Effects of inflammation on the properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent mechanoreceptors in the hindpaw glabrous skin of mice

Abstract

We recently used Nav1.8-ChR2 mice in which Nav1.8-expressing afferents were optogenetically tagged to classify mechanosensitive afferents into Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors. We found that the former were mainly high threshold mechanoreceptors (HTMRs), while the latter were low threshold mechanoreceptors (LTMRs). In the present study, we further investigated whether the properties of these mechanoreceptors were altered following tissue inflammation. Nav1.8-ChR2 mice received a subcutaneous injection of saline or Complete Freund’s Adjuvant (CFA) in the hindpaws. Using the hind paw glabrous skin-tibial nerve preparation and the pressure-clamped single-fiber recordings, we found that CFA-induced hind paw inflammation lowered the mechanical threshold of many Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors but heightened the mechanical threshold of many Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors. Spontaneous action potential impulses were not observed in Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors but occurred in Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors with a lower mechanical threshold in the saline goup, and a higher mechanical threshold in the CFA group. No significant change was observed in the mechanical sensitivity of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors and Nav1.8-ChR2-positive C-fiber mechanoreceptors following hind paw inflammation. Collectively, inflammation significantly altered the functional properties of both Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors, which may contribute to mechanical allodynia during inflammation.

Introduction

Mechanoreceptors in the skin are involved in sensing mechanical stimuli, such as a gentle touch or a strong pinch to the skin. Under pathological conditions, such as tissue inflammation, the properties of mechanoreceptors may be altered, contributing to mechanosensory dysfunctions, including mechanical hyperalgesia and allodynia.^1–3 Previous studies have shown that mechanoreceptors of nociceptive C- and Aδ-afferent fibers become sensitized in hindpaws following inflammation, nerve injury, and skin incision,^1–4 suggesting that peripheral sensitization of mechanoreceptors in nociceptive afferent fibers is an underlying mechanism of mechanical hyperalgesia and allodynia in these pathological conditions. However, numerous studies have also shown a lack of peripheral sensitization of mechanoreceptors in nociceptive C- and Aδ-afferent fibers under pathological conditions, including tissue inflammation and nerve injury.^5,6 These studies proposed that central sensitization in the spinal cord dorsal horn, rather than peripheral sensitization of mechanoreceptors, is the underlying mechanism of mechanical allodynia and hyperalgesia. ⁷ Therefore, it remains controversial whether changes occur in the properties of mechanoreceptors in the periphery to contribute to behavioral hypersensitivity to mechanical stimuli, such as mechanical allodynia and hyperalgesia, under pathological conditions.

Mechanoreceptors in the somatosensory system are classified into several subtypes based on afferent fiber conduction velocities (Aβ-, Aδ-, and C-fibers), mechanical thresholds (low threshold mechanoreceptors, LTMRs; high threshold mechanoreceptors, HTMRs), and impulse adaptation types (slowly adapting, SA; rapidly adapting, RA) to sustained mechanical stimulation. ⁸ LTMRs transduce tactile stimuli at the terminals of non-nociceptive afferent fibers. In the glabrous skin of mice, LTMRs are mainly Aβ-fiber SA-LTMRs, which comprise the Merkel cell-neurite complex, and Aβ-fiber RA-LTMRs, which are the terminates of Meissner’s corpuscles.^8–10 In contrast, HTMRs are usually free nerve endings of nociceptive C- and Aδ-fibers that transduce noxious mechanical stimuli.^7,11,12 Additionally, some HTMRs are found to be Aβ-fibers, and these Aβ-fiber HTMRs may serve as nociceptors for mechanical tissue damage.^12,13 The properties of different LTMRs and HTMRs, particularly their potential changes under pathological conditions such as tissue inflammation, have not been fully characterized.

The studies on different types of mechanoreceptors have been greatly facilitated by transgenic mouse lines that express Cre recombinase under the control of the promoters of specific sensory molecules. For LTMRs, Aβ-fiber SA-LTMRs of Merkel cell-neurite complexes are genetically tagged in the skins of TrkC^creER mice, ¹⁴ Aβ-fiber RA-LTMR of Meissner corpuscles in the glabrous skin of Ret^CreER and TrkB^CreER mice,^15,16 and C-fiber LTMRs in hair follicles in TH^CreER mice. ¹⁷ This genetic approach helps with structural and functional studies of these LTMRs. ¹⁸ HTMRs of nociceptive afferent fibers have also been tagged genetically using Cre mouse lines, including Mrgprd^cre, CGRP^cre, TRPV1^cre, Nav1.8^cre, and others.^8,19 Nav1.8 are voltage-gated Na⁺ channels expressed mainly in small-sized C-fiber nociceptors involved in mechanical and thermal nociception.^20–22 A recent study characterized electrophysiological properties and immunochemical profiles of subpopulations of nociceptive dorsal root ganglion (DRG) neurons using Nav1.8^cre, CGRP^cre, and TRPV1^cre mouse lines. ¹⁹ The study shows that the Nav1.8^cre mouse line labels almost all C-fibers, many Aδ-fibers, and some Aβ-fibers. ¹⁹ Transgenic Cre mice have also been used to express channel rhodopsin 2 (ChR2) in distinct subpopulations of afferents, allowing for opto-tagged electrophysiological studies on the properties of different mechanoreceptors. ¹² We recently generated Nav1.8^cre/ChR2+ (Nav1.8-ChR2) mice and studied the properties of mechanoreceptors of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent fibers innervating the hindpaws of these transgenic mice. ²³ We show that Nav1.8-ChR2-positive mechanoreceptors are mainly HTMRs, including C-fiber and Aδ-fiber HTMRs and Aβ-fiber HTMRs. ²³ On the other hand, we found that mechanoreceptors of Nav1.8-ChR2-negative afferent fibers are LTMRs. ²³ Our results provide direct evidence that, in the mouse glabrous skin, most Nav1.8-ChR2-negative Aβ- and Aδ-fiber mechanoreceptors are LTMRs involving in the sense of touch, whereas Nav1.8-ChR2-positive Aβ-, Aδ-, and C-fiber mechanoreceptors are mainly HTMRs involving in mechanical pain. Thus, Nav1.8-ChR2 mice provide a useful transgenic mouse model to investigate the properties of different mechanoreceptors. In the present study, we aimed to utilize Nav1.8-ChR2 mice to investigate the properties of different types of mechanoreceptors, with a specific focus on examining the changes in their properties following inflammation in the hindpaw glabrous skin of the Nav1.8-ChR2 mice.

