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JOURNALOF NEUROPHYSIOLOGY
Vol. 72. No. I, July 1994. Printed in U.S.A.
A C-Fiber Reflex Modulated by Heterotopic Noxious Somatic Stimuli
in the Rat
SYLVAIN FALINOWER, JEAN-CLAUDE WILLER, JEAN-LOUIS JUNIEN, AND DANIEL LE BARS
Institut de Recherche Jouveinal, BP 100, 94265 Fresnes Ckdex; Laboratoire de Neurophysiologie, 75013 Paris;
and Institut National de la Sante’ et de la Recherche Wdicale, U 161, 75014 Paris, France
SUMMARY AND CONCLUSIONS
1. Electromyographic recordings were made from the biceps
femoris muscle through a pair of noninsulated platinium/ iridium
needle electrodes in male Sprague-Dawley rats artificially venti-
lated and anesthetized with 0.8% halothane in a N,O-0, mixture
( 2 / 3: 1 / 3 ) . The animals’ ventilation, heart rates, and body temper-
atures were continuously monitored. Electrical stimuli (2-ms dura-
tion, 0.16 Hz) were delivered to the sural nerve territory through a
pair of noninsulated platinium/ iridium needle electrode inserted
subcutaneously in the medial aspect of the 4th and the lateral part
of the 5th toe. Such stimulation elicited a two-component reflex
response in the ipsilateral biceps femoris muscle: The first had a
short latency ( 17.5 t 2.3 ms), short duration (20.7 k 2.6 ms), and
low threshold ( 1.5 & 0.6 mA), whereas the second had a longer
latency ( 162.4 t 5.1 ms), longer duration (202.3 -t 6.2 ms), and
higher threshold ( 5.7 t 0.5 mA ) .
2. Lidocaine (0.02-O. 1%; 0.1 ml), but not saline, injected sub-
cutaneously over the proximal part of the sural nerve, produced a
selective depression of the late component of the reflex response,
whereas the first component remained unchanged. The conduc-
tion velocity of the afferent fibers was estimated from the stimula-
tion needles in the sural nerve territory to the nerve’s projection in
the lumbar spinal cord: it was concluded that the second, late
component of the reflex response was due to afferent signals trans-
mitted via unmyelinated C-fibers, whereas the first component
was related to activation of fine myelinated fibers (A6 group).
3. Electrical stimulation of the sural nerve was still able to elicit
the two-component reflex responses in the ipsilateral biceps fe-
moris muscle of chronic spinal rats, indicating that these responses
were genuine reflex responses, transmitted completely through a
spinal circuit.
4. The C-fiber reflex was recorded when the duration and fre-
quency of the stimuli applied to the sural nerve varied within the
0.5- to 4-ms and 0.02- to l-Hz ranges, respectively. It was con-
cluded that a single 2-ms duration shock at an intensity of 1.2
times the C-fiber reflex threshold, delivered every 6 s (0.16 Hz),
constituted an acceptable and optimal protocol for experiments in
which the C-fiber reflex was studied as a function of time. These
parameters were used throughout the subsequent experiments.
5. The effects of nonnoxious and noxious mechanical and ther-
mal heterotopic conditioning stimuli were studied on the C-fiber
reflex in normal anesthetized nontransected animals: although
nonnoxious stimuli remained ineffective, all the modalities of
noxious conditioning stimuli that were tested produced clear-cut
depressions of C-fiber reflex activity.
6. A noxious (6-8 N/cm”) heterotopic pinch resulted in a
strong inhibition (80-90s) of the C-fiber reflex during a I-min
conditioning period, followed by poststimulus effects lasting 2-4
min. By contrast, light (0.8-l .2 N/cm2) or mild (2.6-3.2 N/cm2)
nonnoxious pressures were totally ineffective.
7. Noxious (52°C) thermal heterotopic stimuli, whether ap-
plied to a paw or to the tail, produced strong inhibitions (80-90s)
of the reflex response, during both the 1-min conditioning period
and the following minutes. Increasing intensities of thermal condi-
tioning stimuli in the 46-52°C range were applied to the contralat-
era1 hindpaw or the tail. No effects were seen after the application
of 46°C but the C-fiber reflex was inhibited in a temperature-
dependent fashion in the 47-52OC range.
8. These effects of noxious mechanical and thermal hetero-
topic conditioning stimuli disappeared when the C-fiber reflex was
recorded in chronic spinal animals. In these animals, a small tran-
sient decrease in the C-fiber reflex was seen only during noxious
heating of the contralateral hindpaw.
9. In animals with a unilateral lesion of the dorsolateral funicu-
lus (DLF), the effects of noxious-conditioning procedures on the
C-fiber reflex depended on the recording site. When the C-fiber
reflex was recorded from the limb contralateral to the lesion,
noxious conditioning stimuli, whether applied to the muzzle, the
paws, or the tail, produced inhibitory effects that were very similar
to those seen in nontransected animals. When the C-fiber reflex
was recorded from the limb ipsilateral to the lesion, none of the
noxious conditioning stimuli elicited inhibitory effects. Surpris-
ingly, the C-fiber reflex was actually facilitated during noxious
pinch of the muzzle.
10. It is concluded that the general behavior of the C-fiber re-
flex described herein has several striking similarities with conver-
gent neurons recorded in the dorsal horn or the trigeminal nucleus
caudalis, notably, the ability to be depressed by noxious stimuli
applied to remote areas of the body via a supraspinal loop that
include the DLF as) the descending pathway. These results are
discussed with reference to the mechanisms and modulation of
pain. It is proposed that such a model is valid and useful in the
study of such pain processes.
INTRODUCTION
The transmission of nociceptive signals can be modu-
lated by powerful controls at as early a stage as the first
spinal relays. These controls include both segmental mecha-
nisms and systems that involve supraspinal structures, and
some of them can be triggered by somesthetic stimuli (see
references in Besson and Chaouch 1987; Dubner and Ben-
nett 1983; Le Bars et al. 1984, 1986, 1989; Wall 1989; Willis
and Coggeshall 199 1; Ziegelgansberger 1986). This is true
not only for nonnoxious stimulation of large diameter cuta-
neous fibers that trigger segmental mechanisms in the
corresponding dermatome, but also for nociceptive stimuli
that can elicit heterotopic, i.e., nonsegmental, inhibitions.
For convenience, the latter phenomenon has been termed
“diffuse noxious inhibitory controls” (DNIC). DNIC con-
cern mainly the dorsal horn convergent neurons that are
activated by both a variety of nociceptive stimuli and weak
194 0022-3077/94 $3.00 Copyright 0 1994 The American Physiological Society
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 195
mechanical stimuli applied to their receptive fields. The
term “convergent neurons” summarizes their main prop-
erty quite well, i.e., that they constitute a strategic site where
various types of excitatory and inhibitory influences con-
verge. Their role in nociception is a cornerstone in our un-
derstanding of pain processes (see references in Besson and
Chaouch 1987; Dubner and Bennett 1983; Le Bars et al.
1986; Willis and Coggeshall 199 1).
In the rat, the cat (Morton et al. 1987) and probably the
monkey (Brennan et al. 1989; Gerhart et al. 198 1 ), the
activity of these cells can be strongly inhibited by noxious
inputs. Such effects do not appear to be somatotopically
organized and affect all convergent neurons, including
those projecting to the thalamus (Dickenson and Le Bars
1983), whether in the dorsal horn of various segments of
the spinal cord (Cadden and Morrisson 199 1; Cadden et al.
1983; Calvin0 et al. 1984; Le Bars et al. 1979a; Ness and
Gebhart 199 1 a,b; Schouenborg and Dickenson 1985; Tom-
linson et al. 1983) or in trigeminal nucleus caudalis (Dick-
enson et al. 1980; Hu 1990). By contrast, DNIC do not
affect the other neuronal types that are found in these struc-
tures, i.e., lamina 1 noxious-specific, nonnoxious-specific,
cold-responsive, and proprioceptive neurons (Dickenson et
al. 1980; Le Bars et al. 1979b). The principal feature of
DNIC is that they can be triggered by conditioning stimuli
applied to any part of the body other than the excitatory
receptive field of the neuron under study, provided that the
stimuli are clearly noxious. DNIC can be triggered by any
heterotopic nociceptive stimulus, whether it be mechanical,
thermal, chemical, or electrical but nonnoxious stimuli are
completely ineffective, at least in nonpathological condi-
tions. With strong stimuli, the inhibitory effects are power-
ful and are followed by long-lasting poststimulus effects
that can persist for several minutes. DNIC are not observed
in animals with high spinal cord sections (Cadden et al.
1983; Le Bars et al. 1979b; Morton et al. 1987) and it is thus
obvious that the mechanisms underlying DNIC are not
confined to the spinal cord and involve supraspinal struc-
tures. Such a system is therefore completely different from
segmental inhibitory systems that work both in intact and
in spinal animals and can be triggered by the activation of
low-threshold afferents. DNIC are also very different from
the propriospinal inhibitory processes that can be triggered
by noxious inputs (Cadden et al. 1983; Fitzgerald 1982;
Gerhart et al. 198 1). We must acknowledge however that
all possible inhibitory mechanisms triggered by heterotopic
conditioning stimuli were not envisaged in the studies cited
above; in this respect, type of anesthesia, spatial and tem-
poral parameters of stimulation, and species differences are
putative factors of variation.
