Of all Aδ-fiber mechanoreceptors, more than half was ChR2 ChR2 ChR2 Nav1.8 -positive. Of all C-fiber mechanoreceptors, almost all were Nav1.8 -positive. Most Nav1.8 -positive Aβ-, Aδ-, and C-fiber mechanoreceptors displayed slowly adapting (SA) impulses in response to sustained mechanical stimulation, and their mechanical thresholds were high in the range of high threshold mechanoreceptors (HTMRs). ChR2 In contrast, sustained mechanical stimulation applied to Nav1.8 -negative Aβ- and Aδ-fiber mechanoreceptors evoked both SA and rapidly adapting (RA) impulses, and their mechanical thresholds were in the range of low thresh- ChR2 old mechanoreceptors (LTMRs). Our results provide direct evidence that in the mouse glabrous skin, most Nav1.8 - ChR2 negative Aβ-, Aδ-fiber mechanoreceptors are LTMRs involving in the sense of touch, whereas Nav1.8 -positive Aβ-, Aδ-, and C-fiber mechanoreceptors are mainly HTMRs involving in mechanical pain. Keywords Nav1.8, Optogenetics, Opto-tagged single-fiber recording, High threshold mechanoreceptors (HTMRs), Low threshold mechanoreceptors (LTMRs), Touch, Mechanical pain, Hindpaw glabrous skin Introduction Mechanical stimuli, such as a gentle touch or a strong Akihiro Yamada and Ayaka I. Yamada equal contribution and co-first author pinch to the skin, activate mechanoreceptors to sig- *Correspondence: nal the sense of touch or mechanical pain, respec- Jianguo G. Gu tively. Mechanoreceptors are classified into several firstname.lastname@example.org Department of Anesthesiology and Perioperative Medicine, School subtypes based on their distinctive mechanical thresh- of Medicine, University of Alabama at Birmingham, Birmingham, AL olds (LTMRs, HTMRs), afferent fiber conduction veloci - 35294, USA 2 ties (Aβ-, Aδ-, and C-fibers), and impulse adaptation Department of Neurophysiology, Hyogo Medical University, Nishinomiya 663-8501, Japan types (slowly adapting, SA; rapidly adapting, RA) to sus- Department of Neuroscience, Perelman School of Medicine, University tained mechanical stimulation . A gentle touch is of Pennsylvania, Philadelphia, PA 19104, USA transduced by low threshold mechanoreceptors (LTMRs) © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://creativecom- mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yamada et al. Molecular Brain (2023) 16:27 Page 2 of 15 consisting terminals of non-nociceptive afferents, label afferent fibers that appear to give rise to Aδ-fiber whereas a strong pinch activates high threshold mecha- HTMRs and Aβ-fiber HTMRs . However, the prop - noreceptors (HTMRs) on nociceptive afferent endings erties of putative Aδ-fiber HTMRs and Aβ-fiber HTMRs [1, 11, 27]. In mouse glabrous skin, there are at least two in these transgenic mice have not been characterized main types of LTMRs, Aβ-fiber slowly adapting type 1 electrophysiologically. LTMRs (Aβ-fiber SA1-LTMRs) that are consist of Mer - These transgenic Cre mice can also be used to express kel cell-neurite complex, and Aβ-fiber rapidly adapting channel rhodopsin 2 (ChR2) in distinct subpopulations of type 1 LTMRs (Aβ-fiber RA1-LTMRs) that terminate in afferents. This allows to perform optogenetic and opto- Meissner’s corpuscles [1, 11, 27]. In contrast to LTMRs, tagged electrophysiological studies on properties of dif- HTMRs are present as free nerve endings of nocicep- ferent mechanoreceptors. For example, a recent study Cre+/ChR2+ tive afferent fibers in the glabrous skin, and they usually have used NPY2r mice to optogenetically label display SA impulses in response to sustained mechani- NPY2r-positive A-fiber HTMRs for investigating their cal stimulation . HTMRs are believed to be mainly electrophysiological properties and functions in acute nerve endings of nociceptive C- and Aδ-fibers [4, 6, 7], mechanical pain . In the present study, we have used cre but Aβ-fiber HTMRs are also present in the skin and may Nav1.8 mice  to drive the expression of ChR2 in ChR2 serve as nociceptors for mechanical tissue damage [4, Nav1.8-positive afferents (Nav1.8 -positive fibers), 9]. Properties of HTMRs, particularly Aβ-fiber HTMRs, and combined optogenetic and electrophysiological ChR2 have not been well characterized because they don’t have approaches to study the properties of Nav1.8 -positive ChR2 well defined structures and molecular markers. and Nav1.8 -negative afferent mechanoreceptors in ChR2 Transgenic mouse lines expressing Cre recombi- the hindpaw glabrous skin of Nav1.8 mice. nase under the control of the promoters of specific sensory molecules allow the expression of fluorescent Materials and methods proteins (e.g. GFP) in distinct subpopulation of affer - Animals ChR2 cre ents. This genetic approach can label afferent subpopu - Nav1.8 mice were generated by crossing Nav1.8 mice cre lations for structural and functional studies . The with Ai32 (RCL-ChR2(H134R)/EYFP) mice. Nav1.8 genetic reporter mice generated with Cre technique mice were gifts from Dr. John Wood at University Col- in combination with electrophysiology has allowed to lege London and transferred to us from Dr. Stephen Wax- characterize properties of different types of mechanore - man’s lab at Yale University. Ai32 mice were purchased ceptors. Cre mouse lines generated for genetic labeling from Jackson Labs. Animal care and use conformed to creER of LTMRs include TrkC for Aβ-fiber SA1-LTMR of NIH guidelines for care and use of experimental animals. CreER Merkel cell-neurite complex in the skins , Ret Experimental protocols were approved by the Institutional CreER and TrkB for Aβ-fiber RA1-LTMR of Meissner cor - Animal Care and Use Committee (IACUC) at the Univer- CreER puscles in glabrous skin [15, 18]; and TH for C-fiber sity of Alabama at Birmingham. LTMRs in hair follicles . For nociceptors includ- ing HTMRs, several Cre mouse lines have been gen- Behavioral assessment erated to allow to genetically label subpopulations of Cotton swab test The cotton swab test was performed in nociceptors for studies on properties and functions of a manner described previously . In brief, each testing these nociceptors. Cre mouse lines for labeling nocic- mouse was covered by a glass cup (7 cm in diameter and cre cre cre eptors include Mrgprd, CGRP , Nav1.8 , and others 8.5 cm in height) on an elevated platform with a perfo- [11, 19]. Nav1.8 are voltage-gated Na channels largely rated metal floor (Ugo Basile). Animals were acclimatized expressed in small-sized C-fiber nociceptors that are to the environment for approximately 1 h. Each cotton involved in both mechanical and thermal nociception