Mechanical sensibility of nociceptive and non-nociceptive fast-conducting afferents is modulated by skin temperature

Animals

Thirty-six male Sprague-Dawley rats (4–6 wk of age) were used. Some of these animals were studied in protocols examining the effect of incision (Boada et al. 2012) or differences between mechanosensory afferents innervating glabrous and hairy skin (Boada et al. 2010). When a cell was obtained meeting criteria for study in the current protocol, it was examined according to the methods below. None of the cells in the current study have been previously reported or were affected by any other experimental manipulation of their receptor field (RF; e.g., incision). Animals were housed together in pairs in a climate-controlled room under a 12-h light-dark cycle. The use and handling of animals were in accordance with guidelines provided by the National Institutes of Health and the International Association for the Study of Pain and the procedures, and experiments were approved by the Institutional Animal Care and Use Committee of Wake Forest School of Medicine.

Electrophysiology

Animals were deeply anesthetized with isoflurane 3%. The trachea was intubated and the rat ventilated using pressure controlled ventilation (Inspira PCV; Harvard Apparatus, Holliston, MA) with humidified oxygen. Heart rate and noninvasive blood pressure were monitored throughout as a guide to depth of anesthesia. Anesthetized animals were immobilized with pancuronium bromide (2 mg/kg) and inspired and end tidal isoflurane concentration maintained at 2% throughout the study (Tevan Pharmaceutics). As illustrated in Fig. 1, a dorsal midline incision was made in trunk skin and either L4, L5, or T11 dorsal root ganglion (DRG) and adjacent spinal cord were exposed by laminectomy as previously described (Boada et al. 2010). The tissue was continuously superfused with oxygenated artificial cerebrospinal fluid [aCSF (in mM): 127.0 NaCl, 1.9 KCl, 1.2 KH₂PO₄, 1.3 MgSO₄, 2.4 CaCl₂, 26.0 NaHCO₃, and 10.0 d-glucose]. The spinal column was secured using custom clamps and the preparation was transferred to a preheated (32–34°C) recording chamber where the superfusate was slowly raised to 37°C (MPRE8; Cell MicroControls, Norfolk, VA). Pool temperature adjacent to the DRG was monitored with a thermocouple (IT-23; Physitemp, Clifton, NJ). Rectal temperature (RET-3; Physitemp) was maintained at 34 ± 1°C with radiant heat.

The electrophysiological recordings from L4, L5, or T11 DRG neurons were limited to a maximum duration of 75 min to diminish the likelihood that experimental manipulation would result in sensitization. DRG neuronal somata were impaled with quartz micropipettes (80–250 MΩ) containing 1 M potassium acetate. All included cells satisfied the following requirements: resting membrane potential more negative than −40 mV, action potential (AP) amplitude ≥30 mV, the presence of afterhyperpolarization (AHP), and appropriate location of receptive field (RF), as described below. DC output from an Axoclamp 2B amplifier (Axon Instruments/Molecular Devices, Sunnyvale, CA) was digitized and analyzed offline using Spike2 (CED, Cambridge, UK). Sampling rate for intracellular recordings was 21 kHz throughout (MicroPower1401; CED).

Cellular Classification Protocol

The procedure used in this study to classify primary sensory afferents in vivo has been described in detail (Boada et al. 2015). Briefly, RFs were located with the aid of a stereomicroscope using increasing mechanical stimulation; the latter progressed from light touch with a fine sable hair paintbrush to searching with blunt probes and ultimately gentle to noxious pinch with fine-tipped forceps. Although cellular RFs were found across the entire dermatome in these intact preparations, only those along the medial portion of the dermatome were used in this study [Fig. 1A; animal flank (T11); posterior-lateral of the animal leg and paws (L4/L5)]. RFs located near the incision site or in areas not accessible to simultaneous mechanical and thermal stimuli were not studied further.

Mechanical thresholds were determined with calibrated von Frey filaments (VFH; Stoelting, Wood Dale, IL) and the area of maximal sensitivity was clearly marked, using a red fine point marker. Adaptation rate was evaluated using suprathreshold probes mounted in a micromanipulator; skin stretch and vibratory stimuli (tuning forks of 256 and 512 Hz; SKLAR Instruments, West Chester, PA) were also tested in many cells.

