1Department of Neurosurgery and 2Applied Physics Laboratory, Johns Hopkins University, Baltimore, Maryland 21287
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ABSTRACT |
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Slugg, R. M.,
R. A. Meyer, and
J. N. Campbell.
Response of Cutaneous A- and C-Fiber Nociceptors in the Monkey to
Controlled-Force Stimuli.
J. Neurophysiol. 83: 2179-2191, 2000.
The goal of this study was to
determine the capacity of primary afferent nociceptive fibers
(nociceptors) to encode information about noxious mechanical stimuli in
primates. Teased-fiber techniques were used to record from 14 A-fiber
nociceptors and 18 C-fiber nociceptors that innervated the hairy skin.
Stimulus-response functions were examined with an ascending series of
force-controlled stimuli. Stimulus-interaction effects were examined
with use of a series of paired stimuli in which the interval between
the stimulus pairs was varied systematically. Both A-fiber and C-fiber
nociceptors exhibited a slowly adapting response to the stepped force
stimuli. The response of the A fibers increased monotonically with
increasing force, whereas the response of the C fibers reached a
plateau at low force levels. The slope of the stimulus-response
function for the A fibers was significantly steeper than that for the C fibers, and the total response was greater. The A fibers also provided
more discriminative information regarding stimulus intensity. The C
fibers demonstrated a significant fatigue in response when the
interstimulus interval between the paired stimuli was 150 s, whereas
the A fibers did not demonstrate a significant fatigue until the
interstimulus interval was
30 s. This fatigue in response was not due
to changes in tissue compliance. These results suggest that A- and
C-fiber nociceptors have different mechanical transduction mechanisms.
A-fiber nociceptors exhibit steeper stimulus-response functions and
less fatigue than C-fiber nociceptors.
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INTRODUCTION |
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The neurophysiological basis for pain from
mechanical stimulation of normal or injured skin and in patients with
nerve injury is not well understood. This is due in part to the lack of
suitable mechanical stimulators for delivering controlled noxious
stimuli. Many previous studies of nociception relied on hand-held
stimuli such as forceps with strain gauges (Burgess and Perl
1967), arterial clamps (e.g., Willis et al.
1974
), and von Frey probes (von Frey, 1896
) to
establish threshold for activation of nociceptors (e.g., Barasi
and Lynn 1986
; Bessou and Perl 1969
;
Kress et al. 1992
; Lynn 1994
;
Meyer et al. 1981a
; Perl 1968
;
Reeh et al. 1987
; Tanelian and Beuerman
1984
). Feedback controlled mechanical stimulator systems used
in tactile research (e.g., Chubbuck 1966
) can generate precise forces and/or displacements, but these devices usually have a
very limited range of motion (e.g., 2 mm) and therefore do not produce
pain or activate nociceptors unless the probe tip is very small or the
underlying tissue is noncompliant (Greenspan and McGillis
1991
, 1994
). To overcome these limitations, we recently developed a high-resolution mechanical stimulator for pain research based on a servo-controlled linear motor with a 20-mm range of motion.
The position of the stimulator in space is controlled by a
three-dimensional translation table (Schneider et al.
1995
).
Only a few studies have investigated the stimulus-response functions of
nociceptors to mechanical stimuli (Adriaenson et al. 1984; Campbell et al. 1979
; Cooper et al.
1991
, 1993
; Garell et al. 1996
;
Handwerker et al. 1987
; Khalsa et al.
1997
; Koltzenburg and Handwerker 1994
;
van Hees and Gybels 1981
). In this paper, we present the
first systematic evaluation of mechanically-sensitive nociceptive
afferents in monkey. We compare the stimulus-response functions of A-
and C-fiber nociceptors to controlled-force stimuli and investigate
whether the marked stimulus interaction that occurs when repeated heat
stimuli are presented to the receptive field (e.g., LaMotte and
Campbell 1978
; Tillman 1992
) also occurs for mechanical stimuli presented to a single location. Some results were
presented previously in abstract form (Slugg et al. 1993
, 1994
).
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METHODS |
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Neurophysiological preparation
A standard teased-fiber technique was used to record from single
primary afferent nociceptors (e.g., Campbell and Meyer
1983). Briefly, monkeys were sedated with ketamine and then
anesthetized to a level such that the corneal reflex was absent by
intravenous administration of a mixture of sodium pentobarbital (3 mg · kg
1 · h
1 ) and morphine sulfate (0.5 mg · kg
1 · h
1 ).