swab was made by a piece of cotton that was glued onto a cre [2, 3]. Nav1.8 has also been found in a large popula- wood stick with the cotton part approximately 12 mm in tion of C-fiber nociceptors but they are also observed length. The hindpaw of mice was brushed by the cotton cre in some LTMRs . A recent study has used C GRP , swab in the heel-to-toe direction for 5 times, and the fre- cre cre TRPV1 , and Nav1.8 mouse lines to characterize elec- quency of avoidance responses were measured.von Frey trophysiological properties and immunochemical profiles test Each testing mouse was covered by a glass cup (7 cm cre cre cre of CGRP-positive, TRPV1 -positive, and Nav1.8 - in diameter and 8.5 in height) on an elevated platform positive dorsal root ganglion (DRG) neurons . The with a perforated metal floor (Ugo Basile). Animals were cre study shows that Nav1.8 mouse line labels almost habituated to the environment for approximately 1 h. cre cre all C-fibers, whereas CGRP and TRPV1 mouse The plantar side of the hindpaw was poked by calibrated lines label subpopulations of nociceptive C-afferents. von Frey filaments (North coast medical, NC12775-99). Interestingly, all three Cre mouse lines also genetically The von Frey filaments used are 0.02, 0.04, 0.07, 0.16, 0.4, Y amada et al. Molecular Brain (2023) 16:27 Page 3 of 15 0.6, 1.0, 1.4 g, and the 50% paw withdrawal thresholds and mechanical stimulation. In brief, recording elec- were determined using the Up-Down method . In a trodes for pressure-clamped single-fiber recordings were different von Frey test, the plantar side of hindpaw was made by thin-walled borosilicate glass tubing without fil - poked with 0.07-g and 0.4-g von Frey filaments each for ament (inner diameter 1.12 mm, outer diameter 1.5 mm, 10 times with intervals between stimuli being 1 to 2 min, World Precision Instruments, Sarasota, FL). They were and the percent of avoidence responses were determined. fabricated by using P-97 Flaming/Brown Micropipette Light stimulation Blue laser beam was applied to the Puller (Sutter Instrument Co., Novato, CA) and the tip planter surface with an optical fiber (diameter: 0.2 mm: of each electrode was fire polished by a microforge (MF- Laserglow technologies). The light intensities were cali - 900, Narishige) to final size of 4 to 8 μm in diameter. The brated with an optical power and energy meter (PM100D, recording electrode was filled with Krebs bath solution, Thorlab). The light duration was 50 ms and intensities mounted onto an electrode holder which was connected were 1, 2.5, 5, 10, 20, 50, 100 mW/mm . The light evoked to a high-speed pressure-clamp (HSPC) device (ALA Sci- responses were scored as 0, no response; 1, hindpaw lift; entific Instruments, Farmingdale, NY) for fine controls 2, hindpaw flinch, flutter, and/or hold; 3, jump, vocaliza - of intra-electrode pressures. Under a 40 × objective, the tion, lick, and/or guard. end of individual afferent nerve was visualized and sepa - rated by applying a low positive pressure (~ 10 mmHg Ex vivo skin‑nerve preparations or 0.19 Psi) from the recording electrode. The end of ChR2 Nav1.8 mice of both males and females aged a single nerve fiber was then aspirated into the record - 8–11 weeks were used. Animals were anesthetized with ing electrode by a negative pressure at approximately 5% isoflurane and then sacrificed by decapitation. The 10 mmHg. Once the end of the nerve fiber entered into hindpaw glabrous skin including plantar and finger the recording electrode in approximately 10 µm, the regions together with medial planter nerve and tibial electrode pressure was readjusted to − 3 ± 2 mmHg and nerve before the branch from sciatic nerves were dis- maintained at the same pressure throughout the experi- sected out. The skin-nerve preparation was then placed ment. Nerve impulses on the single afferent fiber were in a Sylgard Silicone-coated bottom of a 60-mm record- recorded under the I configuration and amplified using ing chamber. The fat, muscle and connective tissues on a Multiclamp 700B amplifier (Molecular Devices, Sunny - the nerves and the skin were carefully removed with a vale, CA). Electrical signals were amplified 500 times and pair of forceps. The skin was affixed to the bottom of the sampled at 25 kHz with AC filter at 0.1 Hz and Bessel fil - chamber by tissue pins with epidermis side facing up, and ter at 3 kHz under AC membrane mode (Digidata 1550B, the nerve bundle was affixed by a tissue anchor in the Molecular Devices). All experiments were performed at same recording chamber. The cutting end of the nerve 30 ± 2 °C. bundle was briefly exposed to a mixture of 0.05% dispase To determine conduction velocity of recorded affer - II plus 0.05% collagenase for 30–60 s, and the enzymes ent fibers, action potential (AP) impulses were initiated were then washed off by the normal Krebs solution (see by electrical stimulation using a bipolar stimulation elec- below). This gentle enzyme treatment was to help sepa - trode positioned on the tibial nerve bundle. The distance rating individual afferent fibers at the cutting end of the between the electrical stimulation site and the record- nerve bundle so that a single fiber could be aspirated ing site was approximately 12 mm. Electrical stimuli into the recording electrode and pressure-clamped for were monophasic square pulses that were generated by single-fiber recordings (see below). The recording cham - an electronic stimulator (Master-9, A.M.P.I, Israel) with ber was then mounted on the stage of the Olympus a stimulation isolator (ISO-Flex, A.M.P.I, Israel) and BX51WI upright microscope. The skin-nerve prepara - delivered to the stimulation electrode. The duration of tion was superfused with a normal Krebs bath solution each stimulation pulse was 200 μs for A-fibers and 2 ms that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 C aCl , for C-fibers, and the stimulation intensities for evoking 1.2 MgCl , 1.2 NaH PO , 25 NaHCO , and 11 glucose impulses were 0.3–1.7 mA for A-fiber and 0.65–2.5 mA 2 2 4 3 (pH 7.3 and osmolarity 325 mOsm) and was saturated for C-fibers. with 95% O and 5% CO . The Krebs bath solution in the 2 2 recording chamber was maintained at 28–32 °C during Mechanical and light stimulation experiments. For a recorded afferent fiber, its mechanosensitive recep - tive field in the hindpaw glabrous skin was first searched Pressure‑clamped single‑fiber recordings using a glass rod. Poking with the glass rod at the mecha- The pressure-clamped single-fiber recording was per - nosensitive