Response Properties of Mechanoreceptors and Nociceptors in Mouse Glabrous Skin: An In Vivo Study

David M. Cain,1 Sergey G. Khasabov,1 and Donald A. Simone2

 1Department of Preventive Sciences and  2Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cain, David M., Sergey G. Khasabov, and Donald A. Simone. Response Properties of Mechanoreceptors and Nociceptors in Mouse Glabrous Skin: An In Vivo Study. J. Neurophysiol. 85: 1561-1574, 2001. The increasing use of transgenic mice for the study of pain mechanisms necessitates comprehensive understanding of the murine somatosensory system. Using an in vivo mouse preparation, we studied response properties of tibial nerve afferent fibers innervating glabrous skin. Recordings were obtained from 225 fibers identified by mechanical stimulation of the skin. Of these, 106 were classed as Abeta mechanoreceptors, 51 as Adelta fibers, and 68 as C fibers. Abeta mechanoreceptors had a mean conduction velocity of 22.2 ± 0.7 (SE) m/s (13.8-40.0 m/s) and a median mechanical threshold of 2.1 mN (0.4-56.6 mN) and were subclassed as rapidly adapting (RA, n = 75) or slowly adapting (SA, n = 31) based on responses to constant force mechanical stimuli. Conduction velocities ranged from 1.4 to 13.6 m/s (mean 7.1 ± 0.6 m/s) for Adelta fibers and 0.21 to 1.3 m/s (0.7 ± 0.1 m/s) for C fibers. Median mechanical thresholds were 10.4 and 24.4 mN for Adelta and C fibers, respectively. Responses of Adelta and C fibers evoked by heat (35-51°C) and by cold (28 to -12°C) stimuli were determined. Mean response thresholds of Adelta fibers were 42.0 ± 3.1°C for heat and 7.6 ± 3.8°C for cold, whereas mean response thresholds of C fibers were 40.3 ± 0.4°C for heat and 10.1 ± 1.9°C for cold. Responses evoked by heat and cold stimuli increased monotonically with stimulus intensity. Although only 12% of tested Adelta fibers were heat sensitive, 50% responded to cold. Only one Adelta nociceptor responded to both heat and cold stimuli. In addition, 40% of Adelta fibers were only mechanosensitive since they responded neither to heat nor to cold stimuli. Thermal stimuli evoked responses from the majority of C fibers: 82% were heat sensitive, while 77% of C fibers were excited by cold, and 68% were excited by both heat and cold stimuli. Only 11% of C fibers were insensitive to heat and/or cold. This in vivo study provides an analysis of mouse primary afferent fibers innervating glabrous skin including new information on encoding of noxious thermal stimuli within the peripheral somatosensory system of the mouse. These results will be useful for future comparative studies with transgenic mice.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Functional properties of cutaneous somatosensory receptors have been well studied in various mammalian species, the rat being most extensively investigated (for review, Besson and Chaouch 1987; Greenspan 1997; Millan 1999; Raja et al. 1988; Treede et al. 1992). However, the ongoing development of transgenic mice for the study of pain mechanisms necessitates thorough functional characterization of the somatosensory system in this species. By overexpressing specific gene products in transgenic mice, or by ablating the expression of these molecules in knockout mice, transgenic mutagenesis in whole animals has become a valuable tool to study gene function. Recent studies with transgenic mice have employed several methods to study the role of neurotrophins, cytokines, and tachykinins in nociception. For example, Northern blotting, in situ hybridization, and immunocytochemistry approaches have shown that overexpression of epidermal nerve growth factor (NGF) correlates with a hypertrophy of peripheral sensory and sympathetic nerves (Albers et al. 1994). Using behavioral methods, Davis et al. (1993) correlated overexpression of NGF in the skin of mice with a profound hyperalgesia to noxious mechanical stimulation. Electrophysiological studies using an in vitro preparation have indicated increased sensitivity of Adelta nociceptors to mechanical stimuli and enhanced sensitivity of C nociceptors to heat in transgenic mice overexpressing NGF (Stucky et al. 1999). A recent study involving intracellular recordings from the dorsal root ganglion in vitro found very few consistent differences between the electrical properties of neurons from wild-type and NGF overexpressing mice (Ritter et al. 2000). In addition, the cytokine tumor necrosis factor-alpha (TNF-alpha ), when overexpressed in transgenic mice, can trigger complex disease phenotypes associated with pain such as arthritis (Georgopoulos et al. 1996; Keffler et al. 1991), insulitis and diabetes (Guerder et al. 1994; Higuchi et al. 1992; Picarella et al. 1993), and cachexia (Probert et al. 1993; for reviews see Douni et al. 1995; Kollias et al. 1999; Probert et al. 1997). Finally, in mutant mice lacking the NK-1 receptor for the tachykinin substance P, electromyography has shown the absence of the characteristic amplification ("wind up") and the loss of intensity coding of nociceptive reflexes (De Felipe et al. 1998). Mice with a targeted deletion of the gene encoding substance P display reduced behavioral responses to moderate to intense stimuli (Cao et al. 1998), exhibit no nocifensive responses following formalin injection, and have an increased withdrawal threshold in the hotplate test (Zimmer et al. 1998).

In addition to its use in transgenic models, the mouse has been used traditionally in the study of cancer. Specific tumor cell lines will induce tumorigenesis only in strains of mice with targeted mutations. Importantly, a model of cancer pain in animals has been developed recently and relies on a specific murine model that involves injecting osteolytic fibrosarcoma cells into the femur or subcutaneously in the plantar hindpaw of the C3H strain of mouse (Schwei et al. 1999; Wacnik 1999a,b). This model permits, for the first time, investigation of potential tumor/peripheral nerve interactions that may contribute to cancer pain. Characterization of primary afferent fibers in the normal mouse is necessary for future studies of how various tumors affect peripheral nerve function.