The outputs of dorsal horn convergent neurons are nu-
merous and include many supraspinal targets as well as,
presumably, motoneurons. The aim of the present work
was to investigate electrophysiologically in the rat, the be-
havior of a nociceptive flexion reflex, during and after the
application of heterotopic stimuli. It was found that the
electromyographic responses of the biceps femoris related
to the activation of unmyelinated afferent fibers in the sural
nerve were strongly inhibited by noxious mechanical or
thermal stimuli when these were applied to the tail, one of
the other paws, or the muzzle. Such inhibitions were not
found in spinal animals and completely disappeared after a
lesion of the dorsolateral funiculus (DLF) ipsilateral to the
recording site.
A preliminary account of this work has appeared in ab-
stract form (Falinower et al. 199 1).
MATERIALS AND METHODS
Experiments were performed on male Sprague-Dawley rats
weighing 350-400 g. During surgery (tracheotomy and cannula-
tion of a vein and an artery) they were deeply anesthetized with
2.5% halothane in a N20-O2 mixture (2/ 3: 1 / 3). The animals
were artificially ventilated through a tracheal cannula; the rate ( 55
strokes/min) and volume of ventilation were adjusted to maintain
a normal acid:base equilibrium as assessed with a capnometer
(Capnomac II; Datex instruments, Helsinki, Finland) that contin-
uously measured the end-tidal COZ, 02, N20, and halothane lev-
els throughout the experiments. The measurements of COZ, 02,
and N20 were performed by infrared absorption and the O2 levels
with a fast paramagnetic analyzer. These parameters were dis-
played digitally, and each was under the control of an alarm. Heart
rate was monitored continuously and core temperature was main-
tained at 37 t 0.5OC by means of a homeothermic blanket system.
During the recording sessions, the animals were retained in a ven-
tral decubitus position with the legs and tail hanging.
Test procedures
The electrophysiological methods for recording reflex activity
elicited by electrical stimulation of the sural nerve and from the
biceps femoris muscle (a knee-flexor) were based on those de-
scribed previously in man ( Willer 1977) and animals (Strimbu-
Gozariu et al. 1993). Briefly, the sural nerve was stimulated at a
rate of 0.16 Hz via a pair of noninsulated platinium/iridium nee-
dle electrodes that had been inserted subcutaneously into the sural
nerve receptive field ( Wiesenfeld-Hallin 1988), namely into the
medial aspect of the 4th and the lateral part of the 5th toe (Fig.
1 A). The electrical stimuli consisted of single square-wave shocks
of 0.5-, l-, 2-, or 4-ms duration depending on needs, and these
were delivered from a constant-current stimulation unit. Electro-
myographic responses (EMG) were recorded from the ipsilateral
biceps femoris muscle via a pair of noninsulated platinium/
iridium needle electrodes inserted 0.5 cm apart into the muscle.
Figure 1 A illustrates the general set-up for the test procedures,
whereas Fig. 1 B shows examples of EMG responses to several
intensities of stimulation. The EMG responses and stimulus in-
tensities were fed to a storage oscilloscope to allow continuous
monitoring of the experiments and to a computerized system (No-
tocord, Igny, France) for on-line digitization. The reflex responses
were full-wave rectified and integrated within an appropriate 150-
to 450-ms time-window
(see RESULTS).
The individual reflex re-
sponses were then plotted either against time, to allow the tem-
poral evolution to be studied, or against stimulus intensity to allow
the study of the recruitment curves. In the latter case, the stimuli
were delivered in sequences of either increasing or decreasing in-
tensities. As illustrated with an individual example in Fig. 1 C, the
reflex responses increased monotonically as a function of stimulus
intensity and reached a maximum plateau at the higher stimulus
intensities. The threshold of the reflex response was taken as the
intercept with the abcissa of the recruitment curve fitted by poly-
modal regression.
Conditioning procedures
The effects of noxious and nonnoxious thermal and mechanical
conditioning stimuli applied to various remote areas of the body
(muzzle, forelimb, tail, and contralateral hindlimb) were studied
196 FALINOWER ET AL.
time window
A
m.aluteus suDerf.
ph semitendiiosusb-
biceDs femoris;
cawt vertebralis
c)
j
Stimulation
C
-I-
pVxms
l
m n
IL
B
mA
30.5
8.1
15000
I
v
#
I
l +
I
-
I . 8
mm
mm m
l l m
--- - ----------- ----- ----------- -- - --*
5omv 1
I
0.7 -- ----- ------ -- - - ---- --- -----
ow , I I 1
0 10
20 30
mA 40 I
0 l&O
2b0 3do 4th
I
ms 500
FIG.
1. A : experimental set up. A pair of nonisolated platinium/iridium needle electrodes was inserted subcutaneously
into the 4th and 5th digits of the hindpaw to stimulate the territory of the sural nerve. Electromyograms (EMGs) were
recorded from the biceps femoris, a knee-flexor muscle, via an identical pair of electrodes. B: recruitment of the reflex
responses. EMG responses were recorded during the 500 ms that followed stimulation. At 1.1 mA a small EMG response was
recorded at a latency of 15 ms. This early component increased with the stimulus intensity. At 5.0 mA, a second component
could be seen at a latency of 180 ms. This component increased as the stimulus increased and was characterized as a response
to C-fiber activation (see text). C: recruitment-curve of the C-fiber response. Abscissa: intensity of stimulation (in mA).
Ordinate: integral of the second component. Integration (pV X ms) was performed after full-wave rectification, within an
appropriate time window ( 150-450 ms, as shown by horizontal bar in
B).
When the current intensity increased, the C-fiber
response increased and reached a plateau at the higher currents (>20 mA). The response threshold was taken to be the
intersection of the polymodal regression curve and the abcissa ( 5.3 mA in this example).
on the
C-fiber reflex
response elicited
by
electrical stimulation
of was chosen to
allow the study
of
poststimulus
effects and to avoid
the sural nerve. any phenomenon of sensitization of the skin receptors (Beitel and
Mechanical conditioning stimuli consisted of applying cali- Dubner 1976; Lamotte et al. 1982) that could introduce bias into
brated forceps perpendicularly to the upper articulations of the the results.
digits of the paws or to the muzzle. The strength of the mechanical
stimuli were as follows: light pressure, 0%
1.2
N/cm2 ; mild pres-
sure, 2.6-3.2 N/cm2; and noxious pinch, 6.0-8.0 N/cm”. When
Sections
of
the spinal cord
applied to the investigator’s skin, the first two were not painful
whereas the last clearly was.
Thermal conditioning stimuli were delivered using a thermoreg-
ulated and agitated waterbath into which the contralateral hind-
paw was immersed to the knee, the forepaws to the elbows, or the
tail to 2/3 of its length. Several temperatures, namely, 46,47,48,
50, and 52OC were tested in random order.
The conditioning procedures were applied for 1 min with an
interval of 2 10 min between successive stimuli. Such an interval
To assess the spinal nature of the reflex responses and to investi-
gate whether supraspinal structures were involved in the modula-
tion of the reflex by heterotopic noxious stimuli, experiments were
also performed in acute and chronic spinal rats.
The surgical preparation of the acute spinal animals was made
under deep gaseous anesthesia as described above. After a laminec-
tomy ( T6-T,), the spinal cord was exposed and cut with ophthal-
mic scissors, and homeostatic gauze was inserted into the split.
Decerebration was achieved by suction of the brain.
NOXIOUS
STIMULI-MODULATED C-FIBER REFLEX IN RAT
197
FIG. 2. Example of a histological control after a surgical lesion of the left dorsolateral funiculus (level C,-C,),
The surgical preparation of the chronic spinal animals was
made under pentobarbital sodium anesthesia (60 mg/kg ip): a
catheter was inserted into the urinary bladder and externalized
through the skin of the back. The laminectomy and spinal section
were similar to those for acute preparations. Animals were housed
and nursed for 24 h. Infection was prevented by prophylactic anti-
biotic therapy.
Lesions of the DLF
To determine the descending path mediating the supraspinal
modulation of the reflex triggered by heterotopic stimuli, experi-
ments were carried out in rats after lesioning of the DLF. The
surgical procedure has been described previously (Villanueva et
al. 1986a).
Briefly, animals were fixed in a stereotaxic frame. After a lami-
nectomy at C,-C,, the dura was slit over the spinal cord and a
DLF
lesion was made under a dissecting microscope by cutting the
left cord with a lancet diamond knife (Anton Mayer) without
producing any bleeding. At the conclusion of the experiments, the
animals were killed, and large segment of the cervical spinal cord
around the lesion was removed and fixed by immersion in a 10%
formaldehyde solution. It was then soaked in a 30% buffered su-
crose solution for 48 h. The specimens were frozen, cut in serial
lOO-pm-thick sections, and Nissl-stained with cresyl violet or car-
mine. The cord lesions were reconstructed from camera lucida
drawings of serial sections. Figure 2 shows an example of such a
lesion.
