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The Journal of Neurophysiology Vol. 78 No. 6 December 1997, pp. 3165-3179
Copyright ©1997 by the American Physiological Society
1 Laboratoire de Neurophysiologie, Hôpital Pitié-Salpétriêre, 75013 Paris; and 2 Institut National de la Santé et de la Recherche Médicale U-161, 75014 Paris, France
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Gozariu, Manuela, Dominique Bragard, Jean-Claude Willer, and Daniel Le Bars. Temporal summation of C-fiber afferent inputs: competition between facilitatory and inhibitory effects on C-fiber reflex in the rat. J. Neurophysiol. 78: 3165-3179, 1997. Long-lasting facilitations of spinal nociceptive reflexes resulting from temporal summation of nociceptive inputs have been described on many occasions in spinal, nonanesthetized rats. Because noxious inputs also trigger powerful descending inhibitory controls, we investigated this phenomenon in intact, halothane-anesthetized rats and compared our results with those obtained in other preparations. The effects of temporal summation of nociceptive inputs were found to be very much dependent on the type of preparation. Electromyographic responses elicited by single square-wave electrical shocks (2 ms, 0.16 Hz) applied within the territory of the sural nerve were recorded in the rat from the ipsilateral biceps femoris. The excitability of the C-fiber reflex recorded at 1.5 times the threshold (T) was tested after 20 s of electrical conditioning stimuli (2 ms, 1 Hz) within the sural nerve territory. During the conditioning procedure, the C-fiber reflex was facilitated (wind-up) in a stimulus-dependent fashion in intact, anesthetized animals during the application of the first seven conditioning stimuli; thereafter, the magnitude of the responses reached a plateau and then decreased. Such a wind-up phenomenon was seen only when the frequency of stimulation was 0.5 Hz or higher. In spinal, unanesthetized rats, the wind-up phenomenon occurred as a monotonic accelerating function that was obvious during the whole conditioning period. An intermediate picture was observed in the nonanesthetized rat whose brain was transected at the level of the obex, but the effects of conditioning were profoundly attenuated when such a preparation was anesthetized. In intact, anesthetized animals the reflex was inhibited in a stimulus-dependent manner during the postconditioning period. These effects were not dependent on the frequency of the conditioning stimulus. Such inhibitions were blocked completely by transection at the level of the obex, and in nonanesthetized rats were then replaced by a facilitation. A similar long-lasting facilitation was seen in nonanesthetized, spinal rats. It is concluded that, in intact rats, an inhibitory mechanism counteracts the long-lasting increase of excitability of the flexor reflex seen in spinal animals after high-intensity, repetitive stimulation of C-fibers. It is suggested that supraspinally mediated inhibitions also participate in long term changes in spinal cord excitability after noxious stimulation.
The sensitization of neurons in the spinal cord after repetitive or sustained nociceptive inputs was studied extensively in recent years (see Dougherty et al. 1993 General procedure
Experiments were performed on male Sprague-Dawley rats weighing 300-400 g. During the surgical procedures the rats were deeply anesthetized with 2% halothane in a nitrous oxide-oxygen mixture (2-3:1-3). The animals were artificially ventilated through a tracheal cannula after tracheotomy. Some rats were decerebrated at the midcollicular level by suction of the brain contents rostral to the midcollicular region; they were either transected at the level of the obex or spinalized.
Electrophysiological recordings
This method was described previously (Falinower et al. 1994 Experimental procedure
Usually 20-30 min after the end of the surgical preparation(1 h after spinalization) and decrease in the level of anesthesia, the application of 15-mA stimuli to the sural nerve resulted in stable supramaximal reflex responses with minimal spontaneous fluctuations. This was the preliminary finding for starting the subsequent procedures. The reflex responses increased monotonically with stimulus intensity and reached a plateau at high intensities. The threshold (T) of the C-fiber-evoked response was determined as the intersection of the polymodal regression curve and the abscissa. A constant level of stimulation (1.5T) was then employed. Four series of experiments were performed in the following categories of rats: 1) intact and anesthetized, 2) obex-transected and anesthetized, 3) obex-transected and nonanesthetized, and 4) spinal and nonanesthetized.
Analysis of conditioning stimulation paradigm
EMG responses were expressed as percentages of the mean control value, which was derived from the 20 successive C-fiber reflex responses in the 2-min period preceding the conditioning procedure. During the conditioning period the wind-up phenomenon was analyzed with each individual response being expressed as a percentage of that during the 2-min control period. During the postconditioning period results were finally expressed as means of 10 successive individual responses obtained over a 1-min period. One-way analysis of variance (ANOVA) followed by Fisher posteriori least-significant difference (PLSD) tests were used for analyzing the conditioning and postconditioning periods. During the conditioning procedure each response was compared with both the last test response and the first response of the conditioning period. Data were expressed as means ± SE. Results were considered significant when P < 0.05.