Material and methods

Animals

Nav1.8-ChR2 mice, including males and females, were generated by crossing Nav1.8^cre mice with Ai32 (RCL-ChR2 (H134R)/EYFP) mice. The Nav1.8^cre mice were generously provided by Dr John Wood at the University College London and transferred to our laboratory from Dr Stephen Waxman’s lab. at Yale University. Ai32 mice were purchased from Jackson Labs. We performed a crossbreeding of Nav1.8^Cre mice with Ai32 (RCL-ChR2(H134R)/EYFP) mice to establish the Nav1.8^cre+; ChR2-EYFP^loxP/+ mouse line, referred to as Nav1.8-ChR2 hereafter. The care and use of animals in this study conformed to NIH guidelines for the care and use of experimental animals. The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Alabama at Birmingham.

CFA model of inflammatory pain

Nav1.8-ChR2 mice, consisting of males and females aged 8 to 11 weeks, were utilized in this study. They were divided into two groups: the saline group, in which animals were injected with saline, and the CFA group, in which animals were injected with Complete Freund’s Adjuvant (CFA, Sigma-Aldrich, Cat# F5881). The CFA was prepared in a saline solution. Under isoflurane anesthesia, animals were subcutaneously injected with either saline (10 μM) or CFA (10 μM) into the plantar regions of both hindpaws. Behavioral tests were performed on the right or left hindpaws on 0 (before), 1, 3, and 7 days after saline or CFA injection. Skin-nerve preparations were made for electrophysiology recordings 4 to 7 days after CFA injections.

Behavioral assessment

Von Frey test: Each tested animal was placed on an elevated platform with a perforated metal floor (Ugo Basile) and covered by a glass cup measuring 7 cm in diameter and 8.5 cm in height. The animals were allowed to acclimate to the environment for approximately 1 h. The plantar side of the hindpaw was stimulated with calibrated von Frey filaments (North Coast Medical, NC12775-99). The von Frey filaments used had forces of 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, and 1.4 g, and the 50% paw withdrawal thresholds were determined using the Up-Down method. ²⁴

Cotton swab test: The test was performed as previously described. ²³ Each tested animal was briefly covered by a glass cup (7 cm in diameter and 8.5 in height) on an elevated platform with a perforated metal floor (Ugo Basile). The animals were acclimatized to the environment for approximately 1 h. A piece of cotton approximately 12 mm long was glued onto a wooden stick. The hindpaw of the mice was brushed with the cotton swab in a heel-to-toe direction five times, and the frequency of avoidance responses was measured.

Light stimulation: A blue laser beam was applied to the planter surface using an optical fiber (diameter: 0.2 mm: Laserglow technologies). The light intensities were calibrated with an optical power and energy meter (PM100D, Thorlab). The light duration was 50 ms, and the intensities were 3, 5, 10 mW/mm². Light-evoked responses were scored as follows: 0, no response; 1, hindpaw lift; 2, hindpaw flinch, flutter, and hold; 3, jump, vocalization, lick, and guard. The light stimulation at each intensity was applied 3 times to a hindpaw, and the response frequency to the stimulation intensity was then calculated based on the reflexive responses.

The above behavioral experiments were not conducted in a blinded manner because the paws injected with CFA were obviously swollen, and it was challenging to be blinded in our behavioral assessment.

Ex vivo skin-nerve preparations

Animals were anesthetized with 5% isoflurane and then sacrificed by decapitation. The glabrous skin in the plantar and toe regions of the hindpaw and the medial plantar and tibial nerve were dissected using a pair of dissection scissors under a dissection microscope. The skin-nerve preparation was placed in a 60-mm recording chamber with a Sylgard Silicone-coated bottom. Fat, muscle, and connective tissues on the nerves and the skin were carefully removed using forceps. The skin was affixed to the bottom of the chamber with tissue pins, with the epidermis side facing up, while the nerve bundle was affixed with a tissue anchor within the same recording chamber. The cutting end of the nerve bundle was briefly exposed to a mixture of 0.05% dispase II (Roche, Cat# 11534200) plus 0.05% collagenase (Sigma-Aldrich, Cat# C0130) for 30 to 60 s, after which the enzyme was washed off using the normal Krebs solution (see below). This gentle enzyme treatment aimed to facilitate the separation of individual afferent fibers at the cutting end of the nerve bundle, allowing for the aspiration of a single fiber into the recording electrode for pressure-clamped single-fiber recordings (see below). Subsequently, the recording chamber was mounted on the stage of an Olympus BX51WI upright microscope. The skin-nerve preparation was continuously superfused with a normal Krebs bath solution that contained (in mM): 117 NaCl, 3.5 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose. The pH of the solution was adjusted to 7.3, and the osmolarity was adjusted to 325 mOsm. The Krebs bath solution was saturated with 95% O₂ and 5% CO₂. Throughout the experiments, the Krebs bath solution in the recording chamber was maintained at 28 to 32°C.

Pressure-clamped single-fiber recordings

The pressure-clamped single-fiber recording was conducted similarly as described in our previous studies. ²⁵ for detecting action potential (AP) impulses elicited by mechanical and light stimulation. Briefly, the recording electrodes for pressure-clamped single-fiber recordings were made with thin-walled borosilicate glass tubing without filament (inner diameter 1.12 mm, outer diameter 1.5 mm, World Precision Instruments, Sarasota, FL). These electrodes were fabricated using a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument Co., Novato, CA), and the tip of each electrode was fire-polished to a final size of 4 to 10 μm in diameter using a microforge (MF-900, Narishige). The recording electrode, filled with Krebs bath solution, was mounted onto an electrode holder connected to a high-speed pressure-clamp (HSPC) device (ALA Scientific Instruments, Farmingdale, NY) to allow fine controls of intra-electrode pressures. Under a 40x objective, the end of the individual afferent nerve was visualized and isolated by applying a low positive pressure (approximately 10 mmHg or 0.19 lbf/in²) to the recording electrode. Subsequently, the end of a single nerve fiber was aspirated into the recording electrode under a negative pressure of approximately 10 mmHg. Once the nerve fiber’s end entered the recording electrode at approximately 10 µm, the electrode pressure was readjusted to −3 ± 2 mmHg and maintained at this pressure throughout the experiment. Nerve impulses in the single afferent fiber were recorded under the I₀ configuration and amplified using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA) in the AC recording mode (100 x AC membrane potential, 5 x gain, 0.1 Hz AC filter, 3 kHz Bessel filter). The analog signals were digitized by the Digidata 1550B (Molecular Devices) and sampled at a rate of 25 kHz with the Axon Clampex 11 software (Molecular Devices). All experiments were carried out at 30 ± 2°C.