General experimental protocol
After surgery and before the beginning of each experiment, the
level of halothane was reduced to 0.8% while the mixture N,O-O2
was maintained at 213: l/3. This level of anesthesia was chosen
after we had found in pilot studies, that in addition to meeting
ethical requirements, it provided the best conditions for pure re-
flex responses to be recorded. Indeed, whereas the reflex activity
was greatly depressed at deeper levels of anesthesia, spontaneous
tonic EMG activity was almost constantly present with lower con-
centrations of halothane (e.g., 0.5%).
Usually, 20-30 min after the end of the surgical procedures and
the decrease in the level of anesthesia, 15-mA stimuli applied to
the sural nerve resulted in stable supramaximal reflex responses.
Figure 3 shows a typical example of how, when the depth of anes-
thesia was reduced, the reflex response increased and achieved of a
steady level (with minimal spontaneous fluctuations). This was
the preliminary “sine qua non” before the start of any of the subse-
quent procedures.
EXPERIMENT
I. Experiments to analyze the main features of the
electrophysical reflex activities elicited in the biceps femoris mus-
cle by sural nerve stimulation were designed to provide a careful
and complete neurophysiological analysis. For this purpose, the
effects of stimulus intensity, duration, and frequency were studied
on the threshold, recruitment curve, latency, amplitude, and dura-
tion of the reflex responses. The effects of a selective block (Lido-
Caine) of the sural unmyelinated C-fibers were also studied to
determine accurately in what ways myelinated and unmyelinated
afferents are involved in the reflex activities. Finally, to assess
whether or not the reflex responses recorded in normal animals
are transmitted through an exclusively spinal circuit, similar ex-
periments were performed in chronic spinal rats.
EXPERIMENT 2.
At the beginning of a session, the test stimulus
intensity was adjusted to 1.2 times ( 1.2 T) the threshold for the
C-reflex to be recorded (see
RESULTS),
to elicit a supraliminal
reflex response with minimal fluctuations from one stimulus to
the next. After a 5-min control period, the conditioning procedure
was applied for 1 mitt, while the poststimulus effects were studied
for 5-10 mitt, depending on how long it took for full recovery to
occur.
This protocol was applied in normal, spinalized, and DLF-le-
sioned rats. The last two experimental situations were used to
investigate the possible involvement of supraspinal structures in
198
50 000
p/xms
FALINOWER ET AL.
FIG.
3. Example of the effect of lowering the level
of anesthesia, on the C-fiber response. At time zero,
halothane was decreased from 2.5% to 0.8% and an
electrical stimulus was delivered every 6 s at an inten-
sity of 15 mA. Note that a regular and steady re-
sponse was recorded within 20 min after this change
in the anesthetic regime. Abscissa: time (in min) . Or-
dinate: integral of the C-fiber response ( PV X ms; see
Fig. 1).
min 40
the inhibitory effects elicited by heterotopic noxious conditioning
stimuli in normal rats.
Control experiments
Because it was not feasable to record effects of the heterotopic
stimuli on blood pressure during experiments 1 and 2 (see
DISCUS-
SION),
this was done in six control experiments performed in
strictly identical conditions except that a carotid artery was cannu-
lated during the surgical procedures. The cannula was filled with
heparinized (25,000 IU/ 500 ml) saline and connected to a trans-
ducer. The blood pressure was recorded on a chart recorder. Light
(0.8-1.2 N/cm’) and mild (2.6-3.2 N/cm2) mechanical stimuli
did not affect blood pressure when applied to the tail, a hindpaw, a
forepaw, or the muzzle (variations ~4%). By contrast, noxious
(6-8 N/cm”) pinches or immersion of the tail or a paw in a 52OC
or 50°C waterbath produced significant transient increases in
blood pressure (Table 1). Blood pressure was unchanged at all
lower temperatures tested, including 48OC.
Analysis of results
For each rat, control responses were determined throughout the
2 min preceding the conditioning period. The mean control value
was then calculated, and each individual reflex response was ex-
pressed as a percentage of this mean. The mean of 10 successive
responses was calculated for each minute of the procedure to allow
an overall analysis of pooled data. Statistical analyses were per-
formed with the use of these mean values.
RESULTS
Experiment 1: general characteristics of the reflex responses
Mann-Wittney U test was required as a nonparametric statisti-
cal test. Each serial (each minute) was tested with the control
minute preceding the conditioning stimulus or each minute was
compared with the same minute in the cases of DLF-lesioned or
spinal animals. Significance was expressed as 0.05 ( * ), 0.0 1 ( * * ),
or 0.001 (***).
nent reflex response
in the ipsilateral biceps femoris mus-
cle. In a population of 20 control rats, the first component
was a single shaped di- or triphasic response with a short
latency (
17.5
t 2.3 ms at 1.2 T intensity), short duration
(20.7 t 2.6 ms at 1.2 T intensity) and low threshold ( 1.5 t
0.6 mA). The second component had a longer latency
( 162.4 t 5.1 ms at 1.2 T intensity) and was generally multi-
phasic and of longer duration (202.3 t 6.2 ms at
1.2
T
intensity) with a higher threshold (T = 5.7 t 0.5 mA).
However, this second component was formed by two suc-
cessive EMG discharges that mixed progressively when the
intensity of stimulation increased. These reflex responses
are illustrated in Fig. 1 B with a typical individual example
and in Fig. 4 with the overall pooled data. This latter figure
represents the mean response elicited by a 7-mA stimulus
in a population of 20 rats that were investigated in strictly
similar experimental conditions.
The profile of the mean histogram (Fig. 4A) together
with its low variability (Fig. 4B) revealed a two-component
response in the muscle. These electrophysiological features
clearly suggest that the two components result from activa-
tion of different afferent volleys produced by sural nerve
stimulation. To estimate with more accuracy what type of
afferent fibers were involved in these reflex responses, the
following measures were undertaken.
myelinated afferent fibers should occur at latencies in the 5-
to 43-ms range, whereas responses elicited by the activation
of unmyelinated fibers should be within 85-425 ms. Be-
cause the mean latencies of the maximum firing of each of
the two components of the reflex response were 27.9 t 2.0
and 268.9 t 6.6 ms, the conduction velocities of the affer-
If one considers the velocities of A6 (4-36 m/s) and C-
fibers (0.4-2 m/s) (Gasser and Erlanger 1927) and the
length of the afferent pathway from the stimulating needles
in the territory of the sural nerve to its projection in the
lumbar spinal cord ( - 170 mm), the following can be
stated: neglecting synaptic delays occurring within the spi-
nal cord, then responses elicited by the activation of fine
NORMAL ANIMALS.
Electrical stimulation (2-ms duration,
0.16 Hz) of the sural nerve territory elicited a two-compo-
TABLE 1.
Effects of noxious pinches, 50°C heating and 52OC heating on blood pressure
Tail Hindpaw Forepaw Muzzle
Control, Variation, Control, Variation, Control, Variation, Control, Variation,
mm Hg % mm Hg % mm Hg % mm Hg %
Pinch 132k 8 +21 Z!Z 9* 135t 5 +I1 t8 130 t 12 +37 I!I 10** 128 IL 11 +24 + 3*
50°C Heating 127 k 20 +lO+ 6 123 k 12 +19 * 2 125 k 14 +31* 4
52°C Heating 125 Z!I 11 +22 zk 14 124+ 9 +29 AI 3** 138+ 4 +37 Ik 4***
Values are mean + SD for blood pressure during the 2 min preceding the stimulus; variations in blood pressure during noxious conditioning are
expressed as percentages of the control values. * P -c 0.05; ** P < 0.0 1; *** P -c 0.00 1.
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 199
2hO 3do 460 ms 5ho
O- 0
FIG.
4. Averaged full-wave rectified EMG response elicited by a 7-mA
stimulus. In each individual rat, the recordings of 10 successive individual
responses were accumulated and considered as the representative single
EMG response for that animal. These responses, obtained under strictly
similar experimental conditions were averaged for a population of 20 rats.
Abscissa: poststimulus time ( in ms). Ordinate: EMG signal ( PV; binwidth,
0.5 ms). A : mean response ( n = 20) : The profile of the histogram suggests
the involvement of different afferent volleys in producing the EMG re-
sponse. According to the conduction velocities of the peripheral fibers and
the length of the afferent pathways, the first, early component ( 15-45 ms)
can be attributed to A6 fibers, whereas the second, late component ( HO-
350 ms) can be attributed to C-fibers (see text). B: identical mean re-
sponse ? SE, i.e., for each binwidth, vertical black bars represent 2 SEs
centered on the mean. Note the relatively low interindividual variability
for each bin.
ent volleys responsible for these can be estimated to be 6.1
and 0.6 m/s, respectively. Thus, it seems likely that the first
low-threshold, short-latency reflex response can be elicited
by the activation of A&fibers whereas the second higher
threshold, longer latency component may result from the
activation of the unmyelinated C-fiber afferents.