receptive field of the recorded afferent fiber formed in the similar manner described in our previous would result in the detection of APs by the recording studies [22, 23] to measure impulses evoked by blue light electrode. In the present study, all data were collected Yamada et al. Molecular Brain (2023) 16:27 Page 4 of 15 from mechanosensitive receptive sites, i.e., mechano- significant difference was found between male and female receptors in the hindpaw glabrous skin. Once a mecha- animals. To confirm that impulses evoked by blue light noreceptor was identified, mechanical stimulation was and mechanical stimulators (indenter or von Frey) are applied to the same receptive field with a force-calibrated generated from the same receptive field, the amplitudes mechanical indenter (300C-I, Aurora Scientific Inc., and shapes of the impulses evoked by both blue light and Ontario, Canada) to determine mechanical thresholds. mechanical stimulators were compared to ensure that The tip size of the indenter was 0.8 mm in diameter. The mechanically evoked impulses matched the light-evoked indenter was connected to a Digidata 1550B Digitizer impulses. Conduction velocity (CV) was calculated by to allow generating ramp-and-hold mechanical stimula- the distance between stimulation site and recording site tion using the pClamp 11 software. Prior to the applica- divided by the time latency for eliciting an AP impulse tion of mechanical stimulation, the tip of the indenter following electrical stimulation. Afferent fibers were clas - was lowered to the surface of the receptive field with a sified as Aβ-fibers with CV > 9 m/s, Aδ-fiber with CV 10-mN force and then the 10-mN force was canceled to between 1.2 and 9 m/s, and C-fiber with CV < 1.2 ms 0 so that the tip of the indenter was just in contact with [9, 26]. All data analyses were performed using Graph the receptive field surface. Under the force control mod - Pad Prism (version 8). Unless otherwise indicated, all ule, ramp-and-hold mechanical stimuli were applied to data were reported as individual observations and/or the mechanoreceptor of the glabrous skin. The step force mean ± SEM of n independent observations. Statisti- commanders were calibrated by applying indenter at fin - cal significance was evaluated using the Kruskal–Wal - ger tips, paw pads and other areas of plantar skin, and the lis (nonparametric) test with Dunn’s post hoc tests for actual forces after the calibration were used in experi- multiple group comparison, Mann–Whitney (nonpara- ments. The ramp-and-hold force steps were at 0, 5, 30, metric) test or Student’s t tests for two group compari- and 80 mN. The duration of the ramp (dynamic phase) son. Differences were considered to be significant with was 10 ms, and the duration of the holding (static phase) *p < 0.05, **p < 0.01, ***p < 0.001, and not significant (ns) was 0.98 s. The minimal force at which AP impulses was with p ≥ 0.05. elicited was defined as intender mechanical threshold of the mechanoreceptors. In a different set of experiments Results mechanical stimulation was applied using von Frey fila - Behavioral responses to mechanical and light stimulation ChR2 ments to vertically poke the glabrous skin. The von Frey applied to the hindpaw plantar region of Nav1.8 mice Cre mechanical thresholds were determined by mechani- We crossed Nav1.8 mice with Ai32 (RCL-ChR2(H134R)/ cre+ loxP/+ cal stimulation with von Frey filaments (0.08 ~ 6 g) onto EYFP) mice to generate Nav1.8 ;ChR2-EYFP mouse ChR2 mechanoreceptors. line, hereafter termed Nav1.8 . We first examined behav - ChR2 To determine whether a mechanoreceptor was from ioral responses of Nav1.8 mice to mechanical stimula- ChR2 ChR2 Nav1.8 -positive or Nav1.8 -negative afferent fib - tion by cotton swabs and von Frey filaments applied to the ChR2 ers, the same mechanosensitive receptive field was stim - hindpaw plantar regions of Nav1.8 mice. For the stimu- ulated by a blue LED light (Thorlab; M455L4, 455 nm) lation with cotton swabs, animals withdrew their hindpaws to test light sensitivity. A mechanoreceptor was from at the frequency of 30 ± 4% (n = 23, Fig. 1A) in response ChR2 Nav1.8 -positive afferent fibers if light stimulation to the gentle strikes of their hindpaw plantar regions with evoked impulses. Otherwise, the mechanoreceptor was cotton swabs. For the hindpaw stimulation with von Frey ChR2 from light-insensitive or Nav1.8 -negative afferent fib - filaments of 0.69-mN force and 3.92-mN force, animals ers. The blue Light was applied through a 40 × objective withdrew their hindpaws at the frequency of 19 ± 2% and to a mechanoreceptor with a 1-s light stimulation pulse 38 ± 3% (n = 17, Fig. 1B), respectively. We also used the up- at the intensity of 50 mW. Afferent impulses evoked by down method with von Frey filaments to determine 50% mechanical and light stimulation were recorded using response thresholds. The 50% response thresholds were the pressure-clamped single-fiber recordings, and signals 4.39 ± 0.4 mN (n = 11, Fig. 1C) for evoking hindpaw with- were amplified by the Multiclamp 700B amplifier and draw responses. sampled at 25 kHz with band path filter between 0.1 Hz We examined behavioral responses to blue light and 3 kHz on AC recording mode. stimulation applied to the hindpaw plantar region of ChR2 Nav1.8 mice. In this set of experiments, a blue laser Data analysis beam was applied to the hindpaw plantar region with Electrophysiological data were analyzed using Clampfit the light intensity ranged from 1 to 100 mW/mm . Light 11 (Molecular Devices, Sunnyvale, CA, USA). Data were stimulation evoked nocifensive responses including paw collected from 27 male and 13 female animals and were lift, flinch, flutter, hold, jump, vocalization, lick, and aggregated together for data analysis since no statistically guard. We first analyzed response frequency without Y amada et al. Molecular Brain (2023) 16:27 Page 5 of 15 ChR2 Fig. 1 Behavioral responses to mechanical and light stimulation at the hindpaws of Nav1.8 mice. A Frequency of hindpaw avoidance in ChR2 response to cotton swab strikes in Nav1.8 mice (n = 23). B Frequency of hindpaw avoidance in response to mechanical stimulation by 0.69-mN ChR2 (left bar, n = 17) and 3.92-mN von Frey filaments (right bar, n = 17) in Nav1.8 mice. C The 50% threshold of hindpaw avoidance responses ChR2 assessed by von Frey filaments using the up-down method in Nav1.8 mice (n = 11). D Frequency of hindpaw avoidance in response to the stimulation by blue laser light at intensities from 1 to 100 mW/mm (n = 5 to 22). E Scores of nocifensive responses induced by blue laser light at intensities from 1 to 100 mW/mm (n = 5 to 22). Data represent individual observations and/or mean ± SEM, *p < 0.05 considering the types of responses. The response fre - intensity-dependent manner from 1 to 100 mW/mm (1 2 2 quencies were increased in a light stimulation inten- to 10 mW/mm , n = 22; 20 mW/mm , n = 22; 50 mW/ 2 2 sity-dependent manner from a narrow range of 1 to 10 mm , n = 5; 100 mW/mm , n = 6). Thus, the hindpaw 2 ChR2 mW/mm (n = 22), and the response frequency quickly plantar regions of Nav1.8 mice appeared to have reached 100% with light intensity at 10 mW/mm normal mechanical sensitivities and showed graded (n = 22), and remained the ceiling effect at 20 mW/mm nocifensive behavioral responses to the increased light 2 2 (n = 11), 50 mW/mm (n = 5), and 100 mW/mm (n = 6) stimulation intensity. (Fig. 1D). We next analyzed nocifensive responses by ChR2 scoring light-evoked responses, and determined relation- Characterization of Nav1.8 ‑positive mechanoreceptors ChR2 ship between light stimulation intensity and response We characterized properties of Nav1.8 -positive scores. The following criteria were used for the response mechanoreceptors by using the ex vivo plantar skin-tibial ChR2 scores: score 0, no response; score 1, hindpaw lift; score nerve preparation. In this set of experiments, Nav1.8 - 2, hindpaw flinch, flutter, and/or hold; score 3, jump, positive mechanoreceptors in the plantar skin were vocalization, lick and/or guard hindpaw. As shown in identified by both mechanical stimulation and blue light ChR2 Fig. 1E, the nocifensive scores were increased in a light stimulation. A Nav1.8 -positive mechanoreceptor Yamada et al. Molecular Brain (2023) 16:27 Page 6 of 15 was a receptive field where both mechanical stimulation mechanosensitive receptive fields. The sustained light and blue light stimulation evoked AP impulses which stimulation evoked rapidly adapting (RA) impulses in ChR2 individually had identical shape. In this set of experi- almost all Nav1.8 -positive Aβ-fiber mechanorecep - ments, mechanical stimulation-evoked AP impulses and tors (Fig. 2H, I, n = 23/24). The light stimulation evoked light stimulation-evoked impulses at the same receptive RA impulses in majority (n = 13/18) and SA impulses in ChR2 fields were recorded using the pressure-clamped single- minority (Fig. 2H, I, n = 5/18) of Nav1.8 -positivive fiber recording technique (Fig. 2A). Furthermore, the Aδ-fiber mechanoreceptors. In contrast, the light stim - ChR2 conduction velocity (CV) of each recorded afferent fiber ulation evoked SA impulses in all Nav1.8 -positive was measured, and the afferent fibers were classified into C-fiber mechanoreceptors (Fig. 2H&I, n = 15/15). The Aβ-fibers with CV > 9 m/s [9, 26], Aδ-fibers with CV light-evoked SA impulses showed increased frequency between 9 and 1.2 m/s, and C-fibers with CV < 1.2 m/s in a light stimulation intensity-dependent manner for ChR2 ChR2 (Fig. 2B, C). In addition to Nav1.8 -positive mecha- both Nav1.8 -positive Aδ-fiber SA-mechanoreceptors ChR2 ChR2 noreceptors, we collected data from Nav1.8 -neg- (Fig. 2J, n = 5) and Nav1.8 -positive C-fiber mecha - ative (light-insensitive) mechanoreceptors. Figure 2C noreceptors (Fig. 2J, n = 15). On the other hand, the ChR2 ChR2 shows the CV of individual Nav1.8 -positive mecha- light-evoked impulses in Nav1.8 -positive Aβ-fiber ChR2 ChR2 noreceptors and Nav1.8 -negative mechanorecep- RA-mechanoreceptors (n = 23) and Nav1.8 -positive ChR2 tors. Nav1.8 -poistive mechanoreceptors included Aδ-fiber RA-mechanoreceptors (n = 13) did not shown ChR2 Aβ-, Aδ-, and C-fibers. Our recordings with Nav1.8 - large enhancement of impulse frequency with increased negative mechanoreceptors identified Aβ-, Aδ-, but not stimulation intensity. ChR2 C-fibers (Fig. 2C). Of all Nav1.8 -positive Aβ-fibers ChR2 ChR2 tested, 59% of them were mechanosensitive (Fig. 2D, Properties of Nav1.8 ‑positive and Nav1.8 ‑negative ChR2 E). Of all Nav1.8 -positive Aδ-fiber, 65% of them Aβ‑fiber mechanoreceptors ChR2 ChR2 were mechanosensitive (Fig. 2D, E). Of all Nav1.8 - We compared properties of Nav1.8 -positive Aβ-fiber ChR2 positive C-fibers, 78% of them were mechanosensitive mechanoreceptors with those of Nav1.8 -negative ChR2 (Fig. 2D, E). When Nav1.8 -positivity was examined Aβ-fiber mechanoreceptors. Ramp-and-hold mechanical ChR2 for the mechanoreceptors, of all Aβ-fiber mechanorecep - stimulation evoked SA impulses in all Nav1.8 -posi- ChR2 tors, 33% of them were Nav1.8 -positivity (Fig. 2F). tive Aβ-fiber mechanoreceptors (Fig. 3A, D), but evoked Of all Aδ-fiber mechanoreceptors, 73% of them were both SA (18/26, Fig. 3B, D) and RA (n = 8/26, Fig. 3C, ChR2 ChR2 Nav1.8 -positivity, and of all C-fiber mechanorecep - D) impulses in Nav1.8 -negative Aβ-fiber mecha - ChR2 tors (Fig. 2F), 100% of them were Nav1.8 -positivity noreceptors. The differences in adapting types were ChR2 ChR2 (Fig. 2F). For Nav1.8 -positive Aβ-, Aδ-, and C-fiber significant between Nav1.8 -positive Aβ-fiber mecha - ChR2 mechanoreceptors, mechanical stimulation by ramp- noreceptors and Nav1.8 -negative Aβ-fiber mecha - and-hold indentation applied to the receptive fields in noreceptors (p < 0.001, Fig. 3D). Conduction velocities ChR2 the hindpaw regions evoked slowly adapting (SA) type of of Nav1.8 -positive Aβ-fiber SA-mechanoreceptors, ChR2 impulses in almost all recordings (Fig. 2D, G). Nav1.8 -negative Aβ-fiber SA-mechanoreceptors, ChR2 We examined AP impulses evoked by sustained and Nav1.8 -negative Aβ-fiber RA-mechanorecep - blue light stimulation (50 mW, 1 s) applied to the tors were compared (Fig. 3E). Conduction velocities of (See figure on next page.) ChR2 ChR2 Fig. 2 Properties of Nav1.8 -positive mechanoreceptors in the hindpaw glabrous skin of Nav1.8 mice. A Schematic diagram illustrate ChR2 the experimental setting for characterizing Nav1.8 -positive, i.e., light-sensitive, mechanoreceptors in the hindpaw glabrous skin of the ChR2 ex vivo skin-nerve preparation made from Nav1.8 mice. AP impulses evoked by mechanical stimulation and light stimulation at the same receptive fields were recorded using the pressure-clamped single-fiber recording technique. B Sample traces show examples of Aβ-, Aδ- and C-fiber mechanoreceptors whose conduction velocities (CV ) were determined by electrical stimulation. Arrowhead in each panel indicates electrical stimulation artifact, and the latency between the stimulation artifact and AP impulse was used to