To determine the conduction velocity of the recorded afferent fibers, AP impulses were initiated by electrical stimulation using a bipolar stimulation electrode placed on the tibial nerve bundle. The distance between the site of electrical stimulation and the recording site was approximately 10 to 14 mm. Electrical stimuli consisted of monophasic square pulses generated by an electronic stimulator (Master-9, A.M.P.I, Israel) with a stimulation isolator (ISO-Flex, A.M.P.I, Israel) and delivered to the stimulation electrode. The duration of each stimulation pulse was 200 μs for A-fibers and 2 ms for C-fibers, while the stimulation intensities for evoking impulses were 0.3 - 2.0 mA for A-fiber and 0.65 - 3.0 mA for C-fibers. The spontaneous action potentials were recorded for 1 min.

Mechanical and light stimulation

For a recorded afferent fiber, its mechanosensitive site (receptive field) in the hindpaw glabrous skin was first located using a glass rod. Poking the mechanosensitive receptive field of the recorded afferent fiber with the glass rod would produce APs that the recording electrode could detect. In the present study, all data was collected from mechanosensitive receptive sites, that is, mechanoreceptors, in the glabrous skin of the hindpaw. Once a mechanoreceptor was identified, mechanical stimulation was applied to the same receptive field using a force-calibrated mechanical indenter (300C-I, Aurora scientific) to determine mechanical thresholds. The tip of the indenter had a diameter of 0.8 mm. The indenter was connected to a Digidata 1550B Digitizer to enable the generation of ramp-and-hold mechanical stimulation commands by the pClamp 11 software. Before applying mechanical stimulation, the indenter tip was gently lowered to the surface of the receptive field with a 10-mN force. Subsequently, the 10-mN force was canceled to 0, ensuring that the tip of the indenter contacted the surface of the receptive field without applying force. Under the force control module, ramp-and-hold mechanical stimuli were applied to the mechanoreceptor of the glabrous skin. The step force commanders were calibrated by applying indenter at toe tips, paw pads, and other areas of the plantar skin. The actual forces applied to these skin areas were measured and used. The ramp-and-hold force steps were at 0, 5, 30, and 80 mN. The ramp duration (dynamic phase) was 10 ms, and the holding duration (static phase) was 0.98 s. The minimal force at which AP impulses were elicited was defined as the mechanical threshold of the mechanoreceptors.

To determine whether a mechanoreceptor was from Nav1.8-ChR2-positive or Nav1.8-ChR2-negative afferent fibers, the same mechanosensitive receptive field was stimulated using a blue LED light (Thorlab; M455L4, 455 nm) to test its light sensitivity. If light stimulation also evoked impulses, the mechanoreceptor was from Nav1.8-ChR2-positive afferent fibers. Otherwise, if no impulses were evoked by light stimulation, the mechanoreceptor was from light-insensitive or Nav1.8-ChR2-negative afferent fibers. The blue light was applied to the mechanoreceptor through a 40x objective, with a 1-s pulse of light stimulation at intensities of 1, 10, and 50 mW. Afferent impulses evoked by mechanical and light stimulation were recorded using pressure-clamped single-fiber recordings. The signals were amplified using the Multiclamp 700B amplifier in the AC recording mode (100 x AC membrane potential, 5 x gain, 0.1 Hz AC filter, 3 kHz Bessel filter) and sampled at a rate of 25 kHz with the Axon Clampex 11 software (Molecular Devices, Sunnyvale, CA, USA).

Data analysis

Electrophysiological data were analyzed using Clampfit 11 (Molecular Devices, Sunnyvale, CA, USA). The data were collected from 28 male and 34 female animals and were aggregated for data analysis. To confirm that impulses evoked by blue light and mechanical stimulators (indenter) originated from the same receptive field, the amplitudes and shapes of the impulses evoked by both stimuli were compared to ensure a match between the mechanically evoked impulses and the light-evoked impulses. The conduction velocity (CV) was calculated as the distance between the stimulation and recording sites divided by the time taken to elicit an AP impulse following electrical stimulation. Afferent fibers were classified as Aβ-fibers with CV >9 m/s, Aδ-fibers with CV between 1.2 and 9 m/s, and C-fibers with CV ≤1.2 ms. ²³ All data analyses were performed using Graph Pad Prism (version 8). Unless otherwise indicated, all data were reported as individual observations and mean ± SEM of n independent observations. Statistical significance was evaluated using the Two–way ANOVA with Bonferroni’s post hoc test for multiple group comparison, Mann-Whitney (nonparametric) test, or Student's t-tests for two group comparisons and Chi-square test. Differences were considered significant with *p < .05, **p < .01, ***p < .001, and not significant (ns) with p ≥ .05.

Results

Hindpaw responses to mechanical and light stimulation in Nav1.8-ChR2 mice injected with saline or CFA

We first used the von Frey test and the cotton swab test to determine the mechanical sensitivity of the hindpaws in Nav1.8-ChR2 mice injected with either saline (saline group, control) or CFA (CFA group). In the von Frey test, we measured the 50% threshold. For the saline group, the 50% threshold was 6.1 ± 0.8 mN before saline injection and remained at similar levels 1, 3, and 7 days following saline injection (n = 7, Figure 1(a)). In the CFA group, the 50% threshold was 4.9 ± 0.4 mN before CFA injection and significantly reduced to 1.3 ± 0.4 mN at 1 day, 0.16 ± 0.05 mN at 3 days, and 0.21 ± 0.02 mN at 7 days following CFA injection (Figure 1(a), n = 9). While the von Frey test was primarily used to assess static mechanical allodynia following CFA-induced tissue inflammation in the hindpaws of these Nav1.8-ChR2 mice, we used the cotton swab test to determine dynamic mechanical allodynia in the hindpaws following CFA injection (Figure 1(b)). In the saline group, the frequency of responses to cotton swab strikes on the hindpaws was approximately 40% from day 0 to day 7 following saline injection (n = 7). However, the response frequency significantly increased to over 70% at 1 day after CFA injection and reached a maximum response frequency of 100% at 7 days after CFA injection (n = 9, Figure 1(b)).

Figure 1.

Hindpaw responses to mechanical and light stimulation in Nav1.8-ChR2 mice of the saline group and CFA group. The 50% threshold of hindpaw avoidance responses assessed by the von Frey test performed in Nav1.8-ChR2 mice at 0, 1, 3, 7 days after saline injection (n = 7) or CFA injection (n = 9) into hindpaws. The frequency of hindpaw avoidance responses assessed by the cotton swab test in the saline group (n = 7) and CFA group (n = 9). The frequency of hindpaw avoidance response to blue light stimulations with intensities of 3 (c), 5 (d), and 10 (e) mW/mm². The tests were performed before (day 0) and 1, 3, and 7.days after the injection of saline (n = 7) or CFA (n = 9). Similar to C-E, except that the hindpaw avoidance was scored. Data represent mean ± SEM, **p < .01, ***p < .001, two-way ANOVA with Bonferroni’s post hoc test.

