Mechanical and cold polymodality coexist in tactile peripheral afferents,and it’s not mediated by TRPM8

Abstract

In the mammalian somatosensory system, polymodality is defined as the competence of some neurons to respond to multiple forms of energy (e.g., mechanical and thermal). This ability is thought to be an exclusive property of nociceptive neurons (polymodal C-fiber nociceptors) and one of the pillars of nociceptive peripheral plasticity. The current study uncovered a completely different neuronal sub-population with polymodal capabilities on the opposite mechanical modality spectrum (tactile). We have observed that several tactile afferents (1/5) can respond to cold in non-nociceptive ranges. These cells’ mechanical thresholds and electrical properties are similar to any low-threshold mechano-receptors (LT), conducting in a broad range of velocities (Aδ to Aβ), lacking CGRP and TRPM8 receptors. Due to its density, cold-response range, speed, and response to injury (or lack thereof), we speculate on its role in controlling reflexive behaviors (wound liking and rubbing) and modulation of nociceptive spinal cord integration. Further studies are required to understand the mechanisms behind this neuron’s polymodality, central architecture, and impact on pain perception.

Introduction

Detecting and discriminating different forms of energy is one of the most important functions of the mammalian peripheral somatosensory system. ¹ From a classical perspective, detection and discrimination are thought to be based on different types of afferents (mechanical or thermal sensitive) expressing different receptors per modality. ² This simplistic concept has led us to believe that the somatosensory system can be peeled into its components by eliminating specific receptors.^3,4 Although useful from a pharmacological standpoint, this concept phases two important challenges: (a) disregard for cellular multimodality and (b) disregard for cellular plasticity. It assumes that every component (mechanical and thermal) is modality/receptor-specific and fundamentally stable across stimulation modalities and physiological conditions.

For example, cold sensory neurons are thought to be small-diameter C-fibers and medium-diameter Aδ fibers ⁵ expressing transient receptor potential melastatin 8 (TRPM8) ⁶ to detect mild cooling or transient receptor potential ankyrin 1 (TRPA1) ⁷ to detect noxious cooling. Neither of these receptors seems to be expressed in cells with high mechanical sensibility (tactile afferents). ⁸

To explore the accuracy of these reports, the current study aims to establish the cold sensibility of tactile afferents innervating the thoracic dermatome (T11). Based on the above-mentioned description, we expected to find no thermal sensibility in tactile afferents.

Methods

The present in vivo electrophysiological experiments were conducted on young adult Swiss Webster mice (Swiss OF-1) of either sex, ranging in age from postnatal day (P) 28 to P72 and weighing between 25 and 40 g. The Institutional Animal Care and Use Committee of the University of Wyoming and Wake Forest University Baptist Medical Center approved all procedures used in the present experiments.

Electrophysiology

The in vivo mouse thoracic preparation used in the present experiments has been described in detail. ⁹ Briefly, the animals were anesthetized (ketamine 90%, xylazine 10%, mg/kg, ip), intubated, and ventilated (Minivent, Harvard Apparatus, Holliston, MA). The T11 ganglia were surgically exposed by laminectomy, and the preparation moved to the recording chamber.

DRG cells were impaled with quartz electrodes (80-150 MΩ), filled with 5% Neurobiotin (Vector Laboratories, Burlingame, CA) in 1 M potassium acetate. Following, the T11 dermatome was explored (with a brush) (Figure 1(a)) to locate the mechanical receptor field (RF) of the afferent, the cellular basal properties (CBP) established by injecting current pulse (IcP, 500 ms, ≤0.1 nA). This characterization included active and passive membrane electrical properties (SAP). The mechanical threshold (MT) and response dynamic (rapid-adapting (RA) or slow-adapting (SA)) were measured by the use of calibrated von Frey filaments (Stoelting, Wood Dale, IL), followed by cold stimulation (CP) (pulses: 32 to 0°C or steps: 5°C, approx. 20 s), delivered by a 3 × 5-mm Peltier device (Yale Instruments Repair and Desing Shop, New Heaven). Finally, the afferent conduction velocity (CV) was established by stimulating the afferent RF with a bipolar electrode (0.5 Hz) (eP), recoding the absolute minimal intensity to activate the neuron, and dividing the latency by the distance between the cellular RF and the ganglia (Figure 1(b)). Based on this activation protocol, cells were classified as low threshold mechano receptors (LT), mechano cold receptors (MC), fast conducting high threshold mechano receptors (A-HT, >1.2 m/s) or slow conducting high threshold mechano receptors (C-HT, <1.2 m/s).

Figure 1.