Animals were intubated, and peak expired pCO2 was
maintained at 35-40 Torr using mechanical ventilation. The animals
were paralyzed with pancuronium bromide (0.1 mg/kg) to minimize muscle
artifacts during recordings as well as to facilitate respiratory
control. The rectal temperature was controlled at 38°C by means of a
circulating water heating pad. Five percent dextrose in 0.9% normal
saline was administered intravenously in the course of the experiments to maintain hydration. Adequate depth of anesthesia was ensured by
continuous monitoring of heart rate with an electrocardiogram. The
heart rate was maintained throughout the experiment within 10% of the
baseline heart rate that was recorded before any surgical stimulus. Any
sudden increase in heart rate that was related temporally to a surgical
or test stimulus was treated with an additional bolus of the
intravenous anesthetics. When it became apparent that the animal was
spontaneously breathing (~2-3 h after pancuronium bromide
administration), the absence of motor responses to noxious stimuli was
verified, and an additional bolus dose of pancuronium bromide administered.
The nerves used in this study were the medial antebrachial cutaneous and superficial radial on the forelimb and the sural, saphenous, and superficial peroneal on the hindlimb. None of the nerves had been used in previous studies, and the areas of the receptive fields were free from previous injury. A skin incision was made over the nerve of interest, the nerve was dissected free of connective tissue, and fascicles from the nerve were separated on a dissecting platform and placed on a gold electrode wire for extracellular recordings of action potentials from single nerve fibers. At the end of the recording session, the wound was irrigated repeatedly with saline solution and then sutured. Benzathine penicillin (450,000 U) was injected intramuscularly at the beginning of each experiment. Animal housing conformed to federal regulations and the facilities were accredited by the American Association for Accreditation of Laboratory Animal Care.
The analog action potential activity from the nerve filament was
amplified and passed through a time-amplitude window discriminator. The
discriminator delivered a pulse to the computer whenever a neural event
met the waveshape criteria. Each discriminated action potential was
time-stamped relative to stimulus delivery by the stimulus control
software. To synchronize the neural activity with stimulus delivery,
the conduction delay from the receptive field to the recording
electrode was subtracted in the post hoc analysis. The conduction delay
was determined by electrically stimulating the receptive field through
a saline soaked cotton swab at twice the electrical stimulus threshold
(Meyer et al. 1991).
Receptive fields were located by squeezing of the skin in the distribution of the nerve under study. Calibrated nylon monofilaments (von Frey type, Stoelting asthesiometer set) were used to locate sensitive spots in the receptive field and to determine their response thresholds. The boundary of the receptive field was determined with a suprathreshold von Frey filament. Radiant heat was used to test for thermal sensitivity in C-fiber nociceptors. All receptive fields were located on the distal hairy skin of either the upper or lower extremity.
Fibers were classified as nociceptors if they responded to pinching of
the skin but did not respond to blunt pressure (2 N applied via a
1-cm-diam probe = 2.5 N/cm2) or light stroking.
This criteria has been used to reliably identify A-fiber nociceptors in
the past (e.g., Treede et al. 1995). No attempt was made
to subclassify A fibers into type I and type II AMHs (Meyer et
al. 1994
; Treede et al. 1995
) to avoid thermally induced sensitization. In a previous systematic study of AMH
nociceptors from the hairy skin of monkey (Treede et al.
1998
), very few type II AMHs had thresholds to mechanical
stimuli in the range of the nociceptors reported in this study. Thus it
is likely that most of the A-fiber nociceptors studied here were type I
AMHs. Mechanical thresholds were determined with a set of von Frey
filaments (force range: 0.3-168 mN; pressure range: 1.2-7.8 bar,
diameter range: 178-523 µm). A-fiber nociceptors were studied only
if their von Frey threshold was not lower than 1.9 bar (5 mN) to
eliminate the possible inclusion of low-threshold slowly adapting
mechanoreceptors in the study. C fibers with thresholds <1.9 bar were
included if they had a heat response typical of nociceptors (i.e., heat threshold <49°C).
Mechanical stimulator
We developed a computer-based electromechanical stimulator
system suited for neurophysiological and psychophysical studies of pain
(Schneider et al. 1995). The stimulator consists of a servo-controlled linear motor capable of generating 10 N of force over
a 22-mm range. Forces collinear to the interchangeable probe tip were
calculated by combining the signal from three load cells (1.25-mN
resolution, 2.5-N range) arranged in an equilateral triangle (see
Schneider et al. 1995
). Probe position was measured with an optical encoder (1-µm resolution, 25-mm range). Stimulus probes of
differing size and shape were placed in a receptacle centered at the
end of the motor's shaft. The probe was positioned orthogonal to the
skin by means of an articulated joint. A microprocessor-based digital
control system allowed feedback control to be switched between force or
position at the 1-kHz update rate. An IBM-compatible computer was used
to command stimulus paradigms and to display real-time motor
performance and neural spike-train data.