Next, we determined the light sensitivity of the hindpaws of Nav1.8-ChR2 mice by shining a short pulse (50 ms) of blue laser light at intensities of 3, 5 and 10 mW/mm². In both the saline and CFA groups, animals responded to the light stimulation by rapidly lifting their hindpaws to avoid the light. The frequency of the avoidance response increased in a light intensity-dependent manner (Figure 1(c)–(e)). There were no significant differences in the response frequencies to the light stimulation between the saline and CFA groups during 7 days following the injections of saline and CFA (Figure 1(c)–(e)). Similarly, when the avoidance responses were scored, the average scores for the responses induced by the light stimulation at 3, 5, and 10 mW/mm² did not show a significant difference between the saline and CFA groups (Figure 1(f)–(h)).

Characterization of light-sensitivity and receptive fields of the mechanoreceptors in the hindpaw glabrous skin of Nav1.8-ChR2 mice injected with saline or CFA

To explore the roles of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative afferent fibers in mechanical behavioral responses, we utilized ex vivo skin-nerve preparations made from the hindpaw plantar skin and the tibial nerves of Nav1.8-ChR2 mice in the saline group and CFA group (Figure 2(a)). Pressure-clamped single-fiber recordings were conducted on the tibial nerve fibers to assess various properties, including mechanical sensitivity, conduction velocity, light sensitivity, and the location of the mechanical receptive field (Figure 2(a)). Only mechanosensitive fibers that responded to mechanical stimulation by generating action potential (AP) impulses were included, while mechano-insensitive fibers were excluded. Mechanoreceptors were classified into Aβ-fiber (CV >9 m/s), Aδ-fiber (9 m/s > CV >1.2 m/s), and C-fiber mechanoreceptors (CV <1.2 m/s) based on conduction velocity (Figure 2(b)). Mechanoreceptors were further categorized as Nav1.8-ChR2-positive (light-sensitive) and Nav1.8-ChR2-negative (light-insensitive) mechanoreceptors based on their responsiveness to blue light stimulation (Figure 2(b)). Among all Aβ-fiber mechanoreceptors examined, a small percentage (40% in the saline group vs 27% in the CFA group, no significant difference) were Nav1.8-ChR2-positive, while the majority were Nav1.8-ChR2-negative (Figure 2(b)). For Aδ-fiber mechanoreceptors, most of them (73% in saline group vs 80% in CFA group, no significant difference) were Nav1.8-ChR2-positive, and the remaining were Nav1.8-ChR2-negative (Figure 2(b)). All the C-fiber mechanoreceptors examined in the saline and CFA groups were Nav1.8-ChR2-positive, with a percentage of 100% in both groups (Figure 2(b)). We examined the locations (plantar vs toe) of the mechanically sensitive spots (mechanical receptive fields) for different types of mechanoreceptors in the saline group and CFA group. Among Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors, the majority of mechanical receptive fields were found in the toe region (light red, 12/19 in saline group vs 9/16 in CFA group), with a smaller percentage in the plantar region (dark red, 7/19 in saline group vs 7/16 in CFA group) (Figure 2(c), left panel). The distribution of these mechanoreceptors between the saline group and the CFA group did not show a significant difference. For Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors, 10/18 mechanical receptive fields in the saline group and 5/17 mechanical receptive fields in the CFA group were found in the toe region. In contrast, 8/18 in saline group and 12/17 in CFA group were found in the plantar region (Figure 2(c) middle panel). Although more Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors were observed in the plantar region of the CFA group, there was no statistically significant difference compared to the saline group. For Nav1.8-ChR2-positive C-fiber mechanoreceptors, the saline group displayed an almost equal number in the toe (8/17) and plantar regions (9/17). However, in the CFA group, most mechanical receptive fields were detected in the plantar region (14/16), with only a very small portion (2/16) found in the toe region, which was significantly different from the saline group (Figure 2(c) right panel). We also examined the locations of the mechanical receptive fields of Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors in the saline group and CFA group. For Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors, the majority of them were found in the toe region (16/23 in the saline group vs 22/26 in CFA, not significantly different), with a small portion in the plantar region (Figure 2(d), left panel). Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors were almost equally distributed in the toe and plantar regions in the saline and CFA groups (Figure 2(d), right panel).

Figure 2.

Light-sensitivity and receptive field of mechanosensitive Aβ-, Aδ-, and C-fibers in the hindpaw glabrous skin of Nav1.8-ChR2 mice injected with saline or CFA. Schematic diagram illustrating the experimental procedures and setup for single-fiber recordings from the tibial nerves in the glabrous skin-nerve preparations of Nav1.8-ChR2 mice. Conduction velocities were determined to classify fibers into Aβ-, Aδ-, and C-fibers. Mechanical sensitive receptive sites were first identified, followed by testing light sensitivity at those sites. Nav1.8-ChR2-positive mechanoreceptors were responsive to light, while Nav1.8-ChR2-negative mechanoreceptors were not. Percentage of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative mechanoreceptors in the saline-injected group and CFA-injected group. Left panel: Aβ-mechanoreceptors. Middle panel: Aδ-mechanoreceptors. Right panel: C-mechanoreceptors. The number of recordings is indicated in the bars. Regional distributions (plantar vs toe) of Nav1.8-ChR2-positive mechanoreceptors in the saline and CFA groups. Regional distributions (plantar vs toe) of Nav1.8-ChR2-negative mechanoreceptors in the saline and CFA groups. Saline or CFA was subcutaneously injected into the plantar region of the hindpaws. The number in each bar indicates the number of recorded mechanoreceptors, *p < .05, ns, not significantly different, Chi-squared test.