Somatic Electrical Properties

Active membrane properties of all excitable neurons were analyzed including the amplitude and duration of the AP and AHP of the AP, along with the maximum rates of spike depolarization and repolarization (MRD and MRR, respectively); AP and AHP durations were measured at half-amplitude (D₅₀ and AHP₅₀, respectively) to minimize hyperpolarization-related artifacts. Passive properties were analyzed including membrane resting potential (E_m), input resistance (R_i), time constant (Tau), inward rectification, and, where possible, rheobase; all but the latter were determined by injecting incremental hyperpolarizing current pulses (≤0.1 nA, 500 ms) through balanced electrodes.

Conduction Velocity

Because intact thoracic and lumbar DRGs serve multiple nerves, spike latency was obtained by stimulating the RF at the skin surface using a bipolar electrode (0.5 Hz); this was performed following all natural stimulation to prevent potential alterations in RF properties by electrical stimulation. All measurements were obtained using the absolute minimum intensity required to excite neurons consistently without jitter; this variability (jitter) in the AP generation latency (particularly at significantly shorter latencies), seen at traditional (i.e., 2- to 3-fold threshold) intensity, has been presumed to reflect spread to more proximal sites along axons. Stimuli ranged in duration from 50 to 100 μs; utilization time was not taken into account. Conduction distances were measured for each afferent on termination of the experiment by inserting a pin through the RF (marked with ink at the time of recording) and carefully measuring the distance to the DRG along the closest nerve.

Maximal Instantaneous Frequency Response and Discharge Rate

Since it is extremely difficult to deliver a controlled, sustained mechanical stimulus (both force and duration) we used maximal instantaneous frequency response (IF_max; Hz) in addition to discharge rate (AP/s), as an additional parameter to detect a change in the response characteristics of these neurons. IF_max was calculated as the reciprocal of the minimum interval between impulses meanwhile the discharge rate were obtained as the peak cellular response (with a bin of 1 s), during multimodal (thermal and mechanical) stimulation (for details, see next section).

Cellular RF Multimodal Stimulation Protocol

Once the area of highest sensitivity to mechanical stimulation was established, a thermode (3-mm² Peltier device, feedback-controlled; Yale Instrument Repair and Design Shop, New Haven, CT) attached to a micromanipulator was carefully positioned directly over the center of this area [always perpendicular (90°) to the RF]. This thermode was then brought down in close contact with the skin and displaced laterally (parallel to the skin surface) a few times to assure that its movement was capable of eliciting a consistent cellular response. At this point, no axial (vertical) displacement was allowed to maintain constant force. Immediately thereafter four thermal and multimodal stimulation protocols (SP) were applied in a consecutive sequence (Fig. 1B).

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).

Mechanosensitivity During Rapid Thermal Stimulation

No neuron in the study responded to rapid thermal changes (cold-heat pulse) during SP-I in the absence of mechanical stimulation, except for the LTMR-MC neurons. Mechanical sensibility during rapid thermal changes in the skin temperature was then evaluated by simultaneous mechanical activation during fast thermal stimulation (cold-heat pulses) during SP-II (for details, see SP I: single rapid thermal cold-heat pulse stimulation and SP-II: rapid multimodal stimulation and Fig. 1B).

LTMR-RA

These cells exhibited CV within Aβ range (median: 15.9 m/s; 11–35 m/s; Fig. 2A). Although very sensitive to static mechanical stimulation (median: 0.7 mN, 0.07–1.57 mN; Fig. 2B) these afferents were particularly responsive to rapid changes in mechanical stimulation (vibration), being vigorously activated by both 265 and 512 Hz (1:1) tuning fork application (Boada et al. 2015). All these cells (16/16) remained responsive to mechanical stimulation during and after thermal stimulation [typical responses in Figs. 3A (cold) and 5A (heat)].

LTMR-MC

These cells exhibited CV in the Aβ range (median: 21 m/s; 8.8–31 m/s; Fig. 2A) and were exclusively found innervating hairy skin. They were responsive to static mechanical stimulation (median: 0.19 mN; 0.19–0.39 mN; Fig. 2B) as well as vibration (256 Hz) and cold exposure (median: 17.5°C, 10–19°C). Responses to mechanical stimulation were altered during and after thermal exposure [typical responses in Fig. 3B (cold) and 5B (heat)].