A recent study (Koltzenburg et al. 1997) using an in vitro skin-nerve preparation is the first detailed investigation of the receptive properties of identified cutaneous afferent fibers in the hairy skin of the mouse. The in vitro skin-nerve preparation was also recently utilized to study the effects of genetic disruption of the capsaicin receptor (VR1) in mice on nociception (Caterina et al. 2000). We now present results obtained from responses of single primary afferent fibers innervating the glabrous skin of the mouse hindpaw recorded in vivo. We have used a teased fiber technique to record from identified cutaneous afferent fibers in the tibial nerve and have quantified responses evoked by controlled mechanical and thermal stimuli applied to the glabrous skin of the hindpaw. These studies provide new information on responses properties of cutaneous afferent fibers innervating the glabrous skin of the mouse and may contribute to future studies using genetically altered mice to study the function of specific endogenous mediators and their receptors in peripheral nerve function and primary afferent fiber excitability.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Adult (>6 wk old), male C3H/HeJ mice weighing 20-29 g were used. Animals were obtained from the National Institutes of Health, housed on a 12-h light-dark schedule, and given food and water ad libitum. All protocols involved in this study were approved by the Animal Care Committee of the University of Minnesota.

Compound action potentials

Animals were sedated with acepromazine maleate (20 mg/kg ip) and anesthetized with pentobarbital sodium (Nembutal, 48 mg/kg ip). Supplemental doses of pentobarbital sodium (15 mg/kg) were given as needed to maintain areflexia. Latency measurements of compound action potential components were obtained to determine precise cutoff points among the three major cutaneous afferent fiber types studied, Abeta , Adelta , and C. After surgically exposing a maximal length of sciatic-tibial nerve from the surrounding muscle tissue, the epineurium was removed from the distal portion of the tibial nerve, and the entire length of nerve was kept moist by repeated application of saline. The sciatic nerve was exposed as closely as possible to the point of entry into the pelvic girdle to achieve recording nerve lengths in a range of 19-22 mm for all compound actions potentials. The conduction distance of the nerve was measured to the nearest millimeter before and after each experiment from the proximal segment of the sciatic nerve, which was placed on a stimulating electrode, to the most distal segment of the tibial nerve where a recording electrode was located. The electrical stimuli were produced by a stimulus isolator (A365, World Precision Instruments) which passed current within an empirical range of 10-10,000 µA. Latencies of response onset, maximal response amplitude, and response offset were measured using the LabVIEW data analysis software program.

Single fiber recording

Animals were sedated with acepromazine maleate (20 mg/kg ip) and anesthetized with Nembutal (48 mg/kg ip). Supplemental doses of pentobarbital sodium (15 mg/kg) were given as needed to maintain areflexia. Hair was removed from one hindlimb, and an incision was made on the dorsal aspect of the lower leg in the skin overlying the tibial nerve. After surgically removing the gastrocnemius muscle, the skin was sutured to a metal ring (1.3 cm ID) to form a basin that was filled with warm mineral oil. The tibial nerve was dissected from connective tissue and placed on a small, mirrored platform for separation of nerve fibers. To prevent leakage of oil from the recording basin, a rubber-based polysulfide impression material (COE-FLEX, GC America) was applied externally around the ring to the skin of the hindlimb and allowed to set for ~20 min to form a hard seal. The epineurium of the tibial nerve was opened using a miniature scalpel, and small fascicles were cut to allow the proximal ends to be spread out on the platform for separation with fine jewelers forceps.

Nerve fascicles were teased apart, and fine filaments were placed on a silver-wire recording electrode maneuvered by a micromanipulator. Extracellular recordings were obtained only from single fibers that could be easily discriminated according to amplitude and shape. Action potentials were amplified, audiomonitored, displayed on an oscilloscope, and stored on a VCR before being sent to a PC computer for data acquisition. Evoked responses were analyzed off-line using a customized data analysis program (LabVIEW, version 5.1). An amplitude window discriminator was used to separate action potentials of the fiber under study from those of other fibers and/or from background noise. However, recordings usually consisted of one afferent fiber.

Identification of primary afferent fibers

The receptive fields (RFs) of cutaneous afferent fibers were identified using mechanical stimuli. Mechanical stimulation proceeded by a graduated approach beginning with large and soft stimulation with a cotton swab or the experimenter's fingers, followed by mild pinching with curved serrated forceps. Once a fiber was isolated, the location of its RF was identified using a small glass probe (1 mm diam) and/or a suprathreshold von Frey monofilament. The RF location was then marked on the skin with a felt-tip pen and reconstructed on a drawing of the mouse hindpaw.

Conduction velocity

The conduction velocity of each fiber was determined by electrically stimulating the RF, before recording the conduction latency between the RF and the recording electrode. Two fine needle electrodes (30 gauge) were inserted into the skin on opposite sides adjacent to the RF. Square-wave pulses (duration, 0.2 ms, 0.5 Hz) were delivered at a stimulating voltage 1.5 times the voltage required to evoke a threshold response.

Mechanical stimulation

To ensure that recordings were obtained exclusively from fibers innervating cutaneous RFs rather than from deeper units innervating muscle, the skin surrounding the RF was gently grasped with curved forceps and lifted. Only fibers that discharged primarily while the skin around the RF was lifted above the underlying tissue and lightly squeezed were considered to be cutaneous units. At the time of unit isolation, toes and joints were manipulated to identify proprioceptive units, which were not further studied. Mechanical thresholds were determined using calibrated von Frey monofilaments (Stoelting) and were expressed as the minimum force (mN) needed to evoke a response in >= 50% of the trials. The range of von Frey filaments used in this study exerted bending forces from 0.1 to 177.5 mN. Low-threshold mechanoreceptors (Abeta fibers) were identified as either rapidly adapting (RA) or slowly adapting (SA) by applying constant force using a suprathreshold von Frey monofilament (73 mN bending force) that was secured to a microdrive and manually lowered onto the RF for 10 s.

Thermal stimulation

Stimulus temperature represented the temperature at the interface between skin and thermode (contact area, 1 cm2). The thermode consisted of a feedback-controlled Peltier device, and stimuli were delivered and controlled by a customized software program. The relatively large contact area of the thermode, which was attached to a manipulator, facilitated a firm contact with the RF. For Adelta and C units, the thermode was lowered onto the RF exerting pressure just sufficient to elicit a response thus assuring proper angle and position of the thermode over the RF. Then, the thermode was delicately adjusted (by manipulator) until the mechanically induced response ceased yet a visible indentation of the skin was maintained. Heat stimuli ranging from 35 to 51°C were presented in ascending steps of 2° from a base temperature of 32°C at a ramp rate of 20°C/s. Each stimulus was applied for a 5-s duration, and an interval of 60 s was maintained between each stimulus. Following a period of at least 5 min, cold stimuli of 10-s duration were presented in descending increments of 4° from 28 to -12°C and were applied with a ramp rate of 5°C/s. An interstimulus interval of 160 s occurred between each increment of cold stimulation.