Mechanical stimulation protocols
STIMULUS WAVEFORM. All studies were conducted using a 0.4-mm-diam, stainless steel, cylindrical probe with a flat, right-angled tip. A typical stimulus waveform is shown in Fig. 1. Each stimulus trial started with a 4-s prestimulus period during which the probe was moved under position control from a position ~1 mm above the skin surface to a position at which skin contact was made as detected by an increase in the measured probe force. At this point, the stimulator control automatically switched to force control, and a 5-mN resting force was applied for the remaining time in the prestimulus period. The 3-s stimulus period consisted of a 0.2-s force-controlled ramp to the desired force and a 2.8-s constant force interval. A gradual creep in probe position typically was seen during the constant force stimulus (see Fig. 1, top). At the end of the stimulus, the probe was withdrawn in position-control mode to a position 1 mm above the skin surface.
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STIMULUS-RESPONSE FUNCTIONS. Stimulus-response functions were determined with an ascending series of five 3-s stimuli (Fig. 2A). The ascending series consisted of forces ranging from 20 to 100 mN by 20-mN increments or 40 to 200 mN by 40-mN increments. In general, the lower force range was used for units that had a lower threshold to von Frey stimulation. Some units were studied with both stimulus ranges. A 2-min interstimulus interval was used to minimize stimulus interaction. Forces higher than 200 mN were not used to minimize the potential for damaging the skin or sensitizing the afferents.
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STIMULUS-INTERACTION EXPERIMENTS. In preliminary experiments, we observed that the response to a given stimulus was dependent on the interval of time since the previous stimulus applied to the same location. The time course of this stimulus interaction was investigated systematically by delivering pairs of stimuli to a given position with different times between the conditioning stimulus and the test stimulus (Fig. 2B). For these experiments, the conditioning and test stimuli were of the same intensity, and the stimulus duration was 3 s. The interstimulus intervals were 300, 150, 60, 30, and 15 s and were presented in descending order. A 10-min stimulus-free interval preceded each conditioning stimulus, thus the response to the conditioning stimulus could serve as the reference for what the response to the test stimulus would have been in the absence of stimulus interaction.
The stimulus-response paradigm was presented first. After a 10-min stimulus-free interval, the stimulus-interaction paradigm was presented. The stimulus intensity used in the stimulus-interaction paradigm was the same as the maximum stimulus used in the stimulus-response paradigm. Two or more spots in each receptive field were studied in 11 of 14 A fibers and 14 of 18 C fibers resulting in a total of 28 spots for the A fiber and 42 spots for the C fibers. These spots were >1 mm apart so that different terminal structures were activated by the probe. The results from each of these spots were treated as independent information in our analysis. The 1-mm spacing also was chosen to avoid any sensitization effects that may occur in close proximity to a mechanical injury (Slugg et al. 1996ANALYSIS OF RESPONSE ADAPTATION.
The response of the nerve fibers adapted during the course of the
stimulus. To investigate adaptation, curves were fitted using a
nonlinear regression for two-phase exponential decay (Graphpad Prism,
Graphpad Software) using the equation y = aex + be
x + c, where
a and b represent coefficients of functions which decay at rates
and
, and c represents the plateau response.
Statistical analysis
For data analysis, four different measures of response were
used: the response during the 3-s duration of the stimulus, the response during the first 1 s of the stimulus, the maximum
instantaneous frequency, and the mean instantaneous frequency during
the first 1 s. The data comprised repeat measures of these four
parameters at five forces between 20 and 100 mN (or between 40 and 200 mN). Some fibers were recorded more than once with the stimulator at a
different location. For each response variable, a random-effects linear
regression (Diggle et al. 1994) was used to estimate and compare the typical force response function for the A and C fibers. The
average response was modeled as a quadratic function of force with
intercept and slope that varied across fibers and across the repeated
tests at different locations of a given fiber. Likelihood ratio tests
were used to test the hypothesis that the average curves for the A and
C fibers are the same.
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RESULTS |
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Fourteen A-fiber nociceptors and 18 C-fiber mechanoheat-sensitive nociceptors (CMHs) were included in this study. All receptive fields were located on the hairy skin of the forelimb or hindlimb. The static properties of these afferents are listed in Table 1.
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Stimulus response functions
An ascending series of constant force stimuli was used to assess
the stimulus response function of the nociceptors. The responses of a
typical A- and C-fiber nociceptor (both with a von Frey threshold of 5 mN) to this ascending series are shown in Fig.