calculating CV. C Plots of CV of ChR2 ChR2 individual Nav1.8 -positive mechanoreceptors (solid circles) and Nav1.8 -negative mechanoreceptors (open squares). D Sample traces ChR2 show slowly adapting (SA) impulses evoked by an 80-mN ramp-and-hold stimulation in a Nav1.8 -positive Aβ-fiber mechanoreceptor (top), ChR2 ChR2 ChR2 a Nav1.8 -positive Aδ-fiber mechanoreceptor (middle), and a Nav1.8 -positive C-fiber mechanoreceptors. E Percent of Nav1.8 -positive ChR2 ChR2 afferent fibers that are mechanosensitive. Numbers in the bar represent Nav1.8 -positive mechanoreceptors over total Nav1.8 -positive ChR2 ChR2 afferent fibers tested. F Percent of mechanoreceptors that are Nav1.8 -positive afferent fibers. Numbers in the bar represent Nav1.8 -positive ChR2 mechanoreceptors over total mechanoreceptors. G Percent of Nav1.8 -positive mechanoreceptors displaying SA and rapidly adapting (RA) impulses. Numbers in the bar represent numbers of recordings. H Examples of AP impulses evoked by blue LED light (50 mW, 1 s) applied to the mechanosensitive receptive field of an Aβ- (top), an Aδ- (middle), and a C-fiber mechanoreceptor (bottom). I Percent of recordings showing SA ChR2 or RA for the Nav1.8 -positive Aβ-, Aδ-, and a C-mechanoreceptor. Numbers in the bar represent numbers of recordings. J Frequencies of AP impulses evoked by blue light at 1, 10 and 50 mW in SA Aδ-fiber SA-mechanoreceptors (n = 5), C-fiber SA-mechanoreceptors (n = 15), Aβ-fiber RA-mechanoreceptors (n = 23), and Aδ-fiber RA-mechanoreceptors (n = 13). Data represent individual observations or mean ± SEM Y amada et al. Molecular Brain (2023) 16:27 Page 7 of 15 Fig. 2 (See legend on previous page.) (See figure on next page.) ChR2 ChR2 Fig. 3 Comparison of properties between Nav1.8 -positive and Nav1.8 -negative Aβ-mechanoreceptors. A Sample traces show SA AP ChR2 impulses evoked by indentations (5, 30, and 80 mN) in a Nav1.8 -positive Aβ-fiber SA-mechanoreceptor. B&C) Two sets of sample traces ChR2 show SA (B) and RA (C) AP impulses evoked by indentations (5, 30, and 80 nN) in a Nav1.8 -negative Aβ-fiber SA-mechanoreceptors (B) and a ChR2 ChR2 ChR2 Nav1.8 -negative Aβ-fiber RA-mechanoreceptors. D Percent of Nav1.8 -positive and Nav1.8 -negative Aβ-mechanoreceptors that display ChR2 SA or RA impulses. Recording numbers are indicated in the bars. E Conduction velocity of Nav1.8 -positive Aβ-fiber SA-mechanoreceptors, ChR2 ChR2 Nav1.8 -negative Aβ-fiber SA-mechanoreceptors, and Nav1.8 -negative Aβ-fiber RA-mechanoreceptors. F Frequency of impulses evoked ChR2 ChR2 by different indentation forces in Nav1.8 -positive Aβ-fiber SA-mechanoreceptors, Nav1.8 -negative Aβ-fiber SA-mechanoreceptors, and ChR2 Nav1.8 -negative Aβ-fiber RA-mechanoreceptors. G, H Indenter (G) and von Frey (H) mechanical force thresholds for evoking AP impulses in ChR2 ChR2 ChR2 Nav1.8 -positive Aβ-fiber SA-mechanoreceptors (n = 23), Nav1.8 -negative Aβ-fiber SA-mechanoreceptors (n = 18), and Nav1.8 -negative Aβ-fiber RA-mechanoreceptors (n = 8). Data represent individual observations and/or mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 Yamada et al. Molecular Brain (2023) 16:27 Page 8 of 15 Fig. 3 (See legend on previous page.) ChR2 Nav1.8 -positive Aβ-fiber SA-mechanoreceptors frequency largely increased with enhanced mechanical ChR2 was 11.0 ± 0.3 m/s (n = 14), significantly slower than indentation force in both Nav1.8 -positive Aβ-fiber ChR2 ChR2 the Nav1.8 -negative Aβ-fiber RA-mechanorecep - SA-mechanoreceptors (n = 14, Fig. 3F) and Nav1.8 - tors (13.3 ± 0.9 m/s, n = 8, p < 0.01, Fig. 3E). AP impulse negative Aβ-fiber SA-mechanoreceptors (n = 18, Fig. 3F). Y amada et al. Molecular Brain (2023) 16:27 Page 9 of 15 ChR2 However, Nav1.8 -positive Aβ-fiber SA-mechan - Aδ-fiber mechanoreceptors (n = 9/11, 82%) showed oreceptors displayed relatively lower AP impulse fre- RA responses (Fig. 4B, C), and only a small frac- ChR2 ChR2 quency (n = 14) in comparison with Nav1.8 -negative tion (n = 2/11, 18%) of Nav1.8 -negative Aδ-fiber Aβ-fiber SA-mechanoreceptors (n = 18) (Fig. 3F). For mechanoreceptors displayed SA impulses in response ChR2 Nav1.8 -negative Aβ-fiber RA-mechanoreceptors, to the sustained mechanical stimulation. The patterns there was minimal change in AP impulse frequency with of AP impulses were significantly different between ChR2 ChR2 increased indentation forces (n = 7) (Fig. 3F). Nav1.8 -positive and Nav1.8 -negative Aδ-fiber We examined mechanical thresholds for evok- mechanoreceptors (p < 0.001, Fig . 4C). We compared ChR2 ChR2 ing AP impulses in Nav1.8 -positive Aβ-fiber conduction velocity between Nav1.8 -positive ChR2 ChR2 mechanoreceptors and Nav1.8 -negative Aβ-fiber Aδ-fiber SA-mechanoreceptors and Nav1.8 -neg- ChR2 mechanoreceptors. Nav1.8 -positive Aβ-fiber SA- ative Aδ-fiber RA-mechanoreceptors (Fig. 4D). The ChR2 mechanoreceptors had significantly higher mechani - conduction velocity of Nav1.8 -positive Aδ-fiber ChR2 cal thresholds in comparison with Nav1.8 -negative SA-mechanoreceptors was 5.3 ± 0.5 m/s (n = 13), sig- ChR2 ChR2 Aβ-fiber SA-mechanoreceptors and Nav1.8 -nega- nificantly slower than the Nav1.8 -negative Aδ-fiber tive Aβ-fiber RA-mechanoreceptors as tested by both RA-mechanoreceptors (6.9 ± 0.4 m/s , n = 9, p < 0.05) ChR2 mechanical indenter (Fig. 3G) and von Frey filaments (Fig. 4D). Although presented (Fig. 4D), Nav1.8 - (Fig. 3H). For the test with mechanical indenter, the force positive Aδ-fiber RA-mechanoreceptors (n = 1) and ChR2 ChR2 threshold of Nav1.8 -positive Aβ-fiber SA-mechano - Nav1.8 -negative Aδ-fiber SA-mechanoreceptors receptors was 22.7 ± 4.0 mN (n = 23), significantly higher (n = 2) had too small sample sizes to allow for any ChR2 ChR2 than the force threshold of Nav1.8 -negative Aβ-fiber meaningful statistical comparison. Nav1.8 -pos- SA-mechanoreceptors (3.2 ± 1.0 mN, n = 18, p < 0.001) itive Aδ-fiber SA-mechanoreceptors displayed large ChR2 and Nav1.8 -negative Aβ-fiber RA-mechanoreceptors enhancement of AP impulse frequency with increased (5.8 ± 1.8 mN, n = 8, p < 0.05) (Fig. 3G). For the test with mechanical stimulation forces up to 80 mN (Fig. 4E). ChR2 ChR2 von Frey filaments, the force threshold of Nav1.8 - On the other hand, Nav1.8 -negative Aδ-fiber RA- positive Aβ-fiber SA-mechanoreceptors was 11.0 ± 2.2 mechanoreceptors showed minimal enhancement of mN (n = 23), approximately five times higher than those AP impulse frequency with increased mechanical stim- ChR2 of Nav1.8 -negative Aβ-fiber SA mechanoreceptors ulation forces (Fig. 4E). ChR2 ChR2 (2.1 ± 0.4 mN, n = 18, p < 0.001) and Nav1.8 -negative Nav1.8 -positive Aδ-fiber SA-mechanorecep - Aβ-fiber RA-mechanoreceptors (2.2 ± 0.7 mN, n = 8, tors showed significantly higher mechanical thresh - ChR2 p < 0.001) (Fig. 3H). olds in comparison with Nav1.8 -negative Aδ-fiber RA-mechanoreceptors as tested by both mechanical ChR2 ChR2 Properties of Nav1.8 ‑positive and Nav1.8 ‑negative indenter (Fig. 4F) and von Frey filaments (Fig. 4G). For Aδ‑fiber mechanoreceptors the test with mechanical indenter, the force threshold ChR2 ChR2 ChR2 Properties of Nav1.8 -positive and Nav1.8 - of Nav1.8 -positive Aδ-fiber SA-mechanorecep - negative Aδ-fiber mechanoreceptors were com - tors was 19.3 ± 5.9 mN (n = 13), significantly higher ChR2 pared. Ramp-and-hold mechanical stimulation with than Nav1.8 -negative Aδ-fiber RA-mechanore - indenter evoked AP impulses at the receptive field of ceptors (1.6 ± 0.6 mN, n = 9, p < 0.001) (Fig . 