MC neuron’s polymodal response. (a) Schematic of the T11 in vivo model for physiological intracellular recording. (b) Study and activation protocol (CBP: cellular basal properties, RF: receptor field, SAP: cellular somatic properties, MT: mechanical threshold, CV: conduction velocity). (c) Relation between CV and AP D50 (LT: low threshold mechano-receptors, MC: mechano-cold polymodal receptors, AHT: fast conducting A fiber high threshold mechano-receptors, CHT: slow conducting C fiber high threshold mechano-receptors). (d) Typical MC spike (black) presented with the first derivative of the voltage (red). E and (f) MC response to mechanical stimulation (IFmax: Cellular instantaneous maximal frequency, Stm: applied stimuli, CR: cellular response). G and (h) MC response to cold (same cell as E). (i) Stained MC neuron IHC (NB: Neurobiotin). Scale bars: E (1 s), F (0.25 s), G (20 s), H (10 s), I (60×, 25 μm).

After physiological classification, selected tactile neurons with cold sensibility (MC) were iontophoretically injected with 5% Neurobiotin (+0.5-1 nA, 5 min) (1 per animal) and the animal intracardially perfused with artificial cerebrospinal fluid (aCSF) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). T11 DRGs were dissected free and processed for immunohistochemistry.

Immunohistochemistry (IHC)

T11 DRGs were post-fixed with 4% paraformaldehyde for 2 h at 4°C. Afterward, ganglia were washed with 0.01 M phosphate-buffered saline (PBS) and immersed in 30% sucrose for cryoprotection until sectioned on a cryostat. Free-floating sections (50 µm) were incubated with rabbit anti-TRPM8 1:100 Abcam, guinea pig and anti-CGRP 1:1000, Peninsula followed with fluorescently tagged secondary antibodies (goat anti-rabbit-Cy3 1: 200; donkey anti guinea pig-Cy5, 1:200, Jackson Immunoresearch Labs) and avidin-FITC (1:500, Vector Laboratories). Finally, sections were mounted on plus-slides, air-dried, dehydrated, and coverslipped. Sections were examined, and images were acquired at 60× magnification using a confocal microscope.

Statistical analysis

Before analysis, parametric assumptions were evaluated for all variables using histograms and descriptive statistics. For normally distributed measurements, means ± SD are used. For measurements not normally distributed, descriptive statistics are reported using medians (and range). Standard parametric (Student's t-test, one-way ANOVA, linear regression) or nonparametric (Mann-Whitney U-test, Kruskall-Wallis) tests were used, depending on normality. Statistical tests were carried out using multiple packages (OriginPro 9.5, Northampton, MA; InStat/Prism, San Diego, CA).

Results

During this study, 161 afferents were collected (LT: 62/161, MC: 31/161, A-HT: 22/161, C-HT: 46/161). Except for the MC (24/31), the afferents’ properties in this study have already been reported,^9,10 collected during the experiments where MC cells were characterized and included here for distribution comparison only.

MC neuron’s mechanical sensibility, speed, and electrical profile

Accordingly, 19% of the recorded afferents were classified as MC (approx. 1 for every 5 cells). These cells (31/161) RFs were observed to be relatively medium-small (2.2 mm²), oriented longitudinally to the midline of the T11 dermatome. From the center of their RF, these afferent’s CV was scattered along both Aδ and Aβ ranges but not C (CV mean: 15.3 ± 1.7 m/s) (Figure 1(c)).

Their SAP was also lined up with an LT electrical profile. As presented in Figure 1(d), the APs were narrow (D50 mean: 0.4 ± 0.02 ms) and of small amplitude (AP amp mean: 42.3 ± 1.7 mV) with short afterhyperpolarization (AHP50 mean: 3.9 ± 0.7 ms), with no evidence of inflection it the AP repolarizing phase (Ca²⁺ hump) (Figure 1(d) red trace). Their membrane potential (Em mean: −63 ± 1.5 mV), input resistance (Ri mean: 101 ± 22 MΩ), and time constant (Tau mean: 1 ± 0.2 ms) were indistinguishable from an LT electrical profile.

Their mechanical sensibility was exquisite, reacting to both brushing (Figure 1(e)) and tonic stimulation (VFH) (Figure 1(f)) (MT median: 0.07, range: 0.07 to 0.33 mN), mainly with an RA response and high instantaneous frequency discharge (IFmax: 377 ± 38.1 Hz), also indistinguishable for LT afferents.

MC neuron’s thermal sensibility and cold stimulation

None of the cells classified as MC showed spontaneous activity (with two exceptions). The cold threshold (CT) for the MC afferents was in the non-nociceptive range (CT: 21 ± 1.3°C), step-by-step reduction in temperature triggered a frisky response (IFmax: 32 ± 12.9 Hz) followed by slow adaptation until the temperature was again reduced, following both the change and the duration of the pulse (Figure 1(g)). This response was gradually diminished as the temperature was reduced until no response was observable (≥10°C), with a discrete range of cold activation (activity by cold median: 18.5°C, range: 32 to 2°C). Cold pulse (CPs) activated the afferents in the same range of the step-by-step stimulation, and the cellular discharge gradually reduced its IFmax until it ceased at approximately the same lower threshold (≥2°C) (Figure 1(h)). In some cases (8/31), the re-heating process after the CP induced a paradoxical discharge (Re-heating temperature mean: 13.4 ± 2.4°C), then ended rapidly (approx. 2 s) without generating any spontaneous activity thereafter (data not shown).