3. Both fibers exhibited a slowly
adapting response to the stimulus. Although the C fiber had a larger
response than the A fiber to the 20-mN stimulus (i.e., 5 action
potentials for the C fiber vs. 2 action potentials for the A fiber),
the A fiber responded more vigorously to the 100-mN stimulus than did
the C fiber (43 vs. 15 action potentials). The total evoked response of
the A-fiber nociceptor increased monotonically as the stimulus force
increased (Fig. 3B). In contrast, the total evoked response
of the C fiber reached a plateau for stimuli of 40 mN (Fig.
3D).
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The stimulus response functions for all of the units in this study are shown in Fig. 4. For some fibers, a stimulus response function was obtained at different locations in the receptive field. Receptive field locations with relatively low von Frey mechanical thresholds (2-12 mN; 8 A fibers, 11 C fibers) were studied with stimuli ranging from 20 to 100 mN (20-mN increments), whereas locations with higher mechanical thresholds (5-65 mN; 9 A fibers, 9 C fibers) were studied with stimuli ranging from 40 to 200 mN (40-mN increment). The intention was to use a stimulus range such that the lowest stimulus intensity was near the von Frey threshold.
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There are several ways that neural responses in nociceptors may provide information to the CNS. Because it is not certain which feature of responsiveness is relevant, four measures were analyzed (Fig. 5): number of action potentials during the 3-s stimulus, number of action potentials during the first 1 s of the stimulus, average instantaneous frequency during the first 1 s of the stimulus, and peak instantaneous frequency. Regardless of which response parameter is considered, the magnitude of responses of A-fiber nociceptors was significantly greater than that of the C fibers (all P values <0.001). This difference was associated with a significantly greater slope of the stimulus-response function of the A-fiber nociceptors (Fig. 5). Of note, the response of the C fibers reached a plateau, whereas the A fibers had a monotonically increasing response.
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We sought to determine which of the four response measures would provide the most discrimination between different force levels. As a measure of discrimination, we determined the distance in standard deviation units between the responses to the middle (60 or 120 mN) and highest (100 or 200 mN) force. A discrimination score for each response measure was determined by calculating the difference in the response to these two forces for a given fiber and then dividing the mean of this difference across fibers by the standard deviation of this difference distribution. As indicated in Table 2, for a given force series (i.e., a column in Table 2), the discrimination scores were similar (i.e., within 35% of each other) for each response measure except for the peak instantaneous frequency measure whose score was substantially lower than the others in two of the four instances. The discrimination scores for the A fibers were substantially higher than the scores for the C fibers. This suggests that A-fiber nociceptors provide more discriminative information to the CNS than C-fiber nociceptors regarding the magnitude of intense mechanical stimuli over this force range.
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Adaptation of response
The response of both the A fibers and the C-fiber nociceptors
adapted during the 3-s-duration stimulus. To investigate adaptation, we
plotted the instantaneous frequency as function of time (Fig. 6) for the first conditioning stimulus in
the stimulus-interaction paradigm. The response to this stimulus was
investigated because the stimulus was always above threshold and
because it was always preceded by a 10-min stimulus-free interval so
that stimulus interaction was negligible. As illustrated by the
examples in Fig. 6, peak instantaneous frequency usually occurred at
the beginning of the response, and the response adapted such that the
instantaneous frequency at the end of the stimulus was significantly
lower than at the beginning. The discharge pattern for many fibers
(e.g., Fig. 6, A and B) contained two regions
with different adaptation rates, a period of fast adaptation that
occurred during the first part of the stimulus followed by a period of
slow adaptation. This suggests that two different mechanisms are
involved in the adaptation phenomena, one with a short time constant
and one with a long time constant. The curves in Fig. 6 () were
obtained by fitting the data assuming a two-compartment, exponentially
decaying response. Indeed, the responses in Fig. 6, A and
B, were fitted well by a curve with dual exponential decay.
In contrast, the responses in Fig. 6, C and D,
were fitted well by a curve where the two time constants were almost
identical, suggesting that a single exponentially decaying process
probably was involved for these fibers.
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For most fibers, the response ended at the end of the stimulus.
However, one C fiber showed an afterdischarge that lasted for 7 s.
Tissue compliance
As the stimulus force increased, the position of the probe also changed. The skin displacement for a given stimulus force was defined as the probe position at the end of the stimulus minus the probe position at the start of the stimulus. The amount of displacement is related to the compliance of the tissue. For low compliance tissue (e.g., over bone), displacements of <1 mm were observed at the highest forces. In contrast, for high compliance tissue (e.g., over muscle), displacements of >2 mm were frequently observed at the highest forces. To estimate compliance, we computed the slope of the displacement versus force curve over the force interval of 40-80 mN. These forces were chosen because they are common to both stimulus ranges used in these experiments. The distribution of compliance is shown in Fig. 7A. A- and C-fiber nociceptors were found in both compliant and noncompliant tissue.
