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

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).OPEN IN VIEWER

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).

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)).

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.