Functional classification of primary afferent fibers

Mechanoreceptors were considered RA if they exhibited an abrupt response to the onset (and offset) of mechanical stimuli but failed to maintain discharge during the 10-s trial. SA mechanoreceptors were those that discharged throughout the 10-s period of stimulation.

Nociceptors were characterized according to responses evoked by noxious mechanical, heat, and cold stimuli. In the absence of a response to the applied range of thermal stimuli, Adelta and C nociceptors were classed as mechanonociceptors (AM, CM). Nociceptors excited by heat, but not cold, were classed as mechanoheat nociceptors (AMH, CMH), and those responding to cold but not heat were classed as mechanocold nociceptors (AMC, CMC). Nociceptors excited by both types of thermal stimuli, mechanoheatcold nociceptors, were designated AMHC or CMHC. AMH nociceptors exhibiting response thresholds <= 51°C were subclassed as AMH Type II fibers. Those Adelta nociceptors not responsive during initial heat trials were exposed (<= 3 times) to 53°C for 30 s to induce sensitization to heat. If these nociceptors subsequently responded to heat, they were classified as AMH Type I (Meyer et al. 1985; for review, Raja et al. 1988).

Data analysis

Action potentials and discriminated spikes were stored on videotape and on a laboratory computer for off-line analysis. Thermal stimuli, including ascending and descending ramps and the time at which they were reached, were also digitized and stored. An additional channel stored a digitized trace of voltage from a footswitch used to identify the time of mechanical stimulation for characterization of rapidly and slowly adapting responses.

Differences in mechanical threshold between fiber types were determine using the Kruskal-Wallis ANOVA and Mann-Whitney U tests. The responses of Adelta and C fibers to thermal stimuli were analyzed on the basis of the number of impulses and the frequency, i.e., discharge rate (from first to last evoked impulse) evoked by each stimulus. The LabVIEW software files were reviewed for each thermal stimulus trial. To obtain the discharge frequency, the exact number of spikes was divided by the time difference between the first spike and the last spike in response to each thermal stimulus. The mean was computed from the discharge frequency totals obtained for each temperature stimulus for either Adelta or C fibers. Frequency could not be calculated for responses consisting of a single impulse. Differences in response thresholds for heat and cold between fiber types were analyzed using a one-way ANOVA and Neuman-Keuls post hoc comparisons. A probability of <0.05 was considered significant for statistical comparisons.

To determine stimulus-response relationships, power functions were obtained for fibers responding to thermal stimuli which were normalized by defining the "zero point" as the highest stimulus temperature that did not elicit a response. Stimulus intensities were defined as the difference from the zero point. For example, since heat stimuli were always delivered in 2°C increments, the threshold stimulus was assigned an intensity of 2°C. After log-log transformation, the slopes of the stimulus-response functions were determined with the use of linear regression.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Compound action potentials and derived conduction velocities

Latency measurements of compound action potential peaks were obtained from six sciatic-tibial nerve segments in five mice to determine precise cutoff points among the three major fiber types identified. Figure 1 shows six overlaid traces from a representative compound action potential evoked by different intensities of electrical stimulation. When averaged, conduction velocity calculations from all compound action potentials indicated a cutoff velocity between Abeta and Adelta fibers of 13.6 m/s, i.e., fibers with conduction velocities >13.6 m/s were identified as Abeta fibers. The average latencies at peak response amplitude correlated to conduction velocities of 19.8 m/s for Abeta and 10.7 m/s for Adelta fibers. The maximal conduction velocity for C fibers was calculated to be 1.3 m/s.



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Fig. 1. Examples of 6 overlays of Abeta , Adelta , and C components of compound action potentials evoked by electrical stimulation (time 0) recorded from the same mouse. Dashed lines indicate conduction velocity cutoffs for differentiation between Abeta and Adelta fibers as well as the maximal conduction velocity cutoff for C fibers. Abscissa indicates real time from the beginning of the stimulus artifact. Note difference in vertical scales for A and C components: Abeta -Adelta components were evoked by a 100-µA stimulus; C fibers' response evoked by a 6-mA stimulus.

General properties of identified afferent fibers

Recordings were made from a total of 225 identified mechanosensitive afferent fibers from the tibial nerve of 59 adult C3H mice. A summary of the fiber types and their general response properties is provided in Table 1. Of these 225 fibers, 106 were classed as Abeta fibers, 51 as Adelta fibers, and 68 as C fibers. The mean conduction velocity of Abeta fibers was 22.2 ± 0.7 (SE) m/s and ranged from 13.8 to 40.0 m/s. Adelta fibers exhibited a mean conduction velocity of 7.1 ± 0.6 m/s in a range from 1.4 to 13.6 m/s. The mean conduction velocity of C fibers was 0.7 ± 0.1 m/s with a range from 0.2 to 1.3 m/s. (Table 1). The three histograms in Fig. 2 illustrate the number of fibers plotted as a function of conduction velocity. The peaks indicated in the histograms of Abeta and C fibers (Fig. 2, top and bottom) correlate closely with peak amplitudes observed in compound action potentials as well as results from single fiber recordings, whereas Fig. 2, middle, reflects a flat distribution of Adelta fibers over the range of conduction velocities.


                              
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Table 1. General response characteristics of primary afferent fibers



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Fig. 2. Three histograms showing the number of Abeta (top, n = 106), Adelta (middle, n = 51), and C (bottom, n = 68) fibers distributed across their respective ranges of conduction velocities. The conduction velocity range of each fiber type was determined by calculation of cutoff velocities obtained from compound action potentials. The range of conduction velocities for Abeta fibers (top) extended from 13.3 to 40.0 m/s, for Adelta (middle) from 1.3 to 13.3 m/s, and for C fibers (bottom) the conduction velocity range was 0.2-1.3 m/s. The middle panel reflects the broad distribution conduction velocities for Adelta fibers from 1.3 to 13.3 m/s.