LTMR-Hairs

These afferents were activated by a single hair displacement, conducted in a broad range including both Aβ and Aδ afferents (median: 13.7 m/s; 1.5–24 m/s; Fig. 2A), and had significantly lower thresholds to static mechanical stimulation than LTMR-RA afferents (median: 0.07 mN, 0.07–0.7 mN; P < 0.001; Fig. 2B). While showing a rapid adaptation rate (on-off response to static force), their ability to follow vibration (265 Hz; 1:1) was less consistent than LTMR-RA and several afferents (15/21) failed to accurately (1:1) follow 512 Hz. Responses to mechanical stimulation were altered during and after thermal exposure [typical responses in Figs. 3C (cold) and 4C (heat)].

AHTMR

These cells exhibited CV primarily in the Aδ range (median: 8.4 m/s; 1.5–17 m/s; Fig. 2A) and mechanical threshold one to three orders of magnitude greater than the LTMRs (median: 147 mN; 9.8–980 mN; Fig. 2B). None of these afferents was activated during the initial thermal stimulation (cold-heat pulse; SP-I) nor during simultaneous mechanical (low-threshold displacement) and thermal stimulation (cold-heat pulse; SP-II). This is to be expected as the thresholds for the mechanical stimulation of AHTMR remain well above the mechanical stimulation used in the SP-II as defined in Fig. 2B. Most of these neurons (25/31) remained unresponsive to the application of rapid changes in the skin temperature. However, in some cases, repeated exposure to cold and heat by the end of SP-II resulted in thermal sensitivity (6/31). These thermoresponsive, sensitized cells (6/6) were exposed to multiple thermal stimulations (SP-III). Every thermal exposure generated increasingly lower thresholds and more robust responses suggestive of sensitization (Fig. 6; 6/6, cold threshold median: 0°C; −3–10°C; heat threshold median: 47°C; 46–50°C).

Mechanosensitivity After Constant Cold Stimulation

Sustained exposure to cold (for details see SP-IV: sustained cold exposure) significantly modified (P < 0.001) the RF mechanical threshold of both tactile (LTMR: 12/12) and nociceptive (AHTMR: 17/17) afferents. The tactile afferents had an approximately eightfold increase in mechanical thresholds from the sustained cold compared with baseline temperature response [median: from 0.19 mN (0.078–1.56 mN) at 32°C to 1.56 mN (0.39–39.2 mN) at 2°C; P < 0.001]. In contrast, nociceptive afferents (AHTMRs) showed an approximate 40-fold decrease in mechanical threshold from the same cold exposure [median: from 147 mN (9.8–980 mN) at 32°C to 3.9 mN (1.56–588 mN) at 2°C; P < 0.001; Fig. 4E].

Temperature Modulation of Mechanical Sensitivity in LTMRs

The vulnerability of tactile sensibility to environmental temperature variations is a well-known phenomenon in humans (Weitz 1941; Mackworth 1953; Mills 1956; Provins and Morton 1960; Stevens et al. 1977; Green 1977; Green et al. 1979). However, its occurrence in other mammals (in this case, rats) and its relationship with nociceptive neuronal activity remain unclear. In the current study, we have shown via simultaneous multimodal skin stimulation that the balance of input from fast-conducting mechanosensitive afferents shifts dramatically with skin cooling or heating in rodents. Tactile inputs appear to rapidly decrease their mechanical sensitivity (and thereby activity) to mechanical stimulation at temperatures where thermally sensitive nociceptive inputs should become active. Extreme insensitivity of these tactile afferents to mechanical stimuli was achieved around 2°C, consistent with the ultimate development of “cold block” in terminal arbors (Paintal 1965; Franz and Iggo 1968). Nevertheless, the latter could not explain the decrease in sensitivity with moderate cooling or heating of the tactile afferents and the rapidity with which changes occurred. This suggests that temperature exerts a more direct and immediate effect on either mechanotransduction properties (Inman and Peruzzi 1961; Ishiko and Lowenstein 1961; Green et al. 1979; Boada and Woodbury 2007), the vascular array around the terminals (Green 1977; Green et al. 1980), the tissue (skin) effects (Lederman and Taylor 1972; Lederman 1976), the temperature effects on ion channel function (Zimmerman et al. 2007), or a combination of these effects. Temperature resistance of specific sodium channels has been proposed (Zimmerman et al. 2007). However, the lack of clear differences in expression of sodium channels in specific neuronal subtypes is less likely to explain the clear differences in tactile and nociceptive responses to cold, but differential expression may at least be a contributing factor (Ramachandra et al. 2005; Shields et al. 2012).