To test this hypothesis further, a selective block of the
C-fibers was performed on the sural nerve using lidocaine
(0.02-o. 1%; 0.1 ml) injected subcutaneously over the prox-
imal part of the sural nerve ( Fig. 54 ). As controls, the ef-
fects of saline (0.1 ml) injections were also investigated. It
was observed that lidocaine but not saline produced a selec-
tive depression of the late component of the reflex response
as early as 4-5 min after injection, whereas the first compo-
nent of the reflex remained unchanged (Fig. 5, B and C) .
Thus, it appears clear that the second, late component of
the reflex was due to afferent signals transmitted via unmy-
elinated C-fibers whereas the first one was related to activa-
tion of thin (Aa) myelinated fibers.
SPINAL ANIMALS.
To assess whether or not the EMG reflex
responses were transmitted through an exclusively spinal
reflex arc, similar experimental procedures were performed
in acute and chronic spinal animals. In acute preparations,
steady reflex responses were obtained before cutting the spi-
nal cord. Immediately after, only one or two responses were
recorded before the reflex vanished. In the example shown
in Fig. 6, a reflex response appeared 2 or 3 min later, which
had the same electrophysiological characteristics. This re-
sponse then disappeared again for a long period of time,
and lowering the level of anesthesia was necessary to re-
cover such a response.
To avoid such effects of spinal shock, which obviously
could have introduced bias into our data, chronic spinal
animals were prepared. In a total of seven spinal animals,
the mean thresholds of the first and second components of
the EMG response were 1.7 t 0.4 and 5.1 $- 0.8 mA, respec-
tively. When the stimulus was applied at 1.2 times the
threshold for the second component, the first component
exhibited a mean latency of 15.8 t 3.3 ms and a mean
duration of 28.3 $- 6.8 ms, whereas the second component
had a mean latency of 176.7 t 17.4 ms and a mean dura-
tion of 196.7 t 17.4 ms. Note that these values were close to
their counterpart in normal animals.
FURTHER ANALYSIS OF THE C-FIBER REFLEX.
Because our
general study dealt mainly with the nociceptive processes
due to C-fiber activation, some additional experiments
were performed to characterize further the electrophysiolog-
ical features of the second component of the reflex re-
sponse, which we termed the C-fiber reflex (see
DISCUS-
SION) .
For this purpose, we studied the behavior of the C-fiber
reflex when either the duration or the frequency of the stim-
uli applied to the sural nerve were varied within the 0.5- to
4-ms and 0.02- to l-Hz ranges, respectively.
As shown in Fig. 7A, the mean recruitment curves dif-
fered depending on the duration of the electrical pulses. As
expected, the lowest threshold was observed with a 4-ms
duration shock and the highest with a 0.5-ms duration
shock. Note that the plateau was reached very early with the
longest shocks (4 ms: - 10 mA). With shorter shocks, the
threshold for reaching the plateau increased in an inverse
relationship with the duration of the shocks. Not surpris-
ingly, when the abscissa was normalized by expressing the
current intensities in terms of the threshold, the recruit-
ment curves exhibited almost identical profiles and clearly
overlapped (Fig. 7 B). In all cases, the application of a
shock at 1.2 times the threshold resulted in a C-fiber re-
sponse with a magnitude of 30-40% of the maximum re-
sponse ( see below).
Varying the stimulation. rate revealed that at low fre-
quencies (0.02-0.25 Hz), the response remained stable
with minimal spontaneous fluctuations. When the rate was
increased (>0.25 Hz), a “wind-up” (Mendell 1966) phe-
nomenon occurred, i.e., the responses increased progres-
sively with the frequency of stimulation up to 1 Hz. At such
200 FALINOWER ET AL.
control
4001 I
2 min after lidocaine 0.02%)
11
10 min
400
.r l
0 100 200 300 400 ms 500
2nd comDonent
1, t 4
min lh
lidocaine (0.02%)
FIG.
5. Characterization of the types of fiber involved in the triggering of the EMG responses by injection of lidocaine. A :
experimental set-up showing the site over the trajectory of the sural nerve, proximal to the site of electrical stimulation, at
which 0.1 ml SC lidocaine or saline was injected. B: example of the evolution of a full-wave rectified response after injection of
lidocaine. Each histogram is the average of 10 individual responses elicited by a 7-mA, 2-ms single square-wave pulse applied
to the sural nerve over a period of 1 min (presentation as in Fig. 4A). The earlier component was not affected by lidocaine,
whereas the second was depressed. C: corresponding temporal evolution of the early (top histogram) and the late (bottom
histogram) responses (presentation as in Fig. 3). The latter was inhibited by -50% for 6 min and full recovery was seen 10
min after the injection.
stimulation rates, a tonic and constant EMG discharge was
observed in the biceps femoris muscle, even during the in-
tervals between successive stimuli.
From these experiments, it appeared that a single 2-ms
duration shock at an intensity of 1.2 times the reflex thresh-
old, delivered to the sural nerve every 6 s (0.16 Hz) consti-
tuted an acceptable and reliable compromise for the study
of the C-fiber reflex over a period of time. These parameters
were used throughout all the subsequent experiments con-
cerning modulatory processes.
the reflex threshold. The effects of nonnoxious and noxious
mechanical and thermal heterotopic conditioning stimuli
were studied on the C-fiber reflex responses produced
under these conditions.
In normal animals, nonnoxious stimuli were ineffective
but all the noxious conditioning procedures produced a
clear-cut depression of the C-fiber reflex. The earlier A&
fiber component of the EMG response was similarly de-
pressed but this effect was not analyzed at a quantitative
level.
Experiment 2: modulation of the C-jber reflex by
heterotopic stimuli
EFFECTS OF MECHANICAL STIMULI.
As illustrated in Fig. 8
with an individual example and in Fig. 9 with pooled data,
no matter what the site of application, (muzzle, paw, or
tail), noxious pinches ( 6-8 N / cm2) resulted in strong inhi-
As described above, experiments were performed that in- bitions (range 80-90%) of the C-fiber reflex during the
l-
volved recordings the C-fiber reflex elicited by stimulation min period of conditioning; this was followed by poststimu-
at a rate of 0.16 Hz and an intensity adjusted to 1.2 times lus effects lasting 2-4 min. By contrast, light (0.8-l .2 N/
A
30000-
pVxms
15 ooo-
B
FIG.
6. Typical individual example showing
-1 min
the effects of acute spinalization on the reflex re-
200
pv
d+
L- sponse (presentation as in Fig. 3 ) . Stimuli of 1.2
200 ms
times the threshold for obtaining a C-fiber re-
1. +l min
sponse were applied and at time zero, and the spi-
nal cord was cut at the T,-T, level through a lami-
+- +2.5 min
nectomy previously petiormed during the surgi-
cal procedure. As expected, the reflex responses
disappeared rapidly (spinal shock) and, in spite of
returning for a short period (2-3.5 min), re-
O-
section of the
cord
withdrawal
of N20
+5 min
+18 min
mained suppressed for a long time. The removal
of nitrous oxide from the anesthetic regime was
necessary to regenerate a response similar to that
obtained originally. Individual EMG recordings
are shown in B, with the time after section of the
cord indicated to their right.
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 201
400 1
cm2) or mild (2.6-3.2 N/cm2) pressures were totally inef-
fective in this respect.
EFFECTSOFTHERMALSTIMULI.
Aswiththemechanicalstim-
uli, no matter what the site of application, (a paw or tail),
noxious thermal conditioning stimuli produced strong de-
pressive effects both during the 1-min period of heterotopic
stimulation and in the following minutes. This is illustrated
with an individual example in Fig. 10, and pooled data in
Fig. 11, which show the potent depressive effect on the C-
fiber reflex produced by immersion of a paw or the tail in a
52OC water bath.
Figure 12 shows the effects of increasing intensities of
thermal conditioning stimuli (46-52°C) applied to the con-
tralateral hindpaw or the tail. Note the lack of effects after
1001
809
60-
40-
20-
0.5 ms
1 ms
2ms
4ms I
- I . .
0 lb 2; ’ 3b - 4b
mA
B
120
OO
/r
100 1
80 1
,T
60 1 !
44
1
i
1
I- --
- 0.5 ms
- lms
I
0 , I,,
__c__ 2ms
- 4ms
I I I I
0 1 2
times &e ttwdliold
5
FIG.
7. Effects of variations in stimulus duration on the C-fiber recruit-
ment curves. A single square-wave shock was delivered every 6 s. Four
widths of stimuli were tested: 0.5, 1,2, and 4 ms. Ordinate in A : integral of
the C-fiber reflex expressed as percentages of the maximal response
reached at any intensity. Abscissa in A : intensity of stimulation (in mA).
