1Department of Preventive Sciences and 2Department of Psychiatry, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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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 A mechanoreceptors, 51 as A
fibers, and 68 as C fibers. A
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 A
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 A
and C fibers, respectively. Responses of A
and C fibers
evoked by heat (35-51°C) and by cold (28 to
12°C) stimuli were
determined. Mean response thresholds of A
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 A
fibers were heat sensitive, 50% responded to cold.
Only one A
nociceptor responded to both heat and cold stimuli. In
addition, 40% of A
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.
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INTRODUCTION |
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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 A
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-
(TNF-
), 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.
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METHODS |
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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, A,
A
, 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 (A
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 A 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, A 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 A
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 A 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 A
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.
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RESULTS |
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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 A and A
fibers of 13.6 m/s,
i.e., fibers with conduction velocities >13.6 m/s were identified as
A
fibers. The average latencies at peak response amplitude
correlated to conduction velocities of 19.8 m/s for A
and 10.7 m/s
for A
fibers. The maximal conduction velocity for C fibers was
calculated to be 1.3 m/s.
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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 A fibers, 51 as A
fibers,
and 68 as C fibers. The mean conduction velocity of A
fibers was
22.2 ± 0.7 (SE) m/s and ranged from 13.8 to 40.0 m/s. A
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 A
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 A
fibers over the range of
conduction velocities.
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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
A
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 A
and A
fibers with RFs located on
footpads in contrast to a low number of C fibers.
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Functional properties of A mechanoreceptors
A total of 106 A fibers were differentiated into RA and SA
functional subtypes. As summarized in Table 1, 75 A
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 A
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 A
fibers was 2.1 mN.
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Functional properties of A fibers
A total of 51 A 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 A
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 A
fibers were
spontaneously active.
As illustrated in Fig. 5, the median mechanical threshold of the A
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 A
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 A fibers were distinguished
based on responses evoked by thermal stimuli (Table 1). Of 25 A
fibers tested for sensitivity to heat stimuli, 13 (52%) A
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 A
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 A
fiber responded to cold (0°C) and to heat (39°C) and was classed as
AMHC-Type II. Thus 10 of 20 A
fibers (50%) characterized for heat
and cold sensitivity responded to cold stimuli. In addition to the
AMHC-Type II fiber, one other A
fiber had a heat threshold of
45°C, was insensitive to cold, and was classed as a AMH Type II
fiber. Another A
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 A
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 A
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 A
fibers. Subsequent
recordings in our laboratory for a study in progress provide further
evidence that a majority of A
fibers fit the AM profile.
Responses of a single A 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 A
fibers that responded to heat (n = 3) in our study, an
overall intensity-encoding ability of A
fibers to heat was not
demonstrated. Indeed, one of these A
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 A
fibers excited by heat, and Fig. 6D displays
their mean discharge rate plotted as a function of stimulus intensity.
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Responses of A nociceptors evoked by cold are presented in Fig.
7, which includes responses of a
representative A
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 A
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 A
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 A
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 A
nociceptors for noxious cold stimuli.
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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 A and
A
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 A
and A
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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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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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.
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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 A
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 A 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 A
fibers recorded from fibers innervating the mouse
glabrous skin compares to the 1.0 mN median values obtained from A
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 A
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
A
(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 A fibers, especially those
with higher mechanical thresholds, were type I AMH fibers. The range of
the mechanical thresholds of SA A
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 A
group. In future studies,
characterization of the responses of SA A
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 A 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 A 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 A
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 A
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
).
A and C fibers in mouse glabrous skin
The median threshold of mechanosensitive A 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 A
fibers extended between 0.1 and 111.5 mN. Of the low-threshold A
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 A
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, A
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 A
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 A
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 A 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 A
fibers extended between 0.1 and 111.5 mN. Of these A
fibers, 20 exhibited thresholds <0.10 mN, but 5 of these low-threshold A
fibers responded to noxious ranges of temperature stimuli and thus
exert nociceptive function. The remaining 15 A
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 A
and C
fibers, indicated in Fig. 5, is attributable to the fact that 29% of
the A
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 A
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 A
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 A 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 A and C fibers to heat
Whereas our studies indicate cold sensitivity for nearly
half of the A nociceptors tested with cold, only 12% of A
fibers were excited by noxious heat within the range of stimulus temperatures used. This is lower than the 26% heat-sensitive A
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 A
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 A
fibers from a sample of fibers in the sural and plantar nerves (Leem et al. 1993
). In that study, 15% of A
nociceptors responded to heat, a percentage similar to our results in
the mouse (Table 1). The mean heat threshold for rat A
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 A
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 A fibers were omitted because of the low number of heat-sensitive fibers.
Sensitivity of A 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% A
fibers
were excited by cold. In contrast, only 10% A
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 A
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 A
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 A
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 A
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
A 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.
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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).
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FOOTNOTES |
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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.
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REFERENCES |
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