The mechanosensitive RFs of all fibers were located on the glabrous skin. No formal assessments were made regarding RF size. This was difficult to reliably quantify because RFs typically consisted of a single small spot. Although a small number of D hair cells at the border between hairy and glabrous skin were encountered, these were not studied. No attempt was made to isolate vibration-sensitive mechanoreceptors previously reported in monkey glabrous skin by Perl (1968). RF location of RA and SA mechanoreceptors included the toes (30 for RA and 10 for SA), pads (19 for RA and 7 for SA), and plantar surface (26 for RA and 14 for SA). The RF locations of Adelta fibers were toes (n = 16), pads (n = 14), and plantar surface (n = 21). Finally, RF locations of C fibers were distributed on the toes (n = 43), pads (n = 4), and plantar surface (n = 21). In Fig. 3, the distribution of the three major fiber types is plotted as a function of RF location, i.e., toe, pad, or plantar surface. While the observed distribution of RFs according to fiber type was not associated with any particular region of the hindpaw, the histogram reflects a relatively larger distribution of Abeta and Adelta fibers with RFs located on footpads in contrast to a low number of C fibers.



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Fig. 3. The number of Abeta (n = 102), Adelta (n = 40), and C fibers (n = 66) whose RFs were located on the toes, footpads, and plantar surface of the hindpaw. No significant differences were found among fiber types and RF locations, although relatively few RFs of C fibers were found on the pads.

Functional properties of Abeta mechanoreceptors

A total of 106 Abeta fibers were differentiated into RA and SA functional subtypes. As summarized in Table 1, 75 Abeta fibers (71%) were RA while 31 fibers (29%) discharged impulses throughout the duration of 10-s trials of mechanical stimulation and were thus classed as SA mechanoreceptors. The mean conduction velocity of the RA fibers was 21.4 ± 0.7 m/s, which did not differ significantly from the mean conduction velocity of 24.3 ± 1.4 m/s obtained for the SA subtype. Figure 4A shows the discharge of a representative SA mechanoreceptor evoked by constant force applied with a von Frey filament for 10 s. Also illustrated are examples of the constant latency evoked by electrical stimulation of the RF, which was used to calculate the conduction velocity for representative SA (Fig. 4B) and RA fiber responses (Fig. 4D). Mechanical thresholds of Abeta mechanoreceptors (Fig. 5) extended over a range of 0.4-56.6 mN for RA fibers and 0.4-30.1 mN for SA fibers. For RA subtypes, the median mechanical threshold was 2.1 mN (interquartile range = 9.2 mN), and for SA fibers, the median threshold was 4.4 mN (interquartile range = 23.2 mN). The difference between RA and SA fiber median mechanical thresholds was not statistically significant. The median mechanical threshold for all Abeta fibers was 2.1 mN.



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Fig. 4. Representative examples of evoked responses of Abeta mechanoreceptors evoked by constant force of 10-s duration. A: evoked response of a single slowly adapting (SA) mechanoreceptor. This mechanoreceptor discharged throughout the duration of the stimulus (--- above the oscilloscope trace). B: 3 examples of the constant conduction latency used to calculate the conduction velocity of this fiber. C: evoked responses of a single rapidly adapting (RA) mechanoreceptor. Note that the response occurs only at the onset and offset of the stimulus. D: 3 examples of the constant conduction latency used to calculate this fiber's conduction velocity. Arrows in B and D indicate the stimulus artifact.



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Fig. 5. Median mechanical thresholds (mN) of Abeta , Adelta , and C fibers. Median thresholds were 2.1 (55.5) for Abeta mechanoreceptors, 10.4 for Adelta fibers, and 24.4 for C fibers. Error bars indicate the interquartile ranges for each fiber group. The mechanical threshold differences among the 3 fibers types were significant (*).

Functional properties of Adelta fibers

A total of 51 Adelta fibers were studied, 36 (71%) of which exhibited nociceptive function based on a combination of intensity encoding response to pinch and either high mechanical thresholds (>10 mN) or response to noxious thermal stimuli. The remaining 15 Adelta fibers (29%) were possibly-low threshold mechanoreceptors since their mechanical thresholds were relatively low (<= 6 mN) and they did not respond to noxious thermal stimuli. None of the Adelta fibers were spontaneously active.

As illustrated in Fig. 5, the median mechanical threshold of the Adelta fibers was 10.4 mN (range = 0.1-111.5 mN, interquartile range = 19.9 mN). This was significantly greater than the mechanical threshold of Abeta mechanoreceptors (P < 0.01, Mann-Whitney U test) and significantly lower than the median mechanical threshold of 24.4 mN for C fibers (P < 0.01, Mann-Whitney U test).

Five distinct functional subtypes of Adelta fibers were distinguished based on responses evoked by thermal stimuli (Table 1). Of 25 Adelta fibers tested for sensitivity to heat stimuli, 13 (52%) Adelta fibers did not respond. Of these 13, 8 also failed to respond to noxious cold stimuli and were classed as AM units. A second subclass of nine Adelta fibers were excited by cold but not by heat. These fibers were designated as AMCs and exhibited a wide range of cold thresholds, ranging from 20 to -8°C (mean, 5.3 ± 0.4°C). One other Adelta fiber responded to cold (0°C) and to heat (39°C) and was classed as AMHC-Type II. Thus 10 of 20 Adelta fibers (50%) characterized for heat and cold sensitivity responded to cold stimuli. In addition to the AMHC-Type II fiber, one other Adelta fiber had a heat threshold of 45°C, was insensitive to cold, and was classed as a AMH Type II fiber. Another Adelta mechanonociceptor responded to heat only after 30-s exposure to 53°C, but not to cold, and was classed as a AMH Type I fiber. Thus only three Adelta fibers tested were excited by noxious heat.

The functional subclassification of fibers according to temperature sensitivity in Table 1 describes only fibers exposed to both heat and cold stimuli. The proportion of AM fibers may not be representative, and the numbers do not reflect all of the C fibers that responded to heat but were not tested for cold. From reports of Koltzenburg et al. (1997) and Stucky et al. (1999) evidence supports the view that a majority of mouse Adelta fibers are functionally AM type fibers. However, since this classification required the longest duration of temperature trials (i.e., heat, cold, and then heat injury), we were not able to test all Adelta fibers. Subsequent recordings in our laboratory for a study in progress provide further evidence that a majority of Adelta fibers fit the AM profile.

Responses of a single Adelta nociceptor evoked by heat stimuli are shown in Fig. 6A accompanied by the same fiber's latency measurement evoked by constant intensity electrical stimuli (Fig. 6B). Given the small number of Adelta fibers that responded to heat (n = 3) in our study, an overall intensity-encoding ability of Adelta fibers to heat was not demonstrated. Indeed, one of these Adelta nociceptors only responded to the prolonged application of a 53°C stimulus. Figure 6C shows the number of impulses evoked by heat stimuli for each of the Type II Adelta fibers excited by heat, and Fig. 6D displays their mean discharge rate plotted as a function of stimulus intensity.