Characterization of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors

Our previous studies have suggested that Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors mainly represent the high threshold mechanoreceptors (HTMRs) of Aβ-afferent fibers. ²³ To determine whether tissue inflammation may alter their functional properties, we characterized these Aβ-fiber mechanoreceptors in the saline group and CFA group. In the saline group, the majority of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors (15/18 recordings) displayed slowly adapting AP impulses (SA-mechanoreceptors), while only a small portion exhibited rapidly adapting AP impulses (RA-mechanoreceptors) in response to ramp-and-hold mechanical steps (Figure 3(a)), which is consistent with our previous study. ²³ Similarly, in the CFA group, most Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors (14/16 recordings) were SA mechanoreceptors (Figure 3(a)), and there was no significant change in the proportion of SA-mechanoreceptors and RA-mechanoreceptors compared to the saline group (Figure 3(a)). The conduction velocities of Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors were 12.8 ± 1.2 m/s (n = (3) in the saline group, and 11.5 ± 0.1 m/s (n = (2) in the CFA group, and a comparison could not be made due to the small numbers of these mechanoreceptors encountered. The conduction velocities of Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors were 11.2 ± 0.4 m/s (n = 15) in saline group and 11.4 ± 0.3 m/s (n = 14) in CFA group, and were not significantly different between the two groups. Mechanical thresholds, the minimum step forces of mechanical indenter that induced AP impulses, were measured for Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors and SA-mechanoreceptors. The mechanical thresholds of Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors were 32.3 ± 11.2 mN (n = (3) in the saline group and 14.7 ± 3.4 mN (n = (2) in CFA group. Although the mechanical thresholds of Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors appeared lower in the CFA group than in the saline group, the sample sizes were too small to perform the statistical comparison. The mechanical thresholds of Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors were 34.0 ± 5.2 mN (n = 15) in the saline group and 19.5 ± 4.6 mN (n = 14) in the CFA group, and the mechanical thresholds were significantly lower in the CFA group than in the saline group (p < .05, Figure 3(c)). Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors showed increased AP impulse frequencies in a mechanical force-dependent manner in response to the ramp-and-hold indentation applied to the receptive fields. However, there was no significant difference between the saline group (n = 11) and the CFA group (n = 12, Figure 3(d) and (e)).

Figure 3.

The properties of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors in the saline group and CFA group. Percentage of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors exhibiting SA (dark blue) or RA (light blue) responses in the saline group and CFA group. The number in each bar indicates the number of recorded Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors. Conduction velocity of Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline group and CFA group. Mechanical thresholds for evoking AP impulses in Nav1.8-ChR2-positive Aβ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline and CFA groups. Top panel: Sample traces showing AP impulses evoked by indentation steps with forces of 5 (left), 30 (middle), and 80 mN (right) in a Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptor in the saline group. Bottom panel: Similar to the top panel, recordings were made from a Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptor in the CFA group. Summary data of the frequency of AP impulses evoked by different indentation forces in Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors in the saline group (n = 11) and CFA group (n = 12). Examples of transient AP impulses of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors evoked by blue LED light in the saline group (left) and CFA group (right). The blue light was applied to the mechanosensitive receptive field of Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors for 1.s at a light intensity of 1 (top), 10 (middle), and 50 mW (bottom). Percentage of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptor showing light-evoked transient impulses (1 or 2 impulses at the beginning of light stimulation) in the saline group and CFA group. Summary data of the frequency of AP impulses of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors evoked by blue light at intensities of 1, 10, and 50 mW in the saline group (n = 16) and CFA group (n = 16). Data represent individual observations and mean ± SEM, *p < .05, unpaired Studentt test or two-way ANOVA with Bonferroni’s post hoc test.

We next determined the responses to light stimulation in the Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors in both the saline and CFA groups. In this set of experiments, AP impulses were evoked by a beam of blue LED light that was applied to the mechanical receptive fields of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors. The blue light intensities were 1, 10, and 50 mW, and each stimulation lasted for 1 s (Figure 3(f)). While the majority of Nav1.8-ChR2-positive Aβ-fiber SA-mechanoreceptors exhibited SA impulses in response to the ramp-and-hold mechanical indentation (Figure 3(a)), all of them displayed transient AP impulses (1 or 2 impulses) at the beginning of light stimulation, both in the saline group (n = 16) and CFA group (n = 16, Figure 3(f) and (g)). The blue light at 1 mW was subthreshold primarily. In contrast, the 10 mW and 50 mW intensities elicited a single AP impulse in most cases, both in the saline group (n = 16) and CFA group (n = 16), and there was no significant difference between the two groups (Figure 3(h)).

Characterization of Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors

We characterized Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors in both the saline and CFA groups to investigate whether tissue inflammation affects the properties of these mechanoreceptors. Most Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors were identified as SA-mechanoreceptors, with 17 out of 18 in the saline group and 16 out of 17 in the CFA group displaying this property (Figure 4(a)). Only a small portion of these afferent fibers were RA-mechanoreceptors in both groups (Figure 4(a)). The conduction velocities of the single sample of Nav1.8-ChR2-positive Aδ-fiber RA-mechanoreceptors were 4.4 m/s in the saline group and 6.1 m/s in the CFA group. For Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptors, their conduction velocities were 5.1 ± 0.5 m/s (n = 17) in the saline group and 4.7 ± 0.5 m/s (n = 16) in the CFA group, and there was no significant difference observed between the two groups (Figure 4(b)). The mechanical thresholds of the single sample of Nav1.8-ChR2-positive Aδ-fiber RA-mechanoreceptors were 2.1 mN in the saline group and 85.9 mN in the CFA group. As for Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptors, their mechanical thresholds were 21.0 ± 5.1 mN (n = 17) in the saline group and 19.9 ± 5.6 mN (n = 16) in the CFA group, and no significant difference was observed between the two groups (Figure 4(c)). In response to ramp-and-hold mechanical indentation applied to the mechanical receptive fields, Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptors displayed increased frequencies of AP impulse dependent on mechanical force, with no significant difference between the saline group (n = 12) and CFA group (n = 10) (Figure 4(d) and (e)). While nearly all Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors showed SA AP impulses in response to the mechanical indentation, the majority of them displayed transient AP impulses in response to blue light stimulation, both in the saline group and CFA group (Figure 4(f) and (g)). A small portion of Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors showed multiple AP impulses over the entire period of light stimulation (sustained AP impulses). The frequencies of light-evoked transient AP impulses showed no significant difference between the saline group (n = 9) and CFA group (n = 10) (Figure 4(h)). Similarly, the frequencies of light-evoked sustained AP impulses were also not significantly different between the saline group (n = 4) and CFA (n = 4) (Figure 4(h)).

Figure 4.