The reflex threshold was inversely related to the pulse duration with the
plateau being reached at lower intensities with the longer shocks. B: ordi-
nate as in A but with abscissa as the intensity of stimulation expressed as
multiples of the threshold. Note the overlap of the curves. Dotted lines
indicate the estimated level of response (38%) recorded at 1.2 times the
threshold when a 2-ms pulse was applied.
PV
200
control o /
pinch of
the tail
post-stimulus , 1 _ . __ A _ L. _
effects (O-30 s) I
post-stimulus
effects (120-150 s) d
effects (240-270 s) r I I I
I 1
0 100 200 300 400 ms 500
FIG.
8. Individual example of the effects of a pinch applied to the tail
on the EMG responses elicited by sural nerve stimulation. Each histogram
represents the accumulation of 5 successive full-wave rectified reflex re-
sponses recorded over a period of 30 s. The after successive responses are
presented from top to bottom: control, the first and second 30-s period
during which a noxious pinch (6-8 N/cm’) was applied to the middle part
of the tail and the poststimulus effects recorded over a 6.5-min period.
Note that the I-min heterotopic noxious stimulus strongly inhibited both
the early and the late reflex responses and that this effect lasted several
minutes before there was a full recovery of the basal levels.
the application of 46°C and that the C-fiber reflex was inhib-
ited in a temperature-dependent fashion in the 47-52°C
range, the most powerful effects and aftereffects being ob-
served with the highest temperatures. All these tempera-
tures, were more potent at producing the inhibitory effects
from the contralateral hindpaw than from the tail.
INVOLVEMENT OF SUPRASPINAL STRUCTURES.
Because the
general behavior of the C-fiber reflex described above had
striking similarities with that of dorsal horn convergent neu-
rons, notably in its ability to be depressed by noxious stim-
uli applied to remote areas of the body (see
INTRODUC-
202
FALINOWER ET AL.
100
50
0
Cl c2 M PSI PS2 PS3 PS4 PS5
paw ipsilateral to the recording site
Cl
C2
IF PSI PS2 PS3 PS4 PS5
150
ail
%
lr
1.50 Forepaw contralateral to the recording site
% lr
Cl C2 CF PSl PS2 PS3 PS4 PS5
lsOmndpaw contralateral to the recording site
%
100
50
0
Cl C2
CH PSl PS2 PS3 PS4 PS5
0
Cl c2 T PSI PS2 PS3 PS4 PS5
FIG. 9. Modulation of the C-fiber reflex by heterotopic mechanical stimuli. Each histogram shows the pooled data (n =
6) corresponding to the effects of noxious pinches applied to the muzzle, a paw, or the tail. Abscissa: each column represents
I-min period of time corresponding to 10 stimuli. From left to right are the controls (shaded columns, Cl and C2), the
conditioning period (black columns: M, muzzle; IF, ipsilateral forepaw; CF, contralateral forepaw; CH, contralateral hind-
paw; and T, tail), and the postconditioning period (shaded columns: PS 1, PS2, PS3, PS4, and PS5, corresponding to the 5
successive I-min periods of poststimulus effects). Ordinate: mean C-fiber reflex responses expressed as percentages of the
controls. No matter to which part of the body they were applied, the mechanical noxious stimuli depressed the C-fiber reflex
response strongly for several minutes. *P < 0.05; **P
< 0.0
I ; * *
*P
< 0.00 1, compared with the control periods.
TION),
we hypothetized that common mechanism(s) were disappear in spinal animals. We therefore studied the ef-
involved. The first characteristic of the inhibitory effects fects of noxious conditioning procedures on the C-fiber re-
observed on dorsal horn convergent neurons is that they flex recorded in chronic spinal (T,-T,) animals. As shown
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 203
control
immersion of the
contralateral
hindpaw in a
52°C waterbath
post-stimulus
effects (O-30 s)
post-stimulus
effects (30-60 s)
post-stimulus
effects (60-90 s)
post-stimulus
effects (90-l 20 s)
post-stimulus
effects (120-l 50 s)
in Fig. 13 (rear histograms), neither noxious pinch of the
muzzle nor noxious (52°C) heating of the paws or tail pro-
duced a significant modification of the reflex in these ani-
mals. A small, but significant decrease in the C-fiber reflex
was seen only during heating of the contralateral hindpaw.
In all cases, the differences with data recorded in nontrans-
sected animals ( Fig. 13, front histograms) were highly signif-
icant.
INVOLVEMENT OF THE DLF.
It has been demonstrated that
the integrity of the DLF ipsilateral to the recording site is an
absolute requirement for the inhibitory processes triggered
bv noxious heterotopic stimuli that affect dorsal horn con-
vergent neurons (Villanueva et al. 1986a). We therefore
studied the effects of noxious-conditioning procedures on
the C-fiber reflex in animals with a cervical ( C4-C5) unilat-
eral (left) DLF lesion that had been performed during the
surgical preparation. The extents of the lesions are shown in
the lower left hand corner of Fig. 14. In these animals, re-
cordings were made from the left and right biceps femoris
alternately.
During recordings of the C-fiber reflex from the limb con-
tralateral to the lesion (Fig. 14, front histograms), noxious
conditioning stimuli applied to the muzzle (pinch), the
paws or the tail (immersion in a 52OC waterbath), pro-
duced inhibitory effects very similar to those seen in non-
transected animals (see Figs. 9 and 10). By contrast, during
recordings of the C-fiber reflex from the limb ipsilateral to
the lesion (Fig. 14, rear histograms), only one of the
noxious heterotopic conditioning stimuli elicited inhibitory
effects. A small, but significant decrease in the C-fiber reflex
was seen during and after heating of the contralateral hind-
paw. The C-fiber reflex was facilitated during noxious
pinch of the muzzle or immersion of the forepaws in a 52OC
waterbath.
DISCUSSION
We have described the electromyographic recording
from the biceps femoris muscle of the intact anesthetized
rat, of a reflex, elicited by electrical stimulation of the sural
nerve receptive field ( Wiesenfeld-Hallin 1988 ), which
seems to be related to the activation of peripheral C-fiber
afferents and was thus termed a C-fiber reflex. This re-
sponse was found to be strongly inhibited by both mechani-
cal and thermal noxious heterotopic stimuli no matter
whether these were applied to the muzzle, a paw, or the tail.
These inhibitory effects disappeared almost completely
when the C-fiber reflex was recorded in spinal animals or
after disruption of the ipsilateral DLF.
The flexion reflex is a polysynaptic defence response to.
noxious stimulation of the skin or of cutaneous nerves. Oc-
curring in flexor muscles, it is usually described as an EMG
discharge that consists of several components separated by
periods of more or less pronounced EMG silence (Dimitri-
jevic and Nathan 1970; Hagbarth 1960; Kugelberg et al.
1960; Pedersen 1954). These components are due to the
activation of groups of afferent fibers with different conduc-
tion velocities (Kugelberg 1948; Lloyd 1943). In the spinal
cat, Lloyd ( 1943 ) demonstrated that it was possible to elicit
a two-component reflex in hindlimb flexor muscles: the
first exhibited a short latency and low threshold; the second,
with a longer latency and higher threshold, was evoked only
by strong stimuli and was presumably due to the activation
of high-threshold, slowly conducting fibers. Similarly, hav-
ing studied a group of spastic patients, Kugelberg ( 1948)
concluded that the C-fiber afferents were able to mediate
the late components of the fleyion reflex elicited by intense
stimulations of the plantar or sural nerves. Although reflex
activity in flexor muscles have been observed previously in
the rat, their basic electrophysiological properties have
hardly been scarcely studied. We have carefully analyzed
the main characteristics of such a flexion reflex: our results
are verv similar to those described bv Llovd ( 1943) in that
204 FALINOWER ET AL.
150
Forepaw ipsilateral to the recording site
%
loo -----
50
Cl C2 IF PSl PS2 PS3 PS4 PS5
150
II-
Forepaw contralateral to the recording site
%
Cl C2 CF PSl PS2 PS3 PS4 PS5
‘W”(fkdpaw contralateral to the recording site
Cl C2 CH PSl PS2 PS3 PS4 PS5
Cl C2 T PSI PS2 PS3 PS4 PS5
FIG. II. Modulation of the C-fiber reflex by heterotopic thermal stimuli. Each histogram shows the pooled data (n = 6)
corresponding to the effects of immersion of different parts of the body in a 52°C waterbath. Abscissa: each column
represents a I-min period of time corresponding to IO stimuli. From left to rig/a are the controls (shaded columns, Cl and
C2), the conditioning period (black columns: IF, ipsilateral forepaw; CF, contralateral forepaw; CH, contralateral hindpaw;
T, tail), and the postconditioning periods (shaded columns: PS 1, PS2, PS3, PS4, and PSS, corresponding to the 5 successive
I-min periods of poststimulus effects). Ordinate: mean C-fiber reflex response expressed as percentages of the controls. No
matter to which part of the body they were applied, the heterotopic thermal noxious stimuli depressed strongly the C-fiber
reflex response for several minutes. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the control periods.