Temperature Modulation of Mechanical Sensitivity in AHTMRs

In contrast to the tactile afferents, the fast-conducting mechanonociceptors (AHTMR) rapidly become hypersensitive when exposed to either moderately hot or cold temperatures. These novel findings of a shift in the informational channel input with temperature in the periphery have profound repercussions to sensory perception. Moreover, it is curious that the effects are divergent in the fast-conducting tactile afferents and the fast-conducting high thresholds or nociceptive afferents suggesting that a fundamental difference exists in the vascular array, the mechanotransductional properties, cellular specific ion channels, or sympathetic neuronal interaction to produce divergent responses to temperature in unique subsets of myelinated neurons.

Cold temperature produces loss of activity in the tactile afferents, while simultaneously decreasing the threshold and increasing activity in the nociceptive afferents. The net result is numbness to light touch with simultaneous pain in the human (Weitz 1941; Mackworth 1953; Mills 1956; Provins and Morton 1960; Stevens et al. 1977; Green 1977; Green et al. 1979). The pain component has long been thought to be solely the result of C-fiber-mediated activation, but data from this study suggest that cold pain may be at least in part a result of AHTMR activation, possibly from mechanical activation that would normally activate tactile afferents (as seen in the overlap of thresholds between LTMR and AHTMR at cold temperatures reported in this study). This may be further accentuated by the possibility that the reduction in tactile input results in all input to the spinal cord being nociceptive, increasing signal to noise for nociceptive information. Tactile inputs likely have inhibitory effects on nociceptive throughput (Narikawa et al. 2000; Green and Schoen 2005), and their loss may further impact central pain processing. Under normothermic conditions, substantial numbers of substantia gelatinosa (SG) neurons are activated by light touch (Bennett et al. 1980; Light et al. 1979). Indeed, due to a highly sophisticated repertoire of protective reflexes and voluntary behaviors that work in concert to minimize nociceptor activation, tactile afferents are routinely and preferentially activated under normal conditions and would be expected to provide input, at least indirect input but also likely direct input, to the SG (Boada and Woodbury 2008). The rapid loss of the latter during thermal episodes, however, may imply that the loss or reduction in tactile input leads to more information received by the SG being purely nociceptive. A similar divergent effect of heat occurs in myelinated tactile and nociceptive neurons, inactivation and sensitization, respectively (Fitzgerald and Lynn 1977). This may be an evolutionary biology development to enhance pain perception in the face of extreme cold or even heat, thus permitting a retained or enhanced signal to the organism for the need to prevent tissue damage in the area affected. Nevertheless, although the AHTMR may sensitize and respond to thermal activation or increase activity to mechanical stimulation at various temperatures helping to serve and protect with possible assistance of other fibers, its primary roll remains to provide rapidly conducted, mechanically driven nociceptive input.

The amplification of nociceptor inputs during temperature exposure, coupled with simultaneous release from tactile-driven central inhibition, would further facilitate activation of circuits required for appropriate evasive action. The extent to which these findings can be generalized more broadly throughout the somatosensory system is not yet clear, although hyposensitivity of the vast majority of tactile afferents by noxious heat sheds new light on a paradoxical “touch gate” identified in human psychophysical studies whereby noxious heat pain was found to suppress tactile sensation (Apkarian et al. 1994), the diametric opposite of the classic pain gate (Melzack and Wall 1965). The present findings reveal, however, that gating of touch by noxious thermal stimuli occurs, at least in part, not in the cortex as suggested (Bolanowski et al. 2000, 2001), but at the skin surface via selective change in afferent sensitivity.

Conclusion

The skin temperature plays a fundamental role in the response of the peripheral somatosensory system to mechanical stimulation that greatly simplifies computational demands placed on the nervous system during environmental exigencies.