4F). For ChR2 ChR2 both Nav1.8 -positive (Fig. 4A, C) and Nav1.8 - the test with von Frey filaments, the force threshold of ChR2 negative Aδ-fiber mechanoreceptors (Fig. 4B, C). Nav1.8 -positive SA Aδ-fiber SA-mechanoreceptors ChR2 Almost all the Nav1.8 -positive Aδ-fiber mecha - was 14.2 ± 3.4 mN, (n = 13), significantly higher than ChR2 noreceptors (n = 13/14, 93%) displayed SA impulses Nav1.8 -negative Aδ-fiber RA-mechanoreceptors in response to mechanical indentation stimulation (0.3 ± 0.1 mN, n = 9, p < 0.001) (Fig . 4G). Although the ChR2 (Fig. 4A, C). In contrast, most Nav1.8 -negative mechanical thresholds were presented in Fig. 4F and (See figure on next page.) ChR2 ChR2 Fig. 4 Comparison of properties between Nav1.8 -positive and Nav1.8 -negative Aδ-fiber mechanoreceptors. A, B Two sets of sample traces ChR2 ChR2 show AP impulses evoked by indentations (5, 30, and 80 mN) in a Nav1.8 -positive Aδ-fiber SA-mechanoreceptor (A) and a Nav1.8 -negative ChR2 ChR2 Aδ-fiber RA-mechanoreceptors (B). C Percent of Nav1.8 -positive and Nav1.8 -negative Aδ-fiber mechanoreceptors that display SA impulses ChR2 ChR2 or RA impulses. Recordings numbers are indicated in the bars. D Conduction velocity of Nav1.8 -positive and Nav1.8 -negative Aδ-fiber ChR2 mechanoreceptors. E Frequency of AP impulses evoked by different forces (5, 30, and 80 mN) in Nav1.8 -positive Aδ-fiber SA-mechanoreceptors ChR2 and Nav1.8 -negative Aδ-fiber RA-mechanoreceptors. F, G Indenter (F) and von Frey (G) force thresholds for evoking AP impulses in ChR2 ChR2 ChR2 Nav1.8 -positive Aδ-fiber RA-mechanoreceptors (n = 1), Nav1.8 -positive Aδ-fiber SA-mechanoreceptors (n = 13), Nav1.8 -negative Aδ-fiber ChR2 RA-mechanoreceptors (n = 9), and Nav1.8 -negative Aδ-fiber SA-mechanoreceptors (n = 2). Data represent individual observations and/or mean ± SEM, *p < 0.05, ***p < 0.001 Yamada et al. Molecular Brain (2023) 16:27 Page 10 of 15 Fig. 4 (See legend on previous page.) Y amada et al. Molecular Brain (2023) 16:27 Page 11 of 15 ChR2 response to the ramp-and-hold mechanical stimulation, G, Nav1.8 -positive Aδ-fiber RA-mechanoreceptors ChR2 and AP impulses enhanced with the increased mechani- (n = 1) and Nav1.8 -negative Aδ-fiber SA-mechano - ChR2 cal indentation force (n = 14). Only one Nav1.8 - receptors (n = 2) had too small sample sizes to allow for positive C-fiber mechanoreceptors showed RA impulses any meaningful statistical comparison. ChR2 (Fig. 5B). Conduction velocity of Nav1.8 -positive ChR2 C-fiber SA-mechanoreceptors of the above 14 record - Properties of Nav1.8 ‑positive C‑fiber ings and two other recordings not included in Fig. 5B mechanoreceptors ChR2 was 0.53 ± 0.04 m/s (n = 16), and the single Nav1.8 - All recorded C-fiber mechanoreceptors were found ChR2 ChR2 positive C-fiber RA-mechanoreceptor had the conduc - to be Nav1.8 -positive (Fig. 2F). Of 15 Nav1.8 - tion velocity of 0.85 m/s (n = 1) (Fig. 5C). Tested with positive C-fiber mechanoreceptors tested with graded ChR2 the mechanical indenter, Nav1.8 -positive C-fiber force stimulation, 14 of them displayed SA impulses in ChR2 Fig. 5 Properties of Nav1.8 -positive C-fiber mechanoreceptors. A Sample traces show SA impulses evoked by indentations (5, 30, 80 mN) in ChR2 ChR2 a Nav1.8 -positive C-fiber SA-mechanoreceptor. B Frequency of impulses evoked by different indentation forces in Nav1.8 -positive C-fiber ChR2 ChR2 SA-mechanoreceptors (n = 14) and a Nav1.8 -positive C-fiber RA-mechanoreceptor (n = 1). C Conduction velocity of Nav1.8 -positive C-fiber ChR2 SA-mechanoreceptors (n = 16) and a Nav1.8 -positive C-fiber RA-mechanoreceptor (n = 1). D, E Indenter (D) and von Frey (E) force thresholds for ChR2 ChR2 evoking AP impulses in Nav1.8 -positive C-fiber SA-mechanoreceptors (n = 16) and a Nav1.8 -positive C-fiber RA-mechanoreceptor (n = 1). Data represent individual observations and/or mean ± SEM Yamada et al. Molecular Brain (2023) 16:27 Page 12 of 15 ChR2 Nav1.8 -positive Aβ-fiber SA-mechanoreceptors, SA-mechanoreceptors had a force threshold of 10.0 ± 3.4 ChR2 ChR2 19.3 ± 5.9 mN (n = 13) for Nav1.8 -positive SA mN (n = 16) (Fig. 5D). The single Nav1.8 -positive Aδ-fiber SA-mechanoreceptors, and 10.0 ± 3.4 mN C-fiber RA-mechanoreceptor had a force threshold of ChR2 (n = 16) for Nav1.8 -positive C-fiber SA-mecha - 36.7 mN (Fig. 5D). Tested with von Frey filaments, the ChR2 noreceptors (Fig. 6B). The indenter force thresholds Nav1.8 -positive C-fiber SA-mechanoreceptors had ChR2 between Nav1.8 -positive Aβ-fiber SA-mechano - a force threshold of 5.9 ± 1.2 mN (n = 16, Fig. 5E). The ChR2 ChR2 receptors and Nav1.8 -positive Aδ-fiber SA-mech - single Nav1.8 -positive C-fiber RA-mechanoreceptor anoreceptors were not significantly different. The had a force threshold of 58.8 mN. ChR2 indenter force thresholds between Nav1.8 -positive ChR2 Aδ-fiber SA-mechanoreceptors and Nav1.8 -pos- Comparison of mechanical sensitivity ChR2 itive C-fiber SA-mechanoreceptors were also not sig - among Nav1.8 ‑positive Aβ‑, Aδ‑, and C‑fiber nificantly different. The force thresholds were higher mechanoreceptors ChR2 in Nav1.8 -positive Aβ-fiber SA-mechanoreceptors We compared the mechanical thresholds for evoking ChR2 ChR2 than in Nav1.8 -positive C-fiber SA-mechanorecep - AP impulses among Nav1.8 -positive Aβ-, Aδ-, and tors (p < 0.01, Fig . 