sural nerve stimulation evoked a two-component reflex in
the flexor muscles of the ipsilateral hindlimb in both nor-
mal and spinal rats. We have accumulated electrophysiolog-
ical and pharmacological evidence suggesting that the first
component is triggered by activity in small-diameter my-
elinated afferents of the A6 group, whereas the second com-
ponent is mediated through unmyelinated C-fiber affer-
ents. This assertion is based first on conduction velocity
measurements: the maximal firing of the first and second
components were triggered by peripheral fibers with con-
duction velocities of 6 and 0.6 m/s, respectively. According
to Gasser and Erlanger ( 1927) (see also Burgess and Per1
1973), such values are consistent with A& and C-fibers,
respectively. In addition to the longer latency (-9 times)
and higher threshold ( -4 times) of the second component,
we showed that it was blocked selectively by the local appli-
cation of low doses of lidocain, a local anesthetic known to
block preferentially the unmyelinated C-fibers (Gasser and
Erlanger 1929). We recently observed (unpublished data)
that this late component of the EMG response was virtually
absent in rats pretreated neonatally by capsaicin, a drug
that is known also to affect unmyelinated fibers selectively
( Holzer 199 1) . Furthermore, in common with the C-fiber
responses recorded from neurons in the dorsal horn (Dick-
enson and Sullivan 1986), the late component of the flex-
ion reflex was powerfully depressed, in a naloxone-revers-
ible fashion, by low doses of intrathecal morphine
( Strimbu-Gozariu et al. 1993). Thus it appears very likely
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 205
contralateral hindpaw
150
Cl C2 CH PSl PS2 PS3 PS4 PS5
tail
0
Cl c2 T PSl PS2 PS3 PS4 PS5
Cl C2 CH PSl PS2 PS3 PS4 PSS
100 ___---
48°C
so
Cl C2 CH PSl PS2 PS3 PS4 PS5
Cl c2 T PSl PS2 PS3 PS4 PS5
Cl c2 T PSl PS2 PS3 PS4 Ps5
Cl C2 CH PSl PS2 PS3 PS4 PS5 -- Cl c2 T PSl PS2 PS3 PS4 PSS
Cl C2 CH PSI PS2 PS3 PS4 PSS Cl c2 T PSl PS2 PS3 PS4 PSS
FIG.
12. Effects of various temperatures applied to the contralateral hindpaw (left histograms) or the tail (right histo-
grams). The contralateral hindpaw or the tail was immersed in a water bath at different temperatures (range 46-52°C) in
random order (n = 6). Abscissa: each column represents a I-min period corresponding to 10 stimuli. From leff to right are
the controls (shaded columns, C I and CZ), the conditioning period (black columns: CH, contralateral hindpaw; T, tail) and
the postconditioning periods (shaded columns: PSI, PS2, PS3, PS4, and PS5, corresponding to the 5 successive 1-min
periods of poststimulus effects). Ordinate: mean C-fiber reflex response expressed as percentages of the controls. Although
the responses were not modified at 46’C, the reflex decreased in a direct relationship to the higher temperatures both during
and after the conditioning periods. *P i 0.05; **P < 0.01; * * *P < 0.00 1, compared with the control periods.
206 FALINOWER ET AL.
150 fll-
Muzzle
Cl c2 M PSl PS2 PS3 PS4
150
repaw ipsilateral to the recording site
%
100
0
Cl C2 IF PSl PS2 PS3 PS4
vd--
Forepaw contralateral to the recording site
Cl c2 CF PSl PS2 PS3 PS4
1504--
Hindpaw contralateral to the recording site
_
Cl
C2
CH PSl PS2 PS3 PS4
Fail
Cl C2 T PSl PS2 PS3 PS4
FtG.
13. Comparison of the effects of heterotopic conditioning stimuli on the C-fiber reflex in intact and spinal rats.
Spinal section was performed at the T,-T, level, I day before recording. The animals were tested with mechanical pinch of
the muzzle and with thermal (52°C waterbath) conditioning stimuli delivered to the paws and tail. Abscissa: each column
represents a 1-min period corresponding to 10 stimuli; from left to right are the controls (shaded columns, Cl and C2), the
conditioning period (black columns: IF, ipsilateral forepaw; CF, contralateral forepaw; CH, contralateral hindpaw; T, tail)
and the postconditioning periods (shaded columns: PSI, PS2, PS3, PS4, and PS5, corresponding to the 5 successive’ 1 -min
periods of poststimulus effects). Ordinate: mean C-fiber reflex responses expressed as percentages of the controls. Front
histograms: data obtained in intact animals (n = 6). Rear histograms: data obtained in spinal animals (n = 6). Statistical
significances are shown as follows: asterisks in parentheses indicate the significance ofthe effects of the conditioning stimulus
compared with the control period in spinal animals, whereas asterisks without parentheses (above the front histograms)
indicate the significance of the comparison between the effects obtained in spinal and intact rats: *P < 0.05; **P < 0.01;
***P < 0.00 1. In contrast with the data obtained in the intact animals, there was little evidence of inhibitory processes in the
spinal animals. With immersion of the contralateral hindpaw in a 52°C waterbath, inhibitory effects were observed in the
spinal animals, and these were significant during the conditioning period and the following minute. In every cases the
comparison between the intact and the spinal animals revealed a highly significant difference.
Recording 1
Stimulation
Recording
Stimulation
Cl c2 M PSl PS2 PS3 PS4
mfiorepaw ipsiiaterai to tha recording site
0
Cl C2 IF PSl PS2 PS3 PE4
m
Ill-
Forepaw contralateral to the recording site
0.L -l-l**)
Cl c2 CF PSl PS2 PS3 PS4
tWfindpaw contralateral to the recording site
Cl c2 CH PSl PS2 PS3 PS4
0
Cl C2 T PSl PS2 PS3 PS4
FKX 14. Effects of lesions of the dorsolateral funiculus (DLF) on the modulation of the C-fiber reflex by heterotopic
thermal or mechanical noxious stimuli. Reconstructions of the DLF lesions (C& level) are shown in the bottom lefthand
corner (see text). Each animal was tested with the different recording sites and conditioning stimuli in random order. The
experimental situations are summarized in the top lefthand comer, with the drawings of animals showing that recordings
were made from the hindpaws ipsilateral (left) and contralateral (right) to the lesion. The results are summarized on the right
hand histograms with abscissae and ordinates as follows. Abscissa: each column represents a I-min period corresponding to
IO stimuli. From left to right are the controls (shaded columns, Cl and C2), the conditioning period (black columns: M,
pinch of the muzzle; IF, immersion in a 52°C waterbath of the forepaw ipsilateral to the recording site; CF, immersion in a
52°C waterbath of the forepaw contralateral to the recording site; CH, immersion in a 52°C waterbath of the hindpaw
contralateral to the recording site; T, immersion in a 52°C waterbath of the tail), and the postconditioning periods (shaded
columns: PSI, PS2, PS3, PS4, and PS5, corresponding to the 5 successive I-min periods of poststimulus effects). Ordinate:
mean C-fiber reflex responses expressed as percentages of the controls. In contrast with the data obtained during recordings
contralateral to the lesion (front columns) that were, essentially similar to those from nontransected animals, there was little
evidence of inhibitory processes being elicited during “ipsilateral” recordings (rear columns). The weak inhibitory effects
observed during and after the immersion ofthe contralateral hindpaw in 52°C water were reminiscent ofthose seen in spinal
animals. Noxious conditioning elicited facilitations of the responses when parts of the body rostra1 to the lesion were
stimulated; however these effects were significant only during the first 3 min after pinch of the muzzle.
201
208 FALINOWER ET AL.
that this late component is triggered by activity in unmyelin-
ated fibers and can be termed a “C-fiber reflex” without
excessive speculation. Interestingly, the stimulus-response
recruitment curves were steep with maximal firing being
achieved at two to three times threshold. We have noted
that the C-fiber response of dorsal horn convergent neurons
exhibits identical properties ( unpublished observations).
The general characteristics of the C-fiber reflex recorded
here were very much in keeping with the corresponding
activities recorded by Schouenborg and Sjolund ( 1983)
from the common femoral nerve or motoneurons after
electrical stimulation of the sural nerve. The similarities
include latencies, durations and frequency potentiations
when the interstimulus interval is ~3-4 s. Interestingly,
these authors provided convincing evidence that conver-
gent but not nociceptive-specific dorsal horn neurons were
incorporated in the neuronal circuitry responsible for this
reflex activity. In the present study, the threshold of the
C-fiber reflex was found to be -6 mA, which is slightly
higher than the 3- to 4-mA range for obtaining C-fiber re-
sponses during recordings of convergent neurons in several
experimental studies in our laboratory (e.g., Bouhassira et
al. 1992b; Le Bars et al. 1979a; Villanueva et al. 1986a).
This difference could be due to the anesthetic regime, be-
cause 0.8% instead of 0.5% halothane was used in this study
to avoid spontaneous electromyographic activity. A second
difference lies in the fact that electrical stimuli were applied
over the receptive fields of the neurons under study, i.e., on
the most sensitive area, whereas in the present study, the
stimulus was always applied between the 4th and 5th toes.