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Fig. 6. Responses of Adelta nociceptors to heat stimuli. A: responses of a single Adelta nociceptor evoked by heat stimuli of 39, 41, and 47°C. --- above the oscilloscope traces are analog traces of stimulus temperature, which were each of 5-s duration, and the numbers indicate the stimulus temperatures. Response threshold to heat for this nociceptor was 39°C. B: 3 examples showing constant conduction latency for this same Adelta nociceptor. up-arrow , the stimulus artifact. C: the number of impulses evoked by heat stimuli of 35-51°C for the 2 heat-sensitive Adelta fibers. One other Adelta fiber (not shown) responded only to heat stimuli at 53°C. D: mean (±SE) discharge rate (Hz) of the 2 heat-sensitive Adelta fibers shown above evoked by the same stimulus temperatures.

Responses of Adelta nociceptors evoked by cold are presented in Fig. 7, which includes responses of a representative Adelta nociceptor to cold stimuli (Fig. 7A). The response threshold of this fiber to cold occurred at 8°C, which was similar to the mean cold threshold of 7.6 ± 3.8°C for cold-sensitive Adelta nociceptors. As the intensity of the cold stimuli increased, the discharge of action potentials also increased. Responses typically occurred throughout the 10-s duration of the stimulus. Encoding of cold intensity by Adelta nociceptors was studied by plotting the mean number of impulses (Fig. 7C) and mean discharge rate (Fig. 7D) as a function of stimulus temperature. The maximal number of impulses evoked from an Adelta nociceptor in response to cold was 88 (at 4°C) and the maximal discharge rate was 7.1 Hz (at -12°C). The resulting curves demonstrate intensity coding of Adelta nociceptors for noxious cold stimuli.



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Fig. 7. Responses of Adelta nociceptors to cold stimuli. A: responses of a single Adelta nociceptor evoked by cold stimuli of 8, 4, and 0°C each applied for a duration of 10 s. Analog traces of stimulus temperature are provided above each oscilloscope trace. Response threshold to heat was 8°C. B: conduction latency for this Adelta nociceptor is illustrated by 3 aligned traces that show constant latency. The arrow indicates stimulus artifact. C: mean (±SE) number of impulses evoked by cold stimuli between 28 and -12°C for all cold-sensitive Adelta nociceptors. The mean number of impulses evoked increased with stimulus intensity. D: the mean (±SE) discharge rate (Hz) evoked by the same stimulus temperatures tended to increase with stimulus intensity.

Functional properties of C fibers

Mechanical thresholds were measured for 60 C fibers, of which 46 had mechanical thresholds >10 mN. Most of these (43 of 46) were polymodal in that they also responded to heat (threshold range: 37 to 47°C) and/or cold (threshold range: 16 to -12°C) stimuli. Another 11 C fibers exhibited lower mechanical thresholds (<= 6 mN) but also responded to heat (threshold range: 37 to 41°C) and/or cold (threshold range: 24 to 0°C) stimuli. Of the remaining three C fibers, all had mechanical thresholds of 6 mN; one was unresponsive to both heat and cold stimuli, and the other two were not tested for thermal sensitivity.

The median mechanical threshold of all C fibers is displayed in Fig. 5 and is compared with the median threshold values obtained for Abeta and Adelta fibers. Falling within a range of 1.2-177.5 mN (interquartile range = 14.0 mN), the median mechanical threshold of C nociceptors was 24.4 mN, and this was significantly higher than mechanical thresholds of both Abeta and Adelta fibers (P < 0.01, Mann-Whitney U test). Fifty of the C fibers were characterized for sensitivity to thermal stimuli and displayed a mean heat threshold of 40.3 ± 0.4°C and a mean cold threshold of 10.1 ± 1.9°C. Thirty (60%) C fibers (Table 1) were excited by heat and cold stimuli and were classed as CMHC, the most commonly observed type of C fiber nociceptor. C fibers were also found that responded to heat but not to cold, designated CMH (n = 11, 22%), or to cold but not to heat, classed as CMC (n = 4, 8%). Only five C nociceptors (10%) were designated CM because they responded neither to heat nor to cold stimuli but had relatively high (>10 mN) mechanical thresholds.

The mean number of impulses and the mean discharge rate increased significantly as the intensity of heat stimuli increased. This is illustrated in Fig. 8A, which shows responses to heat of a single CMH nociceptor. The responses represented in Fig. 8A are typical of heat-sensitive C fibers, which generally began their response to heat stimuli near 40°C and increased the number of impulses as stimulus intensity increased. In Fig. 8, C and D, the mean responses of all C nociceptors and their mean discharge rate as a function of heat intensity are presented. Both curves illustrate the capacity of C nociceptors to encode stimulus intensity; however, the dynamic range of intensity coding of these nociceptors for heat may extend up to ~47°C, after which mean evoked responses no longer increased.



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Fig. 8. Responses of C nociceptors to heat stimuli. A: responses of a single C nociceptor evoked by heat stimuli of 43, 45, and 51°C. ---, the analog trace of stimulus temperature. Response threshold to heat was 43°C. B: constant conduction latency for this C nociceptor is illustrated by 3 aligned traces demonstrating constant latency. up-arrow , stimulus artifact. C: mean (±SE) number of impulses evoked by heat stimuli between 35 and 51°C for all heat-sensitive C nociceptors. The mean number of impulses increased significantly with stimulus intensity. D: the mean (±SE) discharge rate (Hz) evoked by the same stimulus temperatures also increased with stimulus intensity <= 47°C.

Responses of C fiber nociceptors evoked by cold stimuli are shown in Fig. 9. Responses of a single CMHC fiber (Fig. 9A) exhibited increased discharge to cold stimuli with increasing stimulus intensity. Analysis of the mean number of impulses for all fibers (Fig. 9C) indicated that cold-sensitive C nociceptors tend to increase their discharge to cold as the stimulus temperature decreases. As shown in Table 1, the mean response threshold of C nociceptors for cold was 10.1 ± 1.9°C but was distributed over a wide range of stimulus temperatures (28 to -12°C). The discharge rate of C nociceptors was generally less than the rate evoked by heat stimuli, as illustrated in Fig. 9D where the discharge rate of C fiber nociceptors is plotted as a function of stimulus temperature.