MC immunohistochemistry

Of these cells, 10/31 were injected with 5% NB. These cells were broadly distributed among small to large diameters (median: 32 µm [range: 17 to 38 µm]). Although clearly visualized, none of these cells were TRPM8 or CGRP positive (Figure 1(i)).

Discussion

Polymodality is not a new property of mammalian sensory neurons. ¹¹ However, it is thought to be a property almost exclusively the domain of slow-conducting nociceptive afferents, ¹² and their capabilities are directly linked to the expression of specific receptors (e.g., TRPM8⁺ neurons for cold, MrgprA3⁺ neurons for itch).^13,14 However, the current study demonstrates that this description is incomplete at best. Some tactile afferents can respond to mechanical and thermal stimulation in a non-nociceptive range and are polymodal neurons by definition. We have also observed that these afferents are not TRPM8⁺ neurons nor strongly express CGRP, validating its non-nociceptive role in the normal functions of the mammalian somatosensory system.

Together, our observations indicate a secondary mild cold detection mechanism independent of TRPM8 and unrelated cold-induced pain perception. Our results also suggest that tactile sensibility and mild cold responses may be linked to add another dimension to the perception of texture, operating in a broad range of conduction velocities. These results allow us to speculate on the potential of these afferents to modulate pain perception by reducing the uncertainty of the nature of the stimuli applied.

Precedent, role, and potential functions of MC sensory neurons

Although we believe this to be the first detailed study of this cellular population (MC), it is certainly not the first time LT polymodality has been reported in several species (cats, rats, and non-human primates).^15–18 First mentioned in 2007 ⁹ in rapid adapting units (RA LT), they have been found in every in vivo study using multimodal stimulation (mechanical and thermal) in both mice and rats across several dermatomes (dorsal root ganglia and trigeminal ¹⁹ ). Importantly, these neurons seem to lose mechanical sensibility during heat stimulation ²⁰ and disappear on chronic pain models (e.g., partial nerve ligation (pSNL)). ²¹

Over the years, cold sensibility has received considerable attention ²² but not polymodality. Our results are consistent with several potential mechanisms to explain cold sensibility other than TRPM8-induced activation. From classical studies showing that the inhibition of Na⁺/K⁺ ATPase affects cold-fiber responses ²³ to more relatively recent findings suggesting 4-AP-sensitive K⁺ current (IKD) acts as an excitability break that controls cold sensibility, ²⁴ all these data indicate a multifactorial response system that controls cold perception. Furthermore, we currently know that a fixture of the development of chronic pain states is the disruption of the tactile system (e.g., mandibular cancer), ²⁵ which also seems consistent with the injury/nociceptive stimulation induction of MC sensory neuron failure.

Based on this description, it is tempting to speculate on the role of the tactile system (and the MC neurons as a part of it) in controlling nociceptive information. Although almost completely unexplored, we know that mild injuries trigger instinctive responses such as wound-licking and rubbing. ²⁶ In the context of the physiological function of the MC sensory neurons, and based on their response, it seems logical to speculate that these neurons may be the peripheral neuronal substrate of these behaviors. Furthermore, components of the somatosensory system (LT and HT) appear to work together, modulating their thresholds in a continuum across both modalities (mechanical and thermal [cold]). The first (LT) responds at non-nociceptive temperatures (±18.5°C) and encodes the decreased temperature by a reduction in activity until the nociceptive threshold is reached (±10°C in mice). ⁹ Obviously, this description takes place simultaneously with cold sensory neuron activation, incorporating all three components into the overall sensation and texture perception (or lack thereof). Unfortunately, we lack three critical pieces of information to elaborate on these neurons’ functions. First, we do not have any molecular marker to address this population. This fact renders our molecular tools useless and highlights the importance of physiological characterization. Second, we know nothing about their central arborization, and therefore, we cannot speculate on how directly or indirectly their information will be integrated into the spinal cord or if these cells have any substantial difference with an LT afferent. Third, little to no research has been done to evaluate the effects of multimodal stimulation on spinal cord second-order neurons, which putatively integrate these cells’ information. Until these three points can be clarified, the importance of this vast cellular population (1/5 of the total of tactile afferents) will remain obscure.

Conclusions

Skin/organ temperature is fundamental to the peripheral sensory system’s response. It modulates peripheral mechanical sensibility and likely contributes to the multimodal integration of peripheral information in the spinal cord. Cells such as the MC neurons seem ideal for explaining specific nocifensive behaviors and are likely to reduce the computational load of the spinal cord circuitry, particularly during a pro-nociceptive event. Its contribution during the injury-induced tactile overall desensitization and its role in cold perception need further exploration.