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A mechanical stimulus to the skin leads to compressive, shear and
tensile stresses in the vicinity of the nociceptor terminals. Although
it is not clear which of these stresses is most important for
activating nociceptors, Khalsa et al. (1997) suggested
that skin tension is a critical factor. We did not control tension directly. However, the amount of tension is related directly to the
amount of displacement that is produced for a given force. In very
compliant skin (e.g., over muscle), there is much more displacement,
and therefore more tension, than in noncompliant skin (e.g., over
bone). To determine whether the neural response was a function of
compliance, we grouped the data into two groups: data from
high-compliance versus low-compliance tissue. The criteria level was
set at a compliance level of 10 µm/mN, which divides the population
approximately in half (see Fig. 7A). The average responses
of A fibers tended to be higher in low-compliance tissue (Fig.
7B). In contrast, the average responses of C fibers were significantly higher (P < 0.05, t-test on
sum of response to force sequence) in high-compliance tissue (Fig.
7C). These results are consistent with the conclusion that
tension may be a greater factor for C-fiber nociceptors than for
A-fiber nociceptors.
Stimulus interaction
A significant fatigue in response is observed when repeated heat
stimuli are applied to C-mechanoheat nociceptors (e.g., LaMotte and Campbell 1978; Tillman 1992
). To see if a
similar phenomenon existed for mechanical stimuli, a
stimulus-interaction paradigm was designed to examine the effect of a
conditioning stimulus on the response to a test stimulus delivered at
specified intervals (see Fig. 2B). The responses of a
typical A- and C-fiber nociceptor to this stimulus-interaction paradigm
are shown in Fig. 8. For the A fiber, the
response to each of the conditioning stimuli was similar both in terms
of the time course and magnitude of evoked response (Fig.
8A). In addition, interaction effects (fatigue) were present
only when the interstimulus interval (ISI) was
30 s.
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As with the A fiber, the response of the C fiber to each of the conditioning stimuli was similar in terms of both time course and magnitude of the evoked response (Fig. 8B). However, stimulus-interaction effects were present for all ISIs <300 s.
Similar results were obtained from the population of A and C fibers that were tested with the stimulus-interaction paradigm. The response to the conditioning stimulus did not vary significantly with time during the stimulus-interaction protocol for either the A or the C fibers. Thus the 10-min interval between conditioning-test pairs provided sufficient time for complete recovery of the response.
To combine data across fibers, the amount of fatigue for each ISI was
determined by computing the ratio of the response to the test stimulus
divided by the response to the immediately preceding conditioning
stimulus. This interaction ratio was dependent on the ISI for the A
fibers (Fig. 9, top) and for
the C fibers (Fig. 9, bottom). The maximum interaction
(i.e., smallest interaction ratio) was observed for the shortest ISI
(i.e., 15 s), and the amount of interaction decreased (i.e., the
interaction ratio approached unity) as the ISI became longer. To avoid
stimulus-interaction effects, ISIs >30 s for the A fibers and 150 s for the C fibers are required. These restrictions apply to mechanical
stimuli applied to a single locus in the receptive field. The
interstimulus interval can be considerably shorter if the mechanical
stimuli are not applied to the same site in the receptive field
(Slugg et al. 1995).
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Because the stimulus-response functions (Fig. 5) were obtained with an ISI of 120 s, some stimulus interaction was present for the C-fiber data. Therefore the possibility exists that the difference in slope between the A- and C-fiber nociceptors described earlier was solely due to the more prominent fatigue evident in the C fibers for the 2-min ISI used in the ascending series. In Fig. 10, we compare the total responses of the A and C fibers to the different forces in the ascending series. On this figure, we also plot the response that was obtained to the first stimulus in the stimulus-interaction paradigm; this stimulus was equivalent to the highest stimulus in the ascending series and was presented 10 min after the completion of the ascending series. This response corresponds to the expected response to the ascending series if stimulus interaction did not occur. As expected, the mean response of the C fibers to this stimulus was significantly lower during the ascending series compared with 10 min later. Thus the fatigue in the C fibers tended to flatten their stimulus response curve. However, this fatigue was not sufficient to make up for the marked difference in slope between the A- and C-fiber stimulus response functions. A new discrimination score was computed for the total response to the 3-s stimulus using the response to the first conditioning stimulus (instead of the response to the last stimulus in the ascending series as done in Table 2). The discrimination scores for both the A fibers (1.31 for low series, 1.93 for high series) and the C fibers (1.00 for the low series, 1.23 for the high series) improved (see for comparison first row in Table 2). However, the discrimination scores for the A fibers were still better than for the C fibers.