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Fig. 9. Responses of C nociceptors to cold stimuli. A: responses of a single C nociceptor evoked by cold stimuli of 8, 4, and 0°C. ---, analog traces of stimulus temperatures. Response threshold to cold was 8°C. B: constant conduction latency for this C nociceptor is illustrated by 3 aligned traces showing identical latency. up-arrow , stimulus artifact. C: mean (±SE) number of impulses evoked by cold stimuli between 28 and -12°C for all cold-sensitive nociceptors. The mean number of impulses increased significantly with stimulus intensity. D: mean (±SE) discharge rate (Hz) evoked by cold stimuli also increased with stimulus intensity.

Power functions were derived for C nociceptors to determine whether the number of impulses evoked by thermal stimuli increased linearly, or in a positively or negatively accelerating fashion relative to incremental changes in stimulus temperature. For responses to heat, power functions were generated for 23 C fibers with a mean slope of 1.09 ± 0.07 (SE) and a range of slopes at 0.44 to 1.66. Of these power functions, 13 were positively accelerating, 8 were negatively accelerating, and 2 were approximately linear (slopes of 0.98 and 1.01). For eight cold-sensitive C nociceptors, power functions were calculated with a mean slope of 1.34 ± 0.16. The range of slopes for these power functions was 0.57-1.94. Five of these power functions displayed positively accelerating functions, one was negatively accelerating, and two were linear (slopes of 1.00 and 1.04).

During the study, two to three high-threshold fibers appeared to exhibit responses indicative of cooling fibers. These fibers were spontaneously active and responded primarily to a cooling stimulus (chilled forceps) applied to the receptive field. A conduction velocity was obtained for only one of these fibers in the C fiber range, and none were systematically studied. Nonetheless, their presence in the mouse is consistent with results from the rat indicating a small number of such high-threshold, nonnociceptive fibers (Fleischer et al. 1983; Leem et al. 1993; Lynn and Carpenter 1982). Neither Koltzenburg et al. (1997) nor Stucky et al. (1999), both using the in vitro approach, reported the presence of cooling fibers in the mouse. Future studies of mouse primary afferents are needed to explore the distribution and functional characteristics of this fiber type more thoroughly. No warm-specific fibers were encountered in this study.


    DISCUSSION
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METHODS
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DISCUSSION
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Thorough electrophysiological studies of the mouse somatosensory system are warranted by the increased development and use of transgenic/knockout mice for the study of pain. A recent report (Koltzenburg et al. 1997) was the first detailed investigation of the receptive properties of unmyelinated and myelinated cutaneous afferent fibers in the mouse. The extracellular recording approach undertaken in that investigation contrasted in three ways to that used in our study. First, an in vitro model was used to record from primary afferent fibers in either the saphenous or sural nerves. Second, all recorded fibers innervated hairy skin. In the present study, conducted in vivo, activity of primary afferent fibers in the tibial nerve was studied, and only responses of fibers innervating glabrous skin were evaluated. Finally, the present study used a contact thermode that enabled precise stimulation with heat and cold stimuli. In the previous study (Koltzenburg et al. 1997), cold stimuli were delivered by injecting a bolus of ice-cold synthetic interstitial fluid solution into the bath of an in vitro prep, thus producing repeatable temperature stimulations of 4-6°C. This approach yielded an exclusively qualitative ("cold-sensitive" vs. "cold-insensitive") result about a fiber's responsiveness. By comparison, the present study utilized a thermode, driven by a Peltier device (see Thermal stimulation) that enabled stimulation in discrete steps of 4° increments from 28 to -12°C, and hence a quantitative analysis of thermal thresholds for cold-sensitive Adelta and C fibers. Similarly, temperature thresholds of heat-sensitive fibers were obtained by delivering heat stimuli in steps of 2° increments from 35 to 51°C. In contrast, earlier studies applied radiant heat via a halogen bulb focused through the translucent bottom of the organ bath, which stimulated the skin as a continuous rise in skin temperature within 15 s from 32 to 47°C at a rate of 1°C/s (Koltzenburg et al. 1997). Consequently, we determined response thresholds of nociceptors for heat and cold as well as encoding properties to thermal stimuli over a wide range of stimulus temperatures.

Low-threshold mechanoreceptors

The prevalence of RA over SA Abeta fibers (71-29%) in the present study agrees with results obtained from fibers innervating the mouse hairy skin (Koltzenburg et al. 1997). D hair cells, which comprise roughly one-third of the myelinated fibers in the hairy skin of the adult mouse, were encountered in the hairy skin adjacent to the glabrous skin but were not studied. The median mechanical threshold of 2.1 mN for Abeta fibers recorded from fibers innervating the mouse glabrous skin compares to the 1.0 mN median values obtained from Abeta fibers in hairy skin. The slightly higher mechanical thresholds reported here may reflect the structural differences between glabrous and hairy skin receptors. In the monkey, Perl (1968) found that SA receptors of glabrous skin required relatively high forces for activation when compared with hairy skin receptors; their thresholds to stimulation with von Frey monofilaments were substantially greater than for other afferent fibers sensitive to gentle mechanical stimuli. On the other hand, Lynn and Shakhanbeh (1988) found no significant difference in mechanical thresholds between hairy and glabrous skin in the rat. The absolute threshold difference for Abeta fibers of 1.1 mN noted in the present study between hairy and glabrous skin, although small, is however consistent with higher median mechanical thresholds also reported for Adelta (10.4 mN) and C (24.4 mN) fibers in the present study. While such differences may be partially due to properties of different nerves tested or to variations attributable to in vitro versus in vivo preparations, the results of this study do support the view that mechanical thresholds of primary afferent fibers in mouse glabrous skin tend to be higher than those found in hairy skin.

It is possible that some of the SA Abeta fibers, especially those with higher mechanical thresholds, were type I AMH fibers. The range of the mechanical thresholds of SA Abeta fibers in this study was 0.4-30.1 mN. Nearly one-third (10/31) of these fibers had mechanical thresholds >24 mN, but all had conduction velocities >14 m/s, which resulted in a classification within the Abeta group. In future studies, characterization of the responses of SA Abeta high-threshold fibers to noxious thermal stimuli and to graded levels of noxious mechanical stimuli are needed to determine whether these are nociceptive fibers.