Control experiments: effects of conditioning stimulation on blood pressure
Blood pressure was recorded in five control experiments performed in intact, anesthetized animals in strictly identical conditions except that a carotid artery was cannulated during the surgical procedures. The cannula was filled with heparinized (25,000IU/500 ml) saline and connected to a pressure transducer (Barovar). The blood pressure and the electrocardiogram were digitized and analyzed by means of a personal computer. The mean control blood pressure and heart rate were 93.0 ± 3.7 mmHg and 315 ± 20 beats/min, respectively. A significant increase was only seen for blood pressure during the last 10 s of conditioning at 5 and 10T (15.8 ± 9.9% and 31.3 ± 9.5%, respectively). Blood pressure returned immediately to basal control level after conditioning at 5T. After conditioning at 10T, blood pressure returned progressively to basal control level within 10 min. Heart rate was unchanged by the conditioning procedure.
In intact, halothane-anesthetized (0.9%) rats, electrical stimulation (2 ms, 0.17 Hz) within the sural nerve territory elicited a two-component reflex response in the ipsilateral biceps femoris muscle. The first component had a short latency (10-20 ms), a short-duration (<50 ms) and a low threshold (0.5- to 2-mA range) and is known to be triggered by activity in myelinated fibers (Falinower et al. 1994 Conditioning at 1 Hz and various intensities in intact, anesthetized animals
The results from an experiment on a single animal are illustrated in Fig. 1 where the insert shows a control individual EMG recording. The temporal evolution of the C-fiber reflex recorded during this individual experiment was analyzed separately during and after the period of conditioning stimulation. Histograms to the left show the pre- and postconditioning periods; histograms to the right show the conditioning periods. As shown on the right, the C-fiber reflex was facilitated during the conditioning period in a stimulus-dependent fashion. The amplitude of the reflex increased over the first few stimuli to a maximum of 299, 568, and 592% of control values for conditioning intensities of 1.5, 5, and 10T, respectively. It then decreased during the second part of the conditioning period to 194, 310, and 259% respectively, at the end of conditioning (20th test stimulus). The left of Fig. 1 also shows that the C-fiber reflex was inhibited after the conditioning period, again in a stimulus-dependent fashion. The maximum postconditioning inhibitory effect occurred during the second minute (40, 15, and 11% of control values for conditioning intensities of 1.5, 5, and 10T, respectively). Note the different time courses of the postconditioning periods; thus after conditioning at 1.5T a facilitation was seen for the first three postconditioning responses and this was followed by inhibition lasting 5 min and a slight facilitation in the 5- to 10-min postconditioning period. After conditioning at 5T, a facilitation was seen during the first postconditioning response followed by inhibition lasting 15 min with a slight recovery of activity during the 5- to 10-min postconditioning period. After conditioning at 10T, the inhibition was long lasting and had still not recovered completely 20 min after the end of conditioning.
Conditioning at 10T at various frequencies in intact, anesthetized animals
Analysis of the mean curves for the conditioning period in Fig. 3A, shows that the C-fiber reflex was facilitated significantly at all frequencies (1, 0.5, 0.25, and 0.17 Hz) of stimulation, but the wind-up phenomenon was seen only at the higher frequencies, namely at 1 and 0.5 Hz. During conditioning at 0.25 or 0.17 Hz, the increase of stimulus intensity from 1.5T before to 10T during conditioning was followed by an increase of the responses to roughly 250% of control levels with no further significant modifications. By contrast, during conditioning at 1 or 0.5 Hz three different periods could be defined within the temporal evolution of the responses: 1) wind-up during the application of the first 10 conditioning stimuli, with the C-fiber reflex increasing progressively from one stimulus to the next; 2) a plateau reached by the magnitude of the C-fiber reflex during the application of the 10th to the 16th stimulus; and 3) a slight decrease in the magnitude of the reflex occurred at the end of the conditioning period. At 1 Hz the reflex reached a maximum of 554.4 ± 55.5% of the control level after the 10th stimulus but was only 392.8 ± 59.1% after the 20th stimulus. The corresponding figures for 0.5 Hz conditioning were 524.0 ± 45.6% and 386.1 ± 52.3%.