The properties of Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors in the saline group and CFA group. Percentage of Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors exhibiting SA (dark blue) or RA (light blue) responses in the saline group and CFA group. The number in each bar indicates the number of recorded Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors. Conduction velocity of Nav1.8-ChR2-positive Aδ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline group and CFA group. Mechanical thresholds for evoking AP impulses in Nav1.8-ChR2-positive Aδ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline and CFA groups. Top panel: Sample traces showing AP impulses evoked by indentation steps with forces of 5 (left), 30 (middle), and 80 mN (right) in a Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptor in the saline group. Bottom panel: Similar to the top panel, but recordings were made from a Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptor in the CFA group. Summary data of the frequency of AP impulses evoked by different indentation forces in Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptors in the saline group (n = 12) and CFA group (n = 10). Sample traces showing AP impulses of Nav1.8-ChR2-positive Aδ-fiber SA-mechanoreceptors evoked by a blue LED light in the saline group (left) and CFA group (right). The blue light was applied to the mechanosensitive receptive field of Aδ-fiber SA-mechanoreceptors for a duration of 1 s at light intensities of 1 (top), 10 (middle), and 50 mW (bottom). Percentage of the Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors showing light-evoked transient impulses (light blue) and sustained impulses (multiple impulses during the entire period of light stimulation) (dark blue) in the saline group and CFA group. The number in each bar indicates the number of recorded Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors. The frequency of AP impulses evoked by blue light at 1, 10, and 50 mW in Aδ-fiber mechanoreceptors of the saline group (dashed lines) and CFA group (solid lines). The light-evoked AP impulses were subdivided into transient type (n = 9 in the saline group, n = 10 in the CFA group) and sustained type (n = 4 in the saline group, n = 4 in the CFA group). Data represent individual observations and mean ± SEM, ns, not significantly different, unpaired Studentt test, Mann-Whitney U test, or two-way ANOVA with Bonferroni’s post hoc test.

Characterization of Nav1.8-ChR2-positive C-fiber mechanoreceptors

Nav1.8-ChR2-positive C-fiber mechanoreceptors were characterized in the saline and CFA groups to explore whether CFA-induced tissue inflammation affected their properties. Most Nav1.8-ChR2-positive C-fiber mechanoreceptors were identified as SA-mechanoreceptors, with only a small portion displaying RA-mechanoreceptors in both the saline and CFA groups (Figure 5(a)). No significant difference in the proportion of SA and RA types was observed between the two groups (Figure 5(a)). The conduction velocity of the single recording of Nav1.8-ChR2-positive C-fiber RA mechanoreceptors was 0.92 m/s in the saline group and 0.62 ± 0.08 m/s (n = 4) in the CFA group (Figure 5(b)). For Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors, their conduction velocities were 0.54 ± 0.04 m/s (n = 16) in the saline group and 0.51 ± 0.07 m/s (n = 11) in the CFA group, with no significant difference observed between the two groups (Figure 5(b)). The mechanical threshold of Nav1.8-ChR2-positive C-fiber RA-mechanoreceptors was 66.4 mN in the single recording in the saline group and 44.2 ± 10.2 mN (n = 4) in the CFA group, and statistical comparison could not be performed due to the small sample sizes. As for Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors, their mechanical thresholds were 15.1 ± 4.5 mN (n = 16) in the saline group and 15.4 ± 4.7 mN (n = 11) in CFA group, with no significant difference observed between the two groups (Figure 5(c)). In response to ramp-and-hold indentation applied to the receptive fields, Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors displayed increased frequencies of AP impulse in a mechanical force-dependent manner in both the saline group (n = 10) and CFA group (n = 8), with no significant difference between the two groups (Figure 5(d) and E). Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors displayed sustained AP impulses in response to blue light stimulation in both the saline group (n = 15) and CFA group (n = 16) (Figure 5(f) and (g)). The frequencies of AP impulses evoked by 10 and 50 mW light stimulation were significantly higher in the CFA group (n = 15) compared to the saline group (n = 11, Figure 5(h)).

Figure 5.

The properties of Nav1.8-ChR2-positive C-fiber mechanoreceptors in the saline group and CFA group. Percentage of Nav1.8-ChR2-positive C-fiber mechanoreceptors exhibiting RA (light blue) or SA (dark blue) responses in the saline group. Conduction velocity of Nav1.8-ChR2-positive C-fiber RA-mechanoreceptors (Left panel) and SA-mechanoreceptors (Right panel) in the saline group and CFA group. Mechanical thresholds for evoking AP impulses in Nav1.8-ChR2-positive C-fiber RA-mechanoreceptors (Left panel) and SA-mechanoreceptors (Right panel) in the saline and CFA groups. Sample traces showing AP impulses evoked by indentations in Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors in the saline group (top) and CFA group (bottom). The indentation forces were 5 (left), 30 (middle), and 80 mN (right). The frequency of AP impulses evoked by different indentation forces (5, 30, and 80 mN) in Nav1.8-ChR2-positive C-fiber SA-mechanoreceptors in the saline group (n = 10) and CFA (n = 8) group. Sample traces showing AP impulses evoked from Nav1.8-ChR2-positive C-fiber mechanoreceptors by blue light at intensities of 1 mW (top), 10 mW (middle), and 50 mW (bottom) in the saline group (left) and CFA group (right). The light stimulation was applied for 1 s to the mechanosensitive receptive fields of Nav1.8-ChR2-positive C-fiber mechanoreceptors. The percentage of Nav1.8-ChR2-positive C-fiber mechanoreceptors showing sustained responses to the light stimulation in the saline group (n = 15) and CFA group (n = 16). The frequency of AP impulses evoked by blue light applied to Nav1.8-ChR2-positive C-fiber mechanoreceptors in the saline group (n = 11) and CFA group (n = 15). The blue light was applied at intensities of 1, 10, and 50 mW for a duration of 1 s in each test. Data represent individual observations and mean ± SEM, **p < .01, ns, not significantly different, Mann-Whitney U test or two-way ANOVA with Bonferroni’s post hoc test.

Spontaneous AP impulses were observed in many Nav1.8-ChR2-positive C-fiber mechanoreceptors (Figure 6(a) and (b)) but were not observed in Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors and Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors (data not shown). In the saline group, spontaneous AP impulses were observed in 10 out of 16 (10/16) Nav1.8-ChR2-positive C-fiber mechanoreceptors, and in the CFA group, they were observed in 10 out of 15 (10/15) Nav1.8-ChR2-positive C-fiber mechanoreceptors. No significant difference in the occurrence of spontaneous AP impulses was found between the saline group and the CFA group (Figure 6(b)). The frequencies of the spontaneous AP impulses recorded from Nav1.8-ChR2-positive C-fiber mechanoreceptors were 0.16 ± 0.06 Hz (n = 10) in the saline group and 0.11 ± 0.05 Hz (n = 10) in the CFA group, and no significant difference was observed between the two groups (Figure 6(c)). When comparing the mechanical thresholds of Nav1.8-ChR2-positive C-fiber mechanoreceptors with spontaneous AP impulses between the saline and CFA groups (Figure 6(d)), it was found that the mechanical threshold in CFA group was marginally higher than that in the saline group (Figure 6(d), (p) = .05).

Figure 6.