A smaller group of Abeta fibers (24/106) had mechanical thresholds >10 mN, including one with a mechanical threshold of 56.6 mN. When tested with degrees of mild pinching, the responses of these fibers to noxious mechanical stimuli did not indicate encoding of stimulus intensity. It is possible that some of these fibers were innervating deep tissue rather than skin since exact differentiation between deep and cutaneous receptor activity is problematical in certain regions of the mouse hindpaw. Unfortunately, these fibers were neither tested with thermal stimuli nor with multiple suprathreshold von Frey filaments, which might have provided more information for their functional identification. In light of these limitations, functional identification of these fibers, including any potentially nociceptive function was not possible.

A difference between the results of present study and previous studies in terms of Abeta mechanoreceptor response characteristics was the mean conduction velocity. For example, Koltzenburg et al. (1997) determined mean velocities of 13.6 ± 0.4 m/s for RA fibers and 15.5 ± 0.5 m/s for SA fibers in the saphenous nerve, whereas, in vivo, we measured tibial nerve mean conduction velocities of 21.4 ± 0.7 m/s for RA fibers and 24.3 ± 1.4 m/s for SA fibers. In another study (Stucky et al. 1999), mean conduction velocities of Abeta mechanoreceptors in normal mice were 16.6 ± 0.8 m/s for RA and 15.2 ± 1.9 m/s for SA mechanoreceptors. By contrast, conduction velocities of Abeta mechanoreceptors in rats exceed 24 m/s and can be as fast as 68 m/s (Handwerker et al. 1991; Leem et al. 1993; Sanders and Zimmermann 1986).

Adelta and C fibers in mouse glabrous skin

The median threshold of mechanosensitive Adelta fibers in the mouse tibial nerve was 10.4 mN, which is higher than the 5.6 mN (saphenous nerve) and 4.0 mN (sural nerve) median threshold values obtained previously in vitro from the mouse (Koltzenburg et al. 1997). In the present study, the range of mechanical thresholds for the 51 Adelta fibers extended between 0.1 and 111.5 mN. Of the low-threshold Adelta fibers, 20 exhibited thresholds <0.10 mN, but 5 of these responded to noxious temperature stimuli and may thus exert nociceptive function. The remaining 15 Adelta fibers (29% of the total) had an average mechanical threshold of 2.9 mN (range: 0.1-6.0 mN) and their function was not clearly definable. In a previous study using fibers from the mouse saphenous nerve, Adelta fibers with mechanical thresholds >1.0 mN were described as high-threshold mechanonociceptors (Airaksinen et al. 1996). That study utilized the in vitro mouse preparation modified from a rat preparation in which the properties of afferent fibers in vitro are essentially the same as in vivo (Kress et al. 1992; Reeh 1986). Even assuming slightly lower mechanical thresholds for cutaneous nociceptors in hairy skin, threshold values of 1-2 mN equate to tactile forces akin to light stroking of the skin with a cotton swab used in the present study as a search technique for low-threshold mechanoreceptors. Some investigators have even used the absence of response to such light brushing as a criterion for classifying a unit as a nociceptor (Andrew and Greenspan 1999). Taking into consideration the differences in mechanical thresholds of cutaneous receptors resulting from skin type and species variation, identification of nociceptive function of Adelta fibers in the glabrous hindpaw skin of the mouse probably requires mechanical thresholds equivalent to ~10 mN (or 1 g/mm2 of stimulus intensity) in the absence of sensitivity to noxious thermal stimuli or differential responses to suprathreshold mechanical stimulations. This view is indirectly supported by comparable studies in the rat that have described cutaneous Adelta nociceptive units in plantar and sural nerves with mean mechanical thresholds of 52.9 and 32.2 mN occurring within a range of 14-100 mN (Leem et al. 1993).

The median mechanical threshold of mechanosensitive Adelta fibers in the mouse tibial nerve was 10.4 mN, which is higher than the 5.6 mN (saphenous nerve) and 4.0 mN (sural nerve) median threshold values obtained previously in vitro from the mouse (Koltzenburg et al. 1997). In the present study, the range of mechanical thresholds for the 51 Adelta fibers extended between 0.1 and 111.5 mN. Of these Adelta fibers, 20 exhibited thresholds <0.10 mN, but 5 of these low-threshold Adelta fibers responded to noxious ranges of temperature stimuli and thus exert nociceptive function. The remaining 15 Adelta fibers, 29% of the total, were presumably low-threshold mechanoreceptors.

Mechanical thresholds of cutaneous C fibers ranged from 1.2 to 177.5 mN. The median mechanical threshold of 24.4 mN was higher than 5.6 mN reported for C fibers by Koltzenburg et al. (1997), who noted quantitatively identical responses of AM and C fiber nociceptors to suprathreshold mechanical stimuli. In the present study, 11 of 14 C fibers displayed mechanical threshold <0.10 mN; however, all but three of these lower threshold C fibers also responded to noxious ranges of temperature stimuli and were considered to be nociceptors. The significant differences in mechanical thresholds between Adelta and C fibers, indicated in Fig. 5, is attributable to the fact that 29% of the Adelta fibers studied were probably low-threshold mechanoreceptors with no nociceptive function, whereas all but 3 of the 68 C fibers exhibited either relatively higher mechanical thresholds (>10 mN) or sensitivity to noxious thermal stimuli. Comparable studies from the sural and plantar nerves of the rat found an average mechanical threshold for Adelta nociceptors of 37.77 ± 18.80 mN with a range of 14-100 mN, while the mechanical threshold of C nociceptors averaged 80.24 ± 59.29 mN with a range of 14-294 mN (Leem et al. 1993). Handwerker et al. (1991) recorded from Adelta nociceptors in the rat sural nerve with mechanical thresholds ranging from 32.0 to 128 mN and from CMH nociceptors with thresholds ranging from 45.0 to 362.0 mN. Collectively, results suggest a substantially lower mechanical threshold of cutaneous nociceptors in mice as compared with rats.

We found a minority (11%) of mechanically sensitive C nociceptors that responded to neither heat nor cold stimuli (classed as CM). This proportion of C fibers not responding to thermal stimuli was <30% found in the saphenous and 5% in the coccygealis nerve of the rat (Fleischer et al. 1983). The variance between results from two nerves in the rat may be attributable to methods of thermal stimulation used (ice and radiant heat).

In addition, a subpopulation of both Adelta and C nociceptors not investigated in the present study are mechanically insensitive and responsive only to noxious heat and/or algesic or pruritic chemicals (Handwerker et al. 1991; Meyer et al. 1991; Schmelz et al. 1997). All fibers in our study were mechanically sensitive since the search strategy employed mechanical stimulation only.