Note however that the receptive field for reflex activity in
the biceps femoris is large (Schouenborg and Kalliomaki
1990; Woolf and Swett 1984), including most, if not all, the
receptive fields of the neurons recorded in the cited studies.
It is also conceivable that a certain degree of convergence
was necessary to trigger the reflex responses, thus increasing
the threshold. Concommittant recordings of both dorsal
horn neurons and reflex activities will be necessary before a
definite conclusion can be drawn concerning these matters.
Note in this respect that in this study, we have dealt with a
lightly interfered-with preparation that appears to be more
physiological for the animals, whereas recording from dor-
sal horn neurons requires a heavy surgical procedure in-
cluding a laminectomy. The wounds caused by surgery
could well be the source of inhibitory processes (Cadden
1985; Clarke and Matthews 1985), as discussed below.
Such factors could possibly increase the threshold for a dor-
sal horn C-fiber response that highlights the difficulties of
such comparisons.
Although we did not investigate in detail the C-fiber re-
flex in chronic spinal animals, the general characteristics,
and notably the threshold, were similar to those recorded in
intact animals. Interestingly Schouenborg et al. ( 1992)
showed changes in excitability, namely an early blockade
(“spinal shock”) followed by a long-lasting facilitation,
during the 8 h after section of the thoracic spinal cord. Note
that our recordings were made 24 h after an identical sec-
tion.
The C-fiber reflex was strongly inhibited by heterotopic
noxious stimuli applied to the tail, a paw, or the muzzle.
These inhibitions had long durations lasting far beyond the
conditioning periods. In addition, they were temperature
dependent when the hindpaw or tail was immersed in a
thermoregulated waterbath and pressure dependent when
mechanical conditioning stimuli were employed. In both
cases, nonnoxious stimuli were ineffective at triggering any
inhibitory effects on the C-fiber reflex. Interestingly similar
observations were made at a qualitative level regarding the
A&fiber reflex. The quantitative analysis of these observa-
tions was not made because we stimulated the sural nerve
receptive field at 1.2 times the threshold for the C-fiber re-
flex. We recently observed similar inhibitory effects during
and after colorectal distension ( Falinower et al. 1993),
which suggests that any area of the body, including the vis-
cera, can be the source of heterotopic inhibitions. Such an
interpretation is in keeping with several earlier reports: the
reflex discharge in the common peroneal nerve after electri-
cal stimulation of the sural nerve in the rat was found to be
inhibited by pinching the muzzle or tail (Schouenborg and
Dickenson 1985 ) ; the gastrocnemious medialis reflex
evoked by sural nerve stimulation in the decerebrate rabbit
was found to be inhibited by electrical stimulation of the
contralateral common peroneal or either ipsi- or contralat-
era1 median nerves (Taylor et al. 199 1); the digastric reflex
evoked by tooth-pulp stimulation in the cat was found to be
inhibited by toe pinch, percutaneous electrical stimulation
of a limb, or electrical stimulation of the saphenous nerve
(Cadden 1985; Clarke and Matthews 1985).
One must consider the possibility that there is a relation-
ship between the inhibitory effects described herein and
neurovegetative reactions. Indeed, with high intensity het-
erotopic stimuli, namely strong pinches and 50-52OC heat-
ing, increases in blood pressure were seen in control experi-
ments (see Table 1). These are classical responses to
noxious stimuli that could have triggered the inhibitory
processes indirectly. This is probably not the case for the
following reasons:
1)
strong inhibitions were seen in the
absence of changes in blood pressure, for instance during
48OC conditioning, 2) in DLF-lesioned animals, increases -
in blood pressure triggered by frankly noxious stimuli oc-
cured both with and without the inhibitory processes de-
pending only on the side from which recordings were being
made; 3) we recently observed similar inhibitory processes
triggered by colorectal distensions but associated with fall in
blood pressure ( unpublished data). It therefore appears
that we are dealing here not with a phenomenon generated
by a global neurovegetative reaction to a noxious stimulus
but rather with a phenomenon triggered by specific control
mechanisms. Such an interpretation is detailed below, on
the basis of studies in both animals and man.
Indeed such inhibitory processes are very reminiscent of
DNIC, which modulate dorsal horn convergent neurons. In
the rat, the cat (Morton et al. 1987), and probably the mon-
key (Brennan et al. 1989; Gerhart et al. 198 1 ), the activity
of convergent neurons can be strongly inhibited by hetero-
topic noxious inputs. Such effects do not appear to be so-
matotopically organized but apply to the whole body and
affect all convergent neurons, whether in the dorsal horn of
various segments of the spinal cord (Cadden and Morrison
199 1; Cadden et al. 1983; Calvin0 et al. 1984; Le Bars et al.
1979a; Ness and Gebhart 199 1 a,b; Schouenborg and Dick-
enson 1985; Tomlinson et al. 1983) or in trigeminal nuclei
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 209
caudalis and oralis (Dallel et al. 1990; Dickenson et al.
1980; Hu 1990). The principal feature of DNIC is that they
can be triggered by conditioning stimuli applied to any part
of the body distant from the excitatory receptive field of the
neuron under study, provided that the stimuli are clearly
noxious. With strong stimuli, the inhibitory effects are pow-
erful and are followed by long-lasting aftereffects that can
persist for several minutes.
In man, exactly analogous results have been obtained in
studies that have combined psychophysical measurements
with the recording of nociceptive reflexes (Willer et al.
1984, 1987, 1989). Electrical stimulation of the sural nerve
at the ankle simultaneously induces a nociceptive reflex in a
flexor muscle of the thigh (the R,,, reflex) and a painful
sensation in the distribution of the nerve ( Willer 1977).
Painful heterotopic conditioning stimuli, no matter
whether they were thermal, mechanical, or chemical in na-
ture, depressed both the reflex and the associated painful
sensation, with stronger effects being observed with more
intense conditioning stimuli. The depressions were seen
both during and after the application of the heterotopic
nociceptive stimuli and were not associated with changes in
autonomic functions, as assessed by recordings of respira-
tory and heart rates. Because nonnoxious conditioning stim-
uli were ineffective whereas noxious thermal conditioning
stimuli produced inhibitory effects in a direct relationship
with the temperatures, it was concluded that activations of
A& and C-nociceptive afferent fibers were probably in-
volved in producing these effects.
In the present study, the C-fiber reflex was inhibited by
heterotopic noxious thermal stimuli in the 46-52OC range.
However there were some slight discrepancies concerning
the thresholds for producing such effects: it was found to be
in the 46-47 and 47-48°C ranges for inhibitions triggered
from the contralateral hindpaw and the tail, respectively.
Several hypotheses can be advanced to explain this:
1)
it is
possible that the synergy of two different mechanisms of
inhibition (see below) resulted in a lowering of the thresh-
old when the conditioned and conditioning stimuli were
applied at the same segmental level; 2) the skin of the paw,
which is glabrous and thin, is different from the skin of the
tail, which is hairy, thick, and heavily keratinized; this
could easily explain a 1 O difference between thresholds. The
thresholds for triggering inhibitions of the R,,, reflex and the
related pain sensation in man, and the activities of conver-
gent neurons in the rat were found to be lower, in the 44-
45 OC range. The most rational explanation for such a 2”
difference probably lies in the experimental conditions, no-
tably in the anesthetic regime: the level of halothane was
0.8% in the present study, whereas it was 0.5% during re-
cordings of convergent neurons in the rat; the present level
of anesthesia was chosen because spontaneous tonic electro-
myographic discharges were observed to be almost continu-
ous at lower concentrations of halothane (e.g., 0.5%). By
contrast, the experiments on the R,,, reflex and related pain
sensations were all performed in awake nonanesthetized,
conscious volunteers. The observation that inhibitions on
dorsal horn convergent neurons triggered by heterotopic
noxious stimuli are highly sensitive to the type and dose of
anesthetic (Alarcon and Cervero 1989; Tomlinson et al.
1983 ) strongly supports this interpretation of the data.
In summary, there are clear relationships between condi-
tioning stimulus intensities in the noxious range, A& and
C-nociceptor discharges and inhibitory effects observed on
1)
the RIII reflex and the related pain sensation in man, 2)
the C-reflex described herein in the rat, and 3) the activities
of convergent neurons, again in the rat. However, it is
worth pointing out that there was a marked contrast in our
experimental protocols in both man and animals, between
the phasic nature of the conditioned stimulus measured in
milliseconds and the long duration of the nociceptive con-
ditioning stimuli measured in seconds or minutes. There
was therefore a clear imbalance between the spatiotemporal
features of the afferent volleys triggered by the conditioned
and the conditioning stimuli.