Conditioning at 1 Hz and 10T in obex-transected and spinal animals
One hour after transection at the level of the obex and lowering of anesthesia from 2 to 0.9 or 0% halothane, there were no obvious signs of rigidity and a C-fiber reflex could be recorded from the biceps femoris. The EMG signal was generally weaker than in the intact animals and the reflex not so clearly time locked (see individual control examples as inserts in Fig. 4, A and B). In the 0.9% halothane-anesthetized rats the C-fiber reflex had a mean threshold of 5.3 ± 0.7 mA that was not significantly different from that in the intact, anesthetized animals. In these animals the reflex evoked by 1.5T stimuli exhibited a mean latency and duration of 121.7 ± 6.5 and 203.3 ± 13.8 ms, respectively, the former being significantly lower than that recorded in intact, anesthetized rats. A significantly lower threshold (1.8 ± 0.5 mA) with a comparable latency (120.0 ± 6.8 ms) and longer (not significant) duration (248.3 ± 13.0 ms) were found in the obex-transected, nonanesthetized rats.
We have studied the effects of temporal summation of C-fiber inputs on a C-fiber reflex in the rat and obtained results that are very dependent on the type of preparation. In intact, anesthetized rats, after a facilitation during the conditioning period, the reflex was inhibited in a stimulus-dependent manner for approximately one-quarter of an hour. In spinal, unanesthetized rats this inhibition was replaced by a facilitation. In obex-transected animals inhibitions were never observed, but postconditioning facilitations were seen only in the absence of anesthesia.
EMG signals and preparations
In 0.9% halothane-anesthetized, intact rats electrical stimulation within the sural nerve territory elicited a two-component reflex response in the ipsilateral biceps femoris muscle. We carefully considered at a quantitative level the second component of the reflex, which results from activation of unmyelinated cutaneous afferent C-fibers (Falinower et al. 1994 Wind-up phenomenon observed during conditioning
During the conditioning procedure the C-fiber reflex was facilitated in a stimulus-dependent fashion in the intact animals. This was particularly obvious during the application of the first seven conditioning stimuli, when the C-fiber reflex progressively increased from one stimulus to the next. This is very reminiscent of the wind-up phenomenon initially described by Mendell (1966) Postconditioning effects
We now consider the effects observed after the conditioning period. In intact animals the reflex was inhibited in a stimulus-dependent manner during the postconditioning period. In contrast with the wind-up phenomenon observed during the conditioning period, the subsequent postconditioning inhibitory effects did not appear to be dependent on the frequency of the conditioning stimulus, at least not within the 0.17- to 1-Hz range. Indeed, there were circumstances (when the conditioning stimuli were applied every 4 or 6 s) when there were no wind-up effects at all, but there were strong postconditioning inhibitions. This observation excludes any clear causal relationship between the excitatory wind-up phenomena and the subsequent inhibitory postconditioning effects.
MECHANISMS INVOLVING SPINAL CORD.
In spinal rats not only were the inhibitions observed during the postconditioning period in intact animals completely blocked, but long-lasting facilitatory effects occurred. This observation is very much in keeping with several other studies. Wall and Woolf (1984) MECHANISMS INVOLVING THE BRAIN.
In any case it is reasonable to propose that facilitatory mechanisms (whether related to LTP or not), present but presumably ineffective in intact animals, were unmasked in the spinal preparation. However, in the intact animal not only were the facilitations completely masked but they were overridden by the inhibitory processes, at least after high-intensity stimulation. The complementary action of opposing effects is suggested by the finding that during the conditioning period in intact animals, wind-up was obvious after the first stimuli but then declined, whereas an increasing response was always seen in spinal preparations over the whole period of conditioning. Thus descending inhibitions from brain origins were triggered with a strong enough efficacy to block the wind-up phenomena as early as after a few stimuli. Thus we are dealing with both facilitatory and inhibitory mechanisms, the former being organized at the spinal level and the latter involving supraspinal mechanisms.
Conclusions and functional implications
It is concluded that in intact, anesthetized rats an inhibitory mechanism counteracted the long-lasting increase in excitability of the flexor reflex seen in spinal, nonanesthetized animals after high-intensity, repetitive stimulation of C-fibers. Supraspinal and spinal origins are suggested for the former and the latter mechanisms, respectively. Our results are in keeping with the enhancement of descending inhibitions during the development of inflammation (Cervero et al. 1991
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
; Dubner and Basbaum 1994). The phenomenon was observed in both acute and chronic animal models of pain and is therefore of potentially great interest for clinicians. For convenience, electrical stimuli have often been used to generate such facilitations and the neuronal or reflex responses have been studied during or after conditioning peripheral stimuli.