Sensitivity of Adelta and C fibers to heat

Whereas our studies indicate cold sensitivity for nearly half of the Adelta nociceptors tested with cold, only 12% of Adelta fibers were excited by noxious heat within the range of stimulus temperatures used. This is lower than the 26% heat-sensitive Adelta fibers as recorded in vitro from the mouse sural and saphenous nerves using noxious heat stimuli 32-47°C delivered by a halogen bulb focused through the translucent bottom of the organ bath onto the receptive field on the epidermal side of the preparation (Koltzenburg et al. 1997). This slightly higher percentage of Adelta fibers responding to heat may have resulted from differences in experimental procedures, e.g., differences in the fiber distribution among the different nerves studied and differences in response characteristics of fibers innervating two types of skin (hairy vs. glabrous). A survey of cutaneous sensory receptors in the rat hindpaw identified 55 Adelta fibers from a sample of fibers in the sural and plantar nerves (Leem et al. 1993). In that study, 15% of Adelta nociceptors responded to heat, a percentage similar to our results in the mouse (Table 1). The mean heat threshold for rat Adelta fibers was 47°C for three sural nerve fibers and ranged from 50 to 52° for five other fibers from sural and plantar nerves (Leem et al. 1993), which was higher than the mean threshold of 45.7 ± 4.06°C in the present study.

The majority (60%) of C fibers in our study were responsive to both heat and cold (CMHC). The proportion of all C fibers described as heat-sensitive in the present study, 82%, is greater than the 41% proportion of heat-sensitive C fibers identified by the Koltzenburg et al. (1997) and the similar 42% reported by Stucky et al. (1999), both of which used the same in vitro skin nerve preparation adapted from previous rat studies (Kress et al. 1992; Reeh 1986). The larger proportion of C fibers responding in our study may simply be attributable to the higher stimulus temperatures (i.e., 51 vs. 47°C) used in our study. The tendency for the predominant proportion of C fibers to be sensitive to heat stimuli has also been documented in studies of rat cutaneous C fibers. Lynn and Carpenter (1982) observed both heat and mechanical sensitivity to be common among C fibers of the saphenous nerve innervating rat hairy skin and noted average heat thresholds of these fibers to be 47°C. Fleischer et al. (1983) classed a majority of unmyelinated nociceptors recorded from the saphenous (56%) and coccygealis (70%) nerve as mechanoheat-sensitive (CMHs). Kress et al. (1992) recorded from the rat saphenous nerve and determined that 55% of C fibers recorded in vitro and 40% recorded in vivo responded to heat. Leem et al. (1993) described 80% of C fibers recorded from rat sural and saphenous nerves as mechanoheat nociceptors.

To ensure that the large size of the thermode surface area relative to the mouse foot did not sensitize fibers studied later in the day, heat thresholds of units studied the same day were compared. Only three Adelta fibers responded to heat, and these were each recorded on different days, but on 10 occasions two to five C fibers were tested with heat on the same day. Statistical analysis (1-tailed ANOVA) of average heat thresholds of same day C fiber recordings showed no significant decrease (P = 0.763) throughout the course of the day's recording.

The majority of power functions calculated for heat-sensitive C fibers were either positively accelerating or approximately linear (slopes of 0.98 and 1.01). These results reflect the intensity encoding of noxious thermal stimuli indicated when the number of impulses and discharge rate are plotted as a function of thermal intensity (Fig. 8, C and D). Power functions for Adelta fibers were omitted because of the low number of heat-sensitive fibers.

Sensitivity of Adelta and C fibers to cold

Cold stimuli were delivered by a thermode placed in constant contact with the skin and stimulus intensity increased in increments of four degrees from 28 to -12°C. Using this approach, 50% Adelta fibers were excited by cold. In contrast, only 10% Adelta nociceptors were characterized as cold sensitive using an in vitro mouse preparation (Koltzenburg et al. 1997). Differences in the proportion of nociceptors sensitive to cold are likely due to the intensity of stimulation used. A comprehensive study of cutaneous nociceptors in rats (Leem et al. 1993) observed that 10% of Adelta fibers were sensitive to cold with thresholds ranging from 22 to 12°C. However, stimulus temperatures applied in that study did not extend <12°C. In contrast, Simone and Kajander (1997) found that Adelta nociceptors were typically excited by cold stimuli from 20 to -12°C. Using these stimulus temperatures, a wide range of cold thresholds was observed for Adelta nociceptors in mouse glabrous skin ranging from 14 to -8°C.

A proportion of C fiber nociceptors also exhibit sensitivity to cold (Bessou and Perl 1969; Burgess and Perl 1967; Georgopoulos 1976, 1977; LaMotte and Thalhammer 1982; Lynn and Carpenter 1982; Perl 1968). We found sensitivity to cold in 79% of C fibers tested with a mean cold threshold of 10.1 ± 1.89°C. Previous studies in the mouse (Koltzenburg et al. 1997) reported that noxious cold stimuli excited 30% of C fibers. Similarly, Leem et al. (1993) found that only 8% of C nociceptors in the rat were excited by cold stimuli using a maximal stimulating intensity of 12°C. As in the case of Adelta fibers, the inclusion of stimulus temperatures extending well into the noxious range (e.g., -12°C) reveals a greater and substantial proportion of C fiber nociceptors responsive to cold than was observed previously with less intense cold stimuli (e.g., >0°C).

Conclusion

In conclusion, we have characterized response properties of mouse cutaneous receptors using an in vivo approach. A large proportion of Adelta and C fiber nociceptors were sensitive to heat and cold stimuli over a broad range of temperatures. The results obtained in this study will be useful for comparing response properties of primary afferent fibers in normal mice to those obtained from transgenic strains of mice developed to determine how specific gene products affect primary afferent function.


    ACKNOWLEDGMENTS

We thank Drs. Glenn Giesler Jr. and Darryl Hamamoto for critically reading an earlier version of the manuscript.

This work was supported in part by National Institutes of Health Grants NS-33908 and DA-11986. D. M. Cain was supported by the Minnesota Pain Research Training Grant (DE07288).


    FOOTNOTES

Address for reprint requests: D. A. Simone, Dept. of Psychiatry, University of Minnesota, Box 392 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: simon003{at}tc.umn.edu).

Received 23 August 2000; accepted in final form 2 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society