The properties of spontaneous AP impulses in Nav1.8-ChR2-positive C-fiber mechanoreceptors in the saline group and CFA group. Sample traces showing spontaneous AP impulses recorded from Nav1.8-ChR2-positive C-fiber mechanoreceptors in the saline group (top) and CFA group (bottom). The percentage of Nav1.8-ChR2-positive C-fiber mechanoreceptors without (grey) and with (black) spontaneous AP impulses in the saline and CFA groups. The number in each bar indicates the number of recordings. The frequency of spontaneous AP impulses recorded from Nav1.8-ChR2-positive C-fiber mechanoreceptors in the saline and CFA groups. Comparison between the saline and CFA groups for the mechanical threshold of Nav1.8-ChR2-positive C-fiber mechanoreceptors with spontaneous impulses. Data represent individual observations and mean ± SEM, *p < .05, ns, not significantly different, Man-Whitney U test or Chi-square test.

Characterization of Nav1.8-ChR2-negative mechanoreceptors

We characterized Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors in both the saline and CFA groups to investigate whether CFA-induced tissue inflammation may affect their properties. Most Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors were SA-mechanoreceptors, and a small portion of them exhibited RA-mechanoreceptor properties in both groups (Figure 7(a)); no significant difference was observed in the proportion of SA and RA types between the saline and CFA groups. The conduction velocities of Nav1.8-ChR2-negative Aβ-fiber RA mechanoreceptors were 13.5 ± 1.2 m/s (n = 8) in the saline group and 13.5 ± 1.0 m/s (n = 10) in the CFA group. The conduction velocities of Nav1.8-ChR2-negative Aβ-fiber SA mechanoreceptors were 13.1 ± 0.4 m/s (n = 15) in the saline group and 12.5 ± 0.5 m/s (n = 16) in the CFA group, with no significant difference observed between the two groups. As for mechanical threshold, the Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors had thresholds of 6.2 ± 2.0 mN (n = 8) in the saline group and 9.6 ± 2.1 mN (n = 10) in the CFA group, with no significant difference between the two groups (Figure 7(c) left). However, Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors had a threshold of 1.4 ± 0.5 mN (n = 15) in the saline group and 2.9 ± 0.7 mN (n = 16) in the CFA group. The thresholds in the CFA group were significantly higher than those in the saline group (Figure 7(c), right). For AP impulse frequencies in response to mechanical steps, Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors displayed slight increases in both the saline group (n = 7) and CFA group (n = 8), with no significant difference observed between the two groups (Figure 7(d) and (e)). Similarly, Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors showed increases in AP impulse frequencies in a mechanical force-dependent manner in response to the ramp-and-hold indentation in both the saline group (n = 11) and CFA group (n = 13), with no significant difference between the two groups (Figure 7(f) and (g)).

Figure 7.

The properties of Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors in the saline group and CFA group. Percent of Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors exhibiting RA (grey) or SA (black) responses in the saline group and CFA group. The numbers of recordings are indicated in the bars. Conduction velocity of Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors (Left panel) and SA-mechanoreceptors (Right panel) in the saline group and CFA group. Mechanical thresholds for evoking AP impulses in Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline and CFA groups. Sample traces showing AP impulses evoked by indentations in Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors in the saline group (top) and CFA group (bottom). Indentation forces are indicated under each sample trace. The frequency of AP impulses evoked by different indentation forces in Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors in the saline group (n = 7) and CFA group (n = 8), with indentation forces of 5, 30, and 80 mN. Similar to D&E, except the recordings were made from Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors in the saline (n = 11) and CFA group (n = 13). Data represent individual observations and mean ± SEM, *p < .05. Man-Whitney U test, unpaired Studentt test, or two-way ANOVA with Bonferroni’s post hoc test.

Spontaneous AP impulses were observed in Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors (Figure 8(a) and (b)), while not in Nav1.8-ChR2-negative Aβ-fiber RA-mechanoreceptors (not shown). In the saline group, spontaneous AP impulses were observed in 8 out of 13 (8/13) Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors. In the CFA group, they were observed in 7 out of 14 (7/14) Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors (Figure 8(b)). No significant difference was observed in the occurrence of spontaneous AP impulses between the saline and CFA groups (Figure 8(b)). The frequencies of the spontaneous AP impulses recorded from Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors were 0.3 ± 0.1 Hz in the saline group and 0.5 ± 0.2 Hz in the CFA group, with no significant difference between the two groups (Figure 8(c)). However, when comparing the mechanical thresholds of Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors with spontaneous AP impulses between the saline and CFA groups (Figure 8(d)), it was found that the mechanical thresholds were significantly higher in the CFA group (1.8 ± 0.5 mN, n = 7, p < .05) compared to the saline group (0.5 ± 0.2 mN, n = 8) (Figure 8(d)).

Figure 8.

The properties of spontaneous activities in Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors in the saline group and CFA group. Sample traces showing spontaneous AP impulses recorded from Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors in the saline group (top) and CFA group (bottom). Percent of Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors with (black) and without (grey) spontaneous AP impulses in the saline and CFA groups. The numbers of recordings are indicated in the bars. The frequencies of spontaneous AP impulses in Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors in the saline and CFA groups. Mechanical thresholds of Nav1.8-ChR2-negative Aβ-fiber mechanoreceptors with spontaneous AP impulses in the saline group (left panel) and CFA group (right panel). Data represent individual observations and mean ± SEM, *p < .05, ns, not significantly different, unpaired Studentt test or Mann-Whitney U test.

Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors were characterized in both the saline group and the CFA group. Most Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors were RA-mechanoreceptors, with a smaller portion being SA-mechanoreceptors in both groups (Figure 9(a)). No significant difference was observed in the proportion of SA-mechanoreceptors and RA-mechanoreceptors between the saline group and the CFA group (Figure 9(a)). The conduction velocities of Nav1.8-ChR2-negative Aδ-fiber RA mechanoreceptors were 6.4 ± 0.5 m/s (n = 9) in the saline group and 6.8 ± 0.4 m/s (n = 9) in the CFA group, with no significant difference between the two groups. The conduction velocities of Nav1.8-ChR2-negative Aδ-fiber SA mechanoreceptors were 6.6 ± 0.7 m/s (n = 3) in the saline group and 8.3 m/s in a single recording in the CFA group. The mechanical thresholds of Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors were 0.7 ± 0.2 mN (n = 9) in the saline group and 1.1 ± 0.3 mN (n = 9) in the CFA group, and no significant difference was found between the two groups. The mechanical thresholds of Nav1.8-ChR2-negative Aδ-fiber SA-mechanoreceptors were 0.3 ± 0.1 mN (n = 3) in the saline group and 1.5 mN in a single recording in the CFA group. Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors exhibited slight increases of AP impulse frequencies with larger mechanical forces in both saline group (n = 9) and CFA group (n = 8), with no significant difference between the two groups (Figure 9(d) and (e)). No spontaneous AP impulses were observed in Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors (not shown).