6B). It was noted that von Frey force C-fiber SA-mechanoreceptors (Fig. 6A). For the experi- ChR2 threshold for most Nav1.8 -positive mechanorecep- ments using von Frey filaments, the force thresholds ChR2 tors showed threshold over 4 mN, which could be con- were 11.0 ± 2.2 mN (n = 23) for Nav1.8 -positive sidered as HTMRs. On the other hand, a small portion Aβ-fiber SA-mechanoreceptors, 14.2 ± 3.4 mN (n = 13) ChR2 ChR2 of Nav1.8 -positive mechanoreceptors displayed for Nav1.8 -positive Aδ-fiber SA-mechanorecep - ChR2 force threshold below 4 mN (Fig. 6A), which could be tors, and 5.9 ± 1.2 mN (n = 16) for Nav1.8 -positive considered as LTMRs. With the above classification, C-fiber SA-mechanoreceptors (Fig. 6A). The force ChR2 von Frey force threshold showed no significant dif - thresholds between Nav1.8 -positive Aβ-fiber SA- ChR2 ChR2 ference among the three types of Nav1.8 -positive mechanoreceptors and Nav1.8 -positive Aδ-fiber HTMRs. We further compared AP impulse frequency SA-mechanoreceptors were not significantly differ - ChR2 in response to increased indentation forces among the ent. The force thresholds between Nav1.8 -positive ChR2 ChR2 Nav1.8 -positive Aβ-fiber SA-mechanoreceptors Aβ-fiber SA -mechanoreceptors and Nav1.8 -pos- ChR2 (n = 18), Nav1.8 -positive Aδ-fiber SA-mechanore - itive C-fiber SA-mechanoreceptors were also not sig - ChR2 ceptors (n = 10), and Nav1.8 -positive C-fiber SA- nificantly different. The force thresholds were higher ChR2 mechanoreceptors (n = 14) (Fig . 6C). While all three in Nav1.8 -positive Aδ-fiber SA-mechanoreceptors ChR2 ChR2 types of Nav1.8 -positive mechanoreceptors showed than in Nav1.8 -positive C-fiber SA-mechanore - nearly linear enhancement of AP impulse frequency ceptors (p < 0.05, Fig . 6A). With mechanical indenter, in response to increased indentation forces, there was the force thresholds were 22.7 ± 4.0 mN (n = 23) for ChR2 Fig. 6 Comparison of mechanosensitivity among Nav1.8 -positive Aβ-fiber mechanoreceptors, Aδ-fiber mechanoreceptors, and C-fiber ChR2 ChR2 mechanoreceptors. A Comparison of von Frey threshold of Nav1.8 -positive Aβ-fiber mechanoreceptors, Nav1.8 -positive Aδ-fiber ChR2 ChR2 mechanoreceptors, and Nav1.8 -positive C-fiber mechanoreceptors. B Comparison of indenter force threshold of Nav1.8 -positive Aβ-fiber ChR2 ChR2 mechanoreceptors, Nav1.8 -positive Aδ-fiber mechanoreceptors, Nav1.8 -positive C-fiber mechanoreceptors. C Comparison of impulse ChR2 ChR2 frequency evoked by different forces (5, 30 and 80 mN) applied by indenter in Nav1.8 -positive Aβ-fiber mechanoreceptors, Nav1.8 -positive ChR2 Aδ-fiber mechanoreceptors, and Nav1.8 -positive C-fiber mechanoreceptors. Data are from Figs. 3, 4, 5 and replotted here for comparison. Data represent individual observations and/or mean ± SEM, *p < 0.05, ns, not significantly different Y amada et al. Molecular Brain (2023) 16:27 Page 13 of 15 ChR2 no significant difference in the frequency-force rela - frequency and Nav1.8 -positive C-afferents may ChR2 tionship among the three types of Nav1.8 -positive account for graded nocifensive scores. mechanoreceptors (Fig. 6C). In the present study, we have characterized properties ChR2 ChR2 of Nav1.8 -positive and Nav1.8 -negative affer - Discussion ent fiber mechanoreceptors in the glabrous skin of the In the present study, we have characterized properties hindpaws using the skin-nerve preparations. We have ChR2 of Nav1.8 -positive afferent fiber mechanoreceptors used pressure-clamped single-fiber recording technique ChR2 and compared with those of Nav1.8 -negative afferent [22, 23] to record AP impulses evoked by mechani- fiber mechanoreceptors in the hindpaw glabrous skin of cal stimulation to mechanoreceptors in the glabrous ChR2 ChR2 Nav1.8 mice. Our main findings are that Nav1.8 - skin. Pressure-clamped single-fiber recording technique positive Aβ-, Aδ-, and C-fiber mechanoreceptors are allows us to record both mechanically and optogeneti- ChR2 mostly HTMRs. In contrast, Nav1.8 -negative Aβ- cally evoked impulses, and also allows to determine the and Aδ-fiber mechanoreceptors are mostly LTMRs, and type of afferents based on their conduction velocity. ChR2 no Nav1.8 -negative C-fiber mechanoreceptors are Different from the teased fiber recordings used previ - ChR2 encountered in the present study. For Nav1.8 -posi- ously by many investigators, our recording method is a tive Aβ-, Aδ-, and C-fiber mechanoreceptors, almost all true single-fiber recording technique particularly suit - of them display SA impulses in response to sustained able for studying properties of opto-tagged afferent fiber mechanical stimulation. In comparison, in response to mechanoreceptors. ChR2 sustained mechanical stimulation, Nav1.8 -negative The present study shows that nearly 30% of Aβ-fiber ChR2 Aβ-fiber mechanoreceptors display both SA and RA mechanoreceptors are Nav1.8 -positive. Among ChR2 ChR2 impulses and Nav1.8 -negative Aδ-fiber mecha - Nav1.8 -positive Aβ-fiber mechanoreceptors, most noreceptors predominantly show RA impulses. Among of them have von Frey threshold near or above 4 mN, ChR2 Nav1.8 -positive Aβ-, Aδ-, and C-fiber mechano - suggesting that most of them are Aβ-fiber HTMRs or receptors, thresholds of C-fiber mechanoreceptors are mechanonociceptors. This finding is consistent with ChR2 significantly lower than those of A-fibers. Nav1.8 - previous studies showing the presence of mechanosen- positive Aβ-, Aδ-, and C-fiber SA-mechanoreceptors all sitive Aβ-fiber nociceptive . The scattered distribu - encode mechanical stimulation intensities by increases of tion of the force thresholds may suggest that Nav1.8 ChR2 impulse frequency in a similarly manner. Our results pro- -positive Aβ-afferent fibers with high mechanical vide new insights into the encoding of low (innocuous) threshold may be further divided into different func - ChR2 and high (noxious) threshold mechanical stimuli by the tional subtypes. Nav1.8 -positive Aβ-fiber HTMRs optogenetically defined subpopulations of mechanore - display SA impulses and graded increases of impulse ChR2 ceptors in the hindpaw glabrous skin of Nav1.8 mice. frequency with increased mechanical stimulation force ChR2 ChR2 Our behavioral assessment shows that Nav1.8 mice intensity. These features may allow Nav1.8 -positive have normal paw withdrawal responses to mechanical Aβ-fiber HTMRs to encode a broad range of high-inten - stimulation examined by the cotton swab test and the sity, noxious mechanical stimuli. It should be noted that ChR2 von Frey test. The response frequency measured by the a small portion of Nav1.8 -positive Aβ-fiber mecha - cotton swab test and the von Frey test as well as the 50% noreceptors have mechanical threshold below 4 mN, von Frey threshold measured by the up-down method are suggesting they are Aβ-fiber LTMRs, not mechanono - consistent with those of the