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A proportion of the decrease in responsiveness of both the A- and
C-fiber nociceptors appeared to be due to a decrease in the
high-frequency discharge that occurred at the onset of the stimulus
(e.g., Fig. 8). To quantitate the amount of fatigue that was observed
in the first, high-frequency part of the stimulus and the second,
low-frequency part of the stimulus, the 3-s duration stimulus period
was divided into the initial 1-s interval and the subsequent 2-s
interval. The evoked response in each interval was calculated for the
conditioning stimulus and the test stimulus. An interaction ratio (as
described in the preceding text) was computed for each stimulus part.
For the A fibers, the interaction ratio at an ISI of 15 s for the
first 1 s (0.68 ± 0.03; mean ± SE) was
significantly lower than the interaction ratio for the last 2 s
(0.81 ± 0.03, P 0.01, paired
t-test). In contrast, no significant difference was observed
for the C fibers.
The stimulus interaction does not appear to be due to changes in biophysical properties of the skin because displacements for a given stimulus were similar throughout the testing. These data strongly suggest that the prominent interaction effects are not due to a change in the transmission of the stimulus because the biophysical properties of the skin do not change throughout the protocol.
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DISCUSSION |
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The objective of this study was to characterize the responses of A- and C-fiber nociceptors in monkey to controlled-force stimuli applied to their receptive fields. We found that the response to stepped force stimuli for both types of fibers is slowly adapting, the stimulus response functions of A-fiber nociceptors are steeper than those of C-fiber nociceptors, greater discriminable information about stimulus intensity was provided by the A fibers, and fatigue effects are greater in C-fiber nociceptors than in A-fiber nociceptors.
Slowly adapting response
Both A- and C-fiber nociceptors exhibited a slowly adapting
response to stepped force stimuli. For many fibers, the adaptation appeared to have two components: a fast adaptation during the first
part of the stimulus followed by a slow adaptation for the rest of the
stimulus. This suggests that two different mechanisms may account for
the adaptation. For some of the fibers, the high-frequency discharge
occurred during the 200-ms initial ramp phase of the stimulus after
which a marked decrease in discharge frequency was observed. Thus one
explanation for the fast adaptation at the onset of the stimulus could
be that the initial response of the nociceptor is dependent on the
rate-of-change of the stimulus, a well-known property of low-threshold
mechanoreceptors. Unfortunately, the paradigms used in this study did
not allow us to examine this possibility systematically. Garell
et al. (1996), in their study of feline nociceptors, did not
observe an adapting response similar to what we observed in the present
study. Although species differences might play a role, differences in
experimental design such as ramp rate and ISI likely account for this discrepancy.
Rate sensitivity fits anecdotally with the psychophysical observation
that a slap stimulus to the skin is painful, whereas comparable forces
applied gradually do not necessarily induce pain. In addition,
Koltzenburg and Handwerker (1994) found that nociceptor
response increased with the velocity of a projectile stimulus.
Similarly C-fiber nociceptors (e.g., Meyer and Campbell 1981b
) and innocuous thermal receptors (e.g., Konietzny
and Hensel 1977
; Molinari and Kenshalo 1977
)
exhibit both a rate- and temperature-sensitive response to thermal stimuli.
Stimulus response functions
An important finding of the present study was the marked
difference in stimulus response functions between A- and C-fiber nociceptors. The A- and C-fibers were similar with regard to responses to low-intensity stimuli. However, the A fibers had a relatively steep
stimulus response function, whereas the response of many of the C
fibers reached a plateau at the higher stimulus intensities. A similar
saturation in the stimulus-response functions of C-fiber nociceptors
has been noted in rat (Khalsa et al. 1997) and cat (Garell et al. 1996
).
A discriminative analysis that considered both the slope of the
stimulus-response function and the standard deviation of the response
revealed that the A-fiber nociceptors provide more information about
noxious mechanical stimuli to the CNS than individual C-fiber nociceptors. Garell et al. (1996) came to similar
conclusions in a study of feline nociceptors.
A direct comparison of the magnitude of the response and shape of the
stimulus-responses functions between this and other studies is
difficult because of different stimulus conditions. For example, in the
Garell et al. (1996) study, the ISI was 7-8 s in
contrast to the 2-min ISI used for comparable experiments in this
study. In addition the stimulus duration and force range were greater
in the Garell study, but the displacement range was restricted to 2 mm.