observed that when recording from dorsal horn convergent neurons, low-frequency (0.5-2 Hz) stimulation of peripheral nerves elicited a progressive increase in the number of action potentials from one stimulus to the next when the stimulus was strong enough to recruit unmyelinated C-fibers. This observation was extended by intracellular recordings from dorsal horn neurons (Jeftinija and Urban 1994; Price et al. 1971; Sivilotti et al. 1993; Wagman and Price 1969; Woolf and King 1987; Yoshimura and Jessel 1989). During repeated stimulation of unmyelinated afferent fibers, most of these cells exhibited slow excitatory postsynaptic potentials (EPSPs), the durations of which were long enough to allow temporal summation to occur, thus eliciting an increase in their excitability. N-methyl-D-aspartate (NMDA) receptors and l-type Ca2+ channels are critical for this summating depolarization (Davies and Lodge 1987; Dickenson and Sullivan 1987, 1990; Russo and Hounsgaard 1994; Thompson et al. 1990). Wind-up can also be observed during recordings of flexion reflexes (Price 1972; Schouenborg and Sjölund 1983).
reported a marked facilitation of a flexion reflex after 20 s of 1 Hz homotopic conditioning stimulation of C-fibers. This result suggested that temporal summation of brief C-fiber afferent inputs within the spinal cord elicits a prolonged hyperexcitability of neurons involved in the transmission of nociceptive signals (see Wall and Woolf 1984; Wiesenfeld-Hallin 1985; Wiesenfeld-Hallin et al. 1990, 1991; Woolf and Wiesenfeld-Hallin 1986). Cook et al. (1986) suggested such a C-fiber mediated facilitation of a flexion reflex in rats was not due to changes in afferent terminal or motoneuron excitability.
; Zieglgänsberger 1986). In particular, many studies have shown that descending inhibitory controls can be activated by the noxious inputs themselves (Basbaum and Fields 1984; Bouhassira et al. 1995b; Fields and Basbaum 1978, 1989, 1994; Le Bars and Villanueva 1988).
; Dubner and Ren 1995; Ren and Dubner 1995; Schaible and Grubb 1993; Schaible et al. 1991). The aim of our study was to gauge the relative contributions of these two phenomena in the final response in the spinal cord. We have studied the effects of temporal summation ofC-fiber inputs on a C-fiber reflex in intact, halothane-anesthetized rats. To compare our results with previous reports, we also analyzed the effects of a similar conditioning procedure on the C-fiber reflex in spinal, nonanesthetized rats. To avoid both spinal shock and the rigidity of decerebration, we completed our study in rats whose brains were transected very caudally, at the level of the obex.
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
; Strimbu-Gozariu et al. 1993). In brief, electrophysiological recordings were made from the ipsilateral biceps femoris muscle of C-fiber-evoked reflex activity, elicited by electrical stimulation within the sural nerve receptive field. The stimuli were applied via a pair of noninsulated platinum-iridium (Pt-Ir) needle electrodes inserted subcutaneously in the medial part of the fourth and lateral part of the fifth toe. The electromyographic (EMG) responses were recorded via another pair of noninsulated Pt-Ir needles, inserted 0.5 cm apart through the skin into the biceps femoris muscle. The test stimuli were single square-wave electrical shocks of 2-ms duration delivered every 6 s (0.17 Hz) from a constant-current stimulator. Such stimulation elicited a two-component reflex response in the ipsilateral biceps femoris muscle. The first, a short-latency (10- to 20-ms range), short-duration (<50 ms), and low-threshold (0.5- to 2-mA range) component, is due to activation of myelinated fibers. As already described and discussed, the second component, which has a longer latency, a longer duration, and a higher threshold (see RESULTS), is mediated through unmyelinated C-fiber afferents. This assertion is first based on conduction velocity measurements; the maximal firing of the second component is triggered by peripheral fibers with conduction velocities of 0.6 m/s. In addition, this response is selectively blocked by a subcutaneous injection of lidocaine around the proximal part of the sural nerve, abolished in capsaicin-pretreated animals, and powerfully depressed in a naloxone-reversible fashion by low doses of intrathecal morphine.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). This component was not analyzed in the present study. By contrast, we carefully considered in a quantitative fashion the second component that exhibited a higher threshold(5.8 ± 0.7 mA), a longer latency (166.7 ± 9.9 ms at1.5T), and a longer duration (232.5 ± 15.9 ms at 1.5T). Such a response is known to result from activation of unmyelinated cutaneous afferent C-fibers (Falinower et al. 1994; Strimbu-Gozariu et al. 1993) and we refer to it as the C-fiber reflex. Each individual C-fiber reflex response was analyzed within a 100- to 450-ms time window after the stimulus onset.
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FIG. 1.
Examples of temporal evolutions of C-fiber reflex recorded from biceps femoris in intact, anesthetized animal after electrical stimulation within the sural