Figure 9.

The properties of Nav1.8-ChR2-negative Aδ-mechanoreceptors in the saline group and CFA group. Percent of Nav1.8-ChR2-negative Aδ-mechanoreceptors exhibiting RA or SA responses in the saline and CFA groups. The numbers of recordings are indicated in the bars. Conduction velocity of Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors (left panel) and SA-mechanoreceptors (right panel) in the saline group and CFA group. Mechanical thresholds required for evoking AP impulses in Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors (Left panel) and SA-mechanoreceptors (Right panel) in the saline and CFA group. Sample traces showing AP impulses (RA type) evoked by mechanical indentations in Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors in the saline group (top) and CFA group (bottom). The frequency of AP impulses evoked by different mechanical indentations in Nav1.8-ChR2-negative Aδ-fiber RA-mechanoreceptors in the saline group (n = 9) and CFA group (n = 8). In D and E, the mechanical indentations were applied at 5, 30, and 80 mN. Data represent individual observations and mean ± SEM, ns, not significantly different, unpaired Studentt test or two-way ANOVA with Bonferroni’s post hoc test.

Discussion

A novel finding of the present study is the significant reduction of the mechanical threshold of Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors following the hindpaw inflammation induced by CFA. Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors typically have high mechanical thresholds, indicating their classification as high threshold mechanoreceptors (HTMRs) and suggesting their potential involvement as Aβ-fiber mechano-nociceptors (also see 23). While most nociceptors are C- and Aδ-afferent fibers,^11,26 previous studies have reported the presence of Aβ-fiber nociceptors.^13,27 These Aβ-fiber nociceptors are considered the first responder to noxious mechanical stimulation, such as pinching and pinpricks on the skin. ²⁷ Recently, through recordings from the somas of large-sized trigeminal neurons, we have identified nociceptive-like Aβ-afferent trigeminal neurons that exhibited very low mechanical sensitivity.^28,29 In the present study, we demonstrate that Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors normally have high mechanical thresholds; these thresholds are significantly reduced following hindpaw inflammation induced by CFA. The reduction in mechanical threshold in these Aβ-fiber HTMRs may contribute to behavioral mechanical hypersensitivity following hindpaw inflammation.

In contrast to Nav1.8-ChR2-positive Aβ-fiber mechanoreceptors, we have found that the mechanical threshold of Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors increases following tissue inflammation. Due to their low mechanical thresholds, nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors are predominantly categorized as LTMRs. The elevation of the mechanical threshold in Aβ-fiber LTMRs would result in reduced mechanical sensitivity under inflammatory conditions. The pathological significance of reduced mechanical sensitivity in Aβ-LTMRs during tissue inflammation remains unclear. However, the decreased mechanical sensitivity in Aβ-LTMRs would diminish the tactile sensory inputs to the spinal cord dorsal horn, potentially leading to a decrease in the gate-control effects of Aβ-fiber LTMRs and contributing to central sensitization of nociceptive signals. Our findings also reveal that some Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors exhibit spontaneous impulses. These fibers exhibit higher mechanical thresholds in the CFA group than the saline group, which aligns with the observed elevation of mechanical thresholds in Nav1.8-ChR2-negative Aβ-fiber SA-mechanoreceptors following CFA-induced inflammation.

In the present study, we did not observe any changes in the functional properties of Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors. Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors typically exhibit high mechanical thresholds and are likely involved in mechanical nociception, while Nav1.8-ChR2-negative Aδ-fiber mechanoreceptors have low mechanical thresholds and function as LTMRs. Our study demonstrates that nearly all Nav1.8-ChR2-positive Aδ-fiber mechanoreceptors are SA in both the saline and CFA groups. We found no significant differences between the saline and CFA groups’ mechanical thresholds or responses to suprathreshold stimulation. Similarly, we observed that the functional properties of Nav1.8-ChR2-positive C-fiber mechanoreceptors were unaffected by CFA-induced hindpaw inflammation. These findings align with previous studies,^5,6 showing that tissue inflammation does not alter the properties of Aδ-fiber and C-fiber mechanoreceptors. It is worth noting that previous studies have demonstrated different results regarding sensitization of C- and Aδ-fiber mechanoreceptors at peripheral sites under various pain models, such as neuropathic pain, skin incision pain model, and inflammatory pain.^1–4 While some studies have reported enhanced suprathreshold responses or mechanical hypersensitivity at peripheral sites of C- and Aδ-fiber nociceptors in these pain models, others have not observed changes in mechanical threshold or suprathreshold mechanical responses.^5,6 The discrepancy may be due to different pathological conditions. Therefore, it would be interesting to investigate whether Nav1.8-ChR2-positive and Nav1.8-ChR2-negative Aδ- and C-fiber mechanoreceptors may exhibit changes in mechanical sensitivity at peripheral sites under other pathological conditions, such as nerve injury.

In contrast to mechanically evoked responses, our study revealed that the frequency of impulses evoked by light stimulation is significantly higher in the CFA group than in the saline group. This suggests that the excitability of C-fiber mechanoreceptors may increase following tissue inflammation. Alternatively, ChR2 expression may be upregulated in C-fiber mechanoreceptors under the inflammatory condition. Further investigation is warranted to elucidate the mechanisms underlying these observations.

Our study observed spontaneous impulses in many Nav1.8-ChR2-positive C-fiber mechanoreceptors in both the saline and CFA groups. Previous studies have also reported the occurrence of spontaneous impulses in nociceptive C-fibers, although they are typically observed in a small subset of nociceptive C-fibers. ¹ However, other studies have observed a relatively higher percentage of C-fibers displaying spontaneous impulses. ⁴ The variations in the occurrence of spontaneous impulses could be attributed to the differences in sample preparations across these studies. Alternatively, the relatively large population of C-fiber mechanoreceptors exhibiting spontaneous impulses in our study may be related to the expression of ChR2 in C-fiber mechanoreceptors. A previous study has demonstrated that the frequency of spontaneous impulses increases in Aδ- and C-fibers under certain conditions such as nerve injury and incision injury. ¹ Our study did not observe significant increases in AP impulse frequency following hindpaw inflammation induced by CFA. However, we did observe that a more substantial proportion of high-threshold Nav1.8-ChR2-positive C-fiber mechanoreceptors displayed spontaneous APs in the CFA group compared with the saline group. These findings suggest that the functional properties of C-fiber mechanoreceptors are also modified by tissue inflammation and may have implications for behavioral mechanical hypersensitivity. It is important to note that further research is necessary to understand these observations’ underlying mechanisms and implications fully.

ORCID iD

Jianguo G Gu https://orcid.org/0000-0002-8404-9850