WT mice shown in previ- ciceptors. Consistently, a previous study has shown that cre ous studies . The 50% von Frey thresholds are above 4 Nav1.8 are not restricted to nociceptors and they are mN, a force above which is consider to be high threshold also expressed in some A- and C-fiber LTMRs . It mechanical forces that may activate mechanonociceptors would be interesting to investigate in future whether the cre . We show that light stimulation evokes nocifensive low threshold Nav1.8 -positive Aβ-afferent fibers may ChR2 responses in a stimulation intensity-dependent manner. be a special type of LTMRs. We show that Nav1.8 - The response frequency is enhanced with increased light negative Aβ-fiber mechanoreceptors mostly display low stimulation intensity and quickly reaches 100% at the mechanical threshold and thereby are LTMRs. A large 2 ChR2 light intensity of 10 mW/mm . However, the nocifensive portion of Nav1.8 -negative Aβ-LTMRs displays SA response scores display graded increases with the light impulses but a small portion of them shows RA impulses. 2 ChR2 stimulation intensity up to 100 mW/mm . u Th s, response These Nav1.8 -negative Aβ-fiber SA-LTMRs and RA- frequency and nocifensive scores are not well correlated, LTMRs are most likely Merkel cell-neurite complex and ChR2 which may be because Nav1.8 -positive A-afferents Meissner’s corpuscles, respectively, in the glabrous skin are the first responder mainly account for the response of the hindpaws. Yamada et al. Molecular Brain (2023) 16:27 Page 14 of 15 We show that the majority of Aδ-fiber mechanorecep - intrinsically firing single AP and the latter firing multiple ChR2 ChR2 tors are Nav1.8 -positive. Among Nav1.8 -positive APs in response to sustained depolarization. This may Aδ-fiber mechanoreceptors, all of them have von Frey also suggest that mechanical stimulation may act on cells threshold near or above 4 mN and thereby can be consid- such as keratinocytes that surround the afferent terminals ChR2 ered as Aδ-fiber HTMRs. Nav1.8 -positive Aδ-fiber of A-fiber HTMRs to tune the afferent terminals to fire ChR2 HTMRs have properties similar to those of Nav1.8 - multiple APs in response to sustained mechanical stimu- ChR2 positive Aβ-fiber HTMRs. Nav1.8 -positive Aδ-fiber lation. Consist with this idea, keratinocytes have been HTMRs and Aβ-fiber HTMRs may be the same popula - indicated to be involved in mechanotransduction [16, ChR2 tion of NPY2r -positive A-fiber mechanonociceptors 17]. It would be highly interesting in future to uncover ChR2 , and are likely to be from large-sized DRG that express the underlying mechanisms by which Nav1.8 -posi- ChR2 CGRP . In contrast to Nav1.8 -positive Aδ-fiber tive Aβ-, Aδ-, and C-fiber HTMRs generate SA impulses ChR2 mechanoreceptors, Nav1.8 -negative Aδ-fiber mech - in response to sustained mechanical stimulation and also anoreceptors all show very low von Frey threshold and to identify molecular sensors of Aβ-, Aδ-, and C-fiber most of them display RA responses. Thus, the proper - HTMRs. ChR2 ties of the Nav1.8 -negative Aδ-fiber LTMRs are con - sistent with D-hair mechanoreceptors . D-hairs and Abbreviations D-hair mechanoreceptors have recently been identified AP Action potential in the glabrous skin of the hindpaws of C57BL/6J (A) and CGRP Calcitonin gene-related peptide ChR2 Channel rhodopsin 2 CBA/J (B) mice and also some North American and Afri- CV Conduction velocity can rodent species . DRG Dorsal root ganglion ChR2 Similar to Nav1.8 -positive Aβ- and Aδ-fiber mech - LTMR Low threshold mechanoreceptor ChR2 HTMR High threshold mechanoreceptor anoreceptors, majority of Nav1.8 -positive C-fiber Mrgprd MAS related GPR family member D mechanoreceptors show high thresholds and can be RA Rapidly adapting considered as C-fiber HTMRs. Consistently, C-fiber SA Slowly adapting TRPV1 Transient receptor potential cation channel subfamily V member 1 mechanoreceptors in DRGs have been shown to be ChR2 ChR2 Nav1.8 -positive in Nav1.8 mice . Almost all Acknowledgements ChR2 Nav1.8 -positive C-fiber mechanoreceptors show SA We thank Dr. Jennifer DeBerry for her scientific advice and technical support. impulses in response to sustained mechanical stimu- Author contributions lation. The impulse frequency linearly enhances with JGG conceived and designed the experiments and wrote the paper. AY, AIY, ChR2 increased stimulation force intensity. Thus, Nav1.8 - and JL performed experiments, and analyzed data. HF and WL participated result interpretation and discussions. All authors read and approved the final positive C-fiber HTMRs have properties similar to those manuscript. ChR2 of Nav1.8 -positive Aβ- and Aδ-fiber HTMRs and they all are suitable for encoding a broad range of high inten- Funding This work was supported by NIH Grants DE018661, DE023090 and NS109059 sity nociceptive mechanical stimuli. It should be noted to J.G.G. ChR2 that a number of Nav1.8 -positive C-fiber mechanore - ceptors display very low mechanical threshold, indicating Availability of data and materials All data generated or analyzed during this study are available from corre- that they are C-fiber LTMRs. This is consistent with an sponding author on reasonable request. ChR2 immunochemical study with Nav1.8. -positive DRG neurons . Declarations ChR2 We show that Nav1.8 -positive Aβ-, Aδ-, and C-fiber HTMRs display SA impulses in response to sus - Ethics approval and consent to participate All experimental procedures were approved by the appropriate institutional tained mechanical stimulation. This is consistent with animal care and use committees of the University of Alabama at Birmingham. the findings that mechanonociceptors are SA-mech - Consent to participate is not applicable for this study. anoreceptors . In contrast, sustained light stimula- Consent for publication tion evokes RA rather than SA impulses in almost all Not applicable. ChR2 Nav1.8 -positive Aβ-fiber HTMRs and most Aδ-fiber HTMRs, and these HTMRs may be the first responders Competing interests ChR2 The authors declare no competing interests. There are no financial or other for light-induced pain. Only Nav1.8 -positive C-fiber relationships that may cause a conflict of interest. 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Molecular Brain – Springer Journals
Published: Mar 7, 2023
Keywords: Nav1.8; Optogenetics; Opto-tagged single-fiber recording; High threshold mechanoreceptors (HTMRs); Low threshold mechanoreceptors (LTMRs); Touch; Mechanical pain; Hindpaw glabrous skin
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