On the basis of the close relationships observed in man
between the inhibitory effects of the noxious conditioning
stimuli on the RIII reflex and on the related sensations, it
was suggested that the inhibitory mechanisms modulate a
single pool of spinal neurons that are responsible for trans-
mitting signals in both nociceptive reflex and ascending
pain pathways (Willer et al. 1984). In addition, we demon-
strated that the monosynaptic H reflex evoked from the
soleus muscle was not inhibited by heterotopic nociceptive
conditioning stimuli, which clearly excludes the possibility
that such stimuli trigger any inhibitory process that acts
directly on the membranes of motoneurons (Willer et al.
1989).
It is very likely that the inhibitory processes that we ob-
served acting on the nociceptive C-fiber reflex result pri-
marily from a depression of activity in a pool of spinal inter-
neurons shared by the polysynaptic reflex arc and the as-
cending pain pathways. This assertion is supported by the
observation that reflex discharges in the common peroneal
nerve and simultaneously recorded responses of convergent
neurons were both depressed by heterotopic noxious stim-
uli (Schouenborg and Dickenson 1985). Our present re-
sults are very much akin to those of Schouenborg and Dick-
enson ( 1985 ), except that during application of heterotopic
noxious stimuli we never observed the “released reflex
nerve discharge,” which is presumably triggered by activity
in lamina I nociceptive-specific neurons. One possible ex-
planation for such a discrepancy lies in the differences in
stimulus intensity used in the two studies: Schouenborg and
Dickenson ( 1985 ) stimulated at 100 times the threshold for
any discharge to be recorded, whereas we stimulated at 1.2
times the threshold for the C-fiber reflex, which was roughly
5 times the threshold of the earlier component. Thus, their
stimulus intensities were presumably much higher than
ours, which leaves open the possibility that they would have
activated lamina I nociceptive-specific neurons more easily
than we did. Because these neurons are not under the influ-
ence of DNIC (Le Bars 1979b), they presumably could
trigger the “released reflex nerve discharge” (Schouenborg
and Dickenson 1985 ), which is unmasked during hetero-
topic noxious stimulation. The stimulus intensity used in
the present study was likely to have activated convergent
neurons more than lamina I nociceptive-specific units,
which would explain why we did not observe the “released
reflex nerve discharge.”
The question arises of whether it is possible to identify
the pathway(s) and mechanism(s) mediating the inhibi-
210 FALINOWER ET AL.
tions of the reflexes or the convergent neurons. Intraspinal
pathways were certainly not involved because the inhibi-
tions were not observed in spinal rats. Interestingly, the in-
hibitory effects of noxious conditioning stimuli observed
on the RIII reflex in normal subjects disappeared in quadri-
plegic patients suffering from traumatic clinically complete
spinal cord transections ( Roby-Brami et al. 1987 ). Further-
more, the inhibitory effects of noxious conditioning stimuli
cannot be elicited from the analgesic side of patients suffer-
ing from Wallenberg’s syndrome, i.e., patients with unilat-
eral lesions of the retro-olivary portion of the medulla; how-
ever, in these patients, inhibitions akin to those in normal
subjects were found when the nonaffected side was stimu-
lated (De Broucker et al. 1990). Reticular structures con-
tain key neuronal links for the production of these effects.
The central mechanisms involved in the triggering of
DNIC acting on dorsal horn convergent neurons have been
investigated in some details: DNIC are not observed in anes-
thetized or decerebrate animals in which the spinal cord has
been sectioned (Cadden et al. 1983; Le Bars et al. 1979b;
Morton et al. 1987 ) . It is therefore obvious that the mecha-
nisms underlying DNIC are not confined to the spinal cord
and that supraspinal structures must be involved.
However, there is evidence, that, at the level of conver-
gent neurons in the spinal rat, some inhibitory processes
can be triggered by heterotopic noxious stimuli. These pro-
priospinal processes appear to be different from these me-
diating DNIC (Cadden et al. 1983; Fitzgerald 1982; Ger-
hart et al. 198 1). It is even possible that these mechanisms
are more powerful in the case of homotopic conditioning
stimuli (Chung et al. 1983, 1984a,b; Shin et al. 1986; Tay-
lor et al. 199 1). The findings regarding heterotopic noxious
stimuli were confirmed in the present study in that the in-
hibitory processes observed during recordings of the C-fiber
reflex disappeared in spinal animals except when the condi-
tioning stimuli were applied to the contralateral hindpaw;
such an effect, albeit weak, was highly significant and con-
sistent with the initial observation of Sherrington and Sow-
town ( 19 I5 ). We never observed any signs of facilitation by
contralateral stimulation as described in unanesthetized
rats studied within a few hours of spinalization (Woolf and
Swett 1984). A predominance of supraspinally mediated
over propriospinal mechanisms in inhibitions triggered by
heterotopic noxious inputs appears very likely. This idea is
further supported by experiments with DLF-lesioned ani-
mals. Indeed, when the C-fiber reflex was recorded ipsilat-
erally to the DLF section, the inhibitions disappeared ex-
cept when the conditioning stimulus was applied to the con-
tralateral hindpaw; in this case the remaining inhibitory
effects were very similar to those observed in spinal ani-
mals. By contrast, when the C-fiber reflex was recorded con-
tralaterally to the DLF-section, the inhibitions were akin to
those observed in untransected rats. Surprisingly, when the
C-fiber reflex was recorded ipsilaterally to the DLF-lesion,
facilitations were seen when the muzzle was pinched or a
forepaw was immersed in a 52OC water bath.
Such effects were never observed during recordings of
dorsal horn convergent neurons (Chitour et al. 1986; Vil-
lanueva et al. 1986a), which suggests that we are dealing
here with facilitatory mechanisms related to the motor part
of the reflex arc, mediated through the ventrolateral quad-
rant (see Holmqvist and Lundberg 196 1; Taylor et al.
1991).
The ascending and descending limbs of the loop-sustain-
ing DNIC travel through the ventrolateral and dorsolateral
funiculi, respectively ( Villanueva et al. 1986a,b). Surpris-
ingly, DNIC were not modified by lesions of the following
structures: the periaqueductal grey (PAG), cuneiform nu-
cleus, parabrachial area, locus coeruleus/ subcoeruleus, ros-
tral ventromedial medulla ( RVM) , including nucleus
raph& magnus and the gigantocellular and paragigantocel-
lular nuclei (Bouhassira et al. 1990, 1992b, 1993). These
negative findings suggest that DNIC are likely to constitute
a system that modulates the spinal transmission of nocicep-
tive signals independently of the descending inhibitory con-
trols originating from those midbrain and medullary struc-
tures that have been implicated in the postulated “endoge-
nous pain inhibitory system( s )” ( see references in
Basbaum and Fields 1984; Fields and Basbaum 1989;
Fields and Besson 1988; Willis 1984; Willis and Coggeshall
1991).
By contrast, lesions of subnucleus reticularis dorsalis
(SRD) in the caudal medulla strongly reduced DNIC (Bou-
hassira et al. 1992a). SRD is an area in the more caudal part
of the brainstem, located ventral to the cuneate nucleus,
between trigeminal nucleus caudalis and the nucleus of the
solitary tract, which contains neurons that have a key role
in processing specifically nociceptive information (Bing et
al. 1990; Roy et al. 1992; Villanueva et al. 1988-l 990).
Both electrophysiological and anatomic (Bernard et al.
1990; Villanueva et al. 199 1) data support the involvement
of SRD neurons in spino-bulbo-spinal loop( s) related to
nociception.
Interestingly, the inhibitions of the C-fiber reflex in the
rat described herein disappeared or remained after trans-
verse sections that were, respectively, caudal or rostra1 to a
region delimited by the area postrema and the nucleus
raph& magnus (Villanueva et al. 1993).
We found in this study, that the descending limb of the
loop was confined to the DLF ipsilateral to the recording
site: after a unilateral lesion of the DLF, the inhibitory ef-
fects triggered by heterotopic noxious inputs disappeared
when the C-fiber reflex was recorded from the limb ipsilat-
era1 to the lesion, but remained, apparently unchanged,
when the C-fiber reflex was recorded from the other side.
This result is in keeping with the proposal that the effects
were mediated by DNIC acting on dorsal horn convergent
neurons because identical unilateral lesions of the DLF
blocked these inhibitory processes when convergent neu-
rons were being recorded in the dorsal horn ipsilateral to the
lesion, but not when they were being recorded from the
other side (Villanueva et al. 1986a).
The following points appear to be common to the RIII
reflex recorded in humans and both the C-fiber reflex and
the responses of convergent neurons recorded in the rat: 1)
All can be inhibited by a variety of heterotopic noxious
stimuli; 2) The distance between the sites of the condi-
tioned and conditioning stimuli is not a critical factor in
determining the strength of the inhibitions; 3) All types of
nociceptive conditioning stimuli, whether electrical, me-
chanical, thermal, or chemical are effective; 4) The condi-
tioning stimuli require a large amount of spatial and tem-
NOXIOUS STIMULI-MODULATED C-FIBER REFLEX IN RAT 211
poral
summation to be fully effective; 5) The magnitude of
the inhibition is directlv related to the intensitv of the con-
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10)
The descending limb of the loop runs
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Y., ENDO,
K., AND WILLIS, W. D. Fac-
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