It would appear that response saturation occurred with much higher
forces in the Garell study. Whether this is a species difference or
relates to stimulus variables remains an open question.
Tissue compliance
Both A- and C-fiber nociceptors innervate tissues that vary widely
in compliance. Highly compliant tissue is present when receptive fields
are located in skin that overlies muscle, for example, and relatively
noncompliant tissue is present when receptive fields overlie bone. By
studying the stimulus-response functions of nociceptors in compliant
and noncompliant tissue, we get some hint as to the importance of
different mechanical stresses in generating neural activity. The
A-fiber nociceptors had a tendency to respond more vigorously when
their receptive fields were in regions of noncompliant tissue. In
contrast, C-fiber nociceptors responded significantly more vigorously
when their receptive fields were in areas of compliant tissue (see Fig.
7). These data support the hypothesis that A- and C-fiber nociceptors
have preferential sensitivities to different types of stresses. The
relative role of compressive versus tensile and shear stresses varies
with tissue compliance. For example, in compliant tissue, the amount of
displacement for a given force applied orthogonal to the skin is
greater. Thus tensile stresses are greater than in relatively
noncompliant tissue where the same force leads to much less
displacement. In noncompliant tissue, compressive stress likely plays a
greater role in relation to the total stress generated by the stimulus.
Our observations that A fibers responded more in noncompliant tissue
and C fibers responded more in compliant tissue suggest that
compressive stress plays a more important role for A-fiber nociceptors,
whereas tensile stresses play a more important role for C-fiber
nociceptors. This is consistent with the observation of Khalsa
et al. (1997) that C-fiber nociceptors respond better to
tensile loading than to compressive loading.
Stimulus interaction
Fatigue (the decrement in response that results from a prior stimulus) was found in this study to be a property of both the A- and C-fiber nociceptors. Fatigue was more prominent in the C fibers than in A fibers. In addition, the time for full recovery of the initial response was longer for C fibers (5 min) than for A fibers (1 min). This fatigue could result from changes in stimulus transmission, stimulus transduction, spike initiation, and spike propagation.
Transmission refers to the transfer of energy from the stimulus contact point (the skin surface in this instance) to the receptor endings. A change in the skin properties from one stimulus to the next may modify stimulus transmission and account for the interaction phenomena. This explanation of course would not explain the differences between A and C nociceptors (assuming that the terminals were similarly located in the skin). However, no change in tissue compliance was evident during these experiments, and therefore a change in stimulus transmission does not appear to account for the fatigue.
Fatigue also could result from changes in the stimulus
transduction apparatus. Transduction refers to the
conversion of the physical energy of the stimulus into an ionic current
in the receptor membrane. One line of evidence that suggests that
transduction may be the site of fatigue is the observation that fatigue
to repeated heat stimuli also is present but is more pronounced than the fatigue to repeated mechanical stimuli. In Fig.
11, the interaction to heat stimuli
from a previous study that used a similar paradigm (Tillman
1992) is compared with the interaction to mechanical stimuli
observed in the present study. The initial response to the heat
stimulus (16.9 ± 5.8 action potentials, n = 13)
was not significantly different from the initial response to the
mechanical stimulus (18.3 ± 1.1 action potentials,
n = 33). Of particular note is the observation that the
interaction ratio for heat stimuli presented 30 s apart (0.39 ± 0.03) was significantly smaller than that for mechanical stimuli
found in this study (0.70 ± 0.03, P
0.001).
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Another potential site for fatigue is the point of spike
initiation. Cross-modality interaction effects would support this hypothesis. Preliminary studies in our laboratory have demonstrated that mechanical stimulation lead to a reduction in the response to a
subsequent heat stimulus (Peng et al. 1997).
Another possibility is that alteration in the propagation of
the action potential could account for the temporal interaction effects. A slowing of propagation after a spike train is readily apparent in C-fiber nociceptors (e.g., Meyer et al.
1985) and has been used by microneurographers to mark fibers
(e.g., Schmelz et al. 1995
). Indeed, a failure of
propagation has been reported in A- and C-fiber nociceptors when the
parent axon was stimulated electrically at relatively high frequencies
(50 Hz for A fibers, 10 Hz for C fibers) for relatively long durations
(i.e., 20 s) (Raymond et al. 1990
). Recovery of
propagation took 3-60 s. Although peak frequencies reached these
levels in this study, they were not maintained for long durations, and
thus propagation failure of the parent axon probably is not a factor
for the fatigue to repeated mechanical stimuli. Whether propagation
failure occurs in the fine branches of the terminal arborization
remains to be determined.
We wondered whether the prominent fatigue in C fibers could account for
the differences in stimulus-response functions between the A and C
fibers because the time between stimuli for the stimulus response
protocol was only 2 min, which is not long enough for full recovery in
the C fibers. In other words, had the stimuli been presented with a
sufficiently long ISI to allow full recovery of response in both the A
and C fibers, perhaps the slope of the stimulus response functions
would not have been different. Because fatigue is related to the
intensity of the preceding stimulus (LaMotte and Campbell
1978; Peng et al. 1997
), the fatigue for an
ascending series is greatest at the higher force levels. This would
tend to flatten the stimulus-response curves for the C fibers. However,
even if the response of the C fibers to the ascending series was
replotted to incorporate fatigue (for example, by using the response to
the maximum stimulus presented at a 10-min ISI), the stimulus-response
function still would plateau at higher stimulus intensities, and the
response still would be lower than that obtained for A fibers (Fig.
10).
Role of nociceptors in pain sensation
For heat stimuli, the threshold for activation and the response to
suprathreshold stimuli of C-fiber nociceptors closely parallels the
pain threshold and stimulus response function for human subjects (e.g.,
LaMotte and Campbell 1978; Meyer and Campbell
1981a
). In contrast, for mechanical stimuli, the threshold for
activation of C fibers is significantly lower than the threshold for
pain (Cooper et al. 1993
; Greenspan and McGillis
1991
), and the response of many of the C fibers has reached a
saturation level at force levels that are below the pain threshold. For
example, the 200-mN mechanical stimulus in this study produces a mean
response of 16 action potentials in the C-fiber nociceptors, but this
stimulus is below the pain threshold in humans (>400 mN)
(Greenspan and McGillis 1991
). In contrast, an initial
45°C 3-s laser thermal stimulus evokes a weaker discharge (mean = 10 action potentials) in C-fiber nociceptors (Campbell and
Meyer 1983
), but this stimulus reliably induces a distinct pain
sensation (LaMotte and Campbell 1978
). What accounts for
this disparity?
Adriaensen et al. (1984) suggested that the concurrent
activation of low-threshold mechanoreceptors (LTMs) centrally
suppressed the pain that would otherwise be evoked by activity in the
nociceptors. Heat stimuli hurt more because no coactivation of LTMs
occurs with a heat stimulus.
Another possible explanation relates to the observation that the laser
heat stimulus activates nociceptors over an 8-mm diameter region,
whereas the number of nociceptors activated by the punctate mechanical
stimulus is considerably less. The disparity may relate to the
psychophysical property of spatial summation which has been shown to be
important for heat pain sensation (Douglass et al. 1992;
Price et al. 1989
).
Another possibility is that mechanically sensitive afferents are not
concerned with pain sensation (Meyer et al. 1991).
Garell et al. (1996)
found that nociceptors with high
mechanical thresholds (i.e., mechanically insensitive afferents, MIAs,
von Frey threshold >6 bar) showed less response saturation and
appeared to have the capacity to encode unambiguously mechanical
stimuli into the noxious range. However, in a study of oral mucosal
afferents in goat (Cooper et al. 1991
), a group of
nociceptors with especially high mechanical thresholds did not encode
the magnitude of pressure stimuli. MIAs were not encountered in the
present study because an electrocutaneous search strategy (Meyer
et al. 1991
) was not employed and intense mechanical stimuli
were not used for searching.
In this study, mechanical stimuli were delivered to the most sensitive region within the receptive field with a small, punctate probe. A saturation in the stimulus response function of C-fiber nociceptors may not occur if the same stimuli were presented to less-sensitive areas of the receptive field or if larger probe diameters were used. Thus the response of the C-fiber population may not exhibit saturation until much higher force levels if spatial recruitment is taken into account. Future studies are needed to determine the stimulus-response functions of nociceptors for stimuli presented to any position within the receptive field.
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ACKNOWLEDGMENTS |
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The authors thank T. V. Hartke for assistance during the electrophysiological experiments and S. L. Zeger and A. Chen for advice about the statistical analysis. We appreciate the useful comments of Drs. Joel Greenspan and Rolf-Detlef Treede on an earlier draft of this manuscript.
This research was supported by National Institute of Neurological Disorders and Stroke Grants NS-14447 and NS-09260.
Present address of R. M. Slugg: Dept. Physiology/Pharmacology, Oregon Health Sciences University, Portland, OR 97201.
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FOOTNOTES |
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Address for reprint requests: R. A. Meyer, Johns Hopkins Applied Physics Laboratory, Johns Hopkins Rd., Laurel, MD 20723.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 February 1999; accepted in final form 10 December 1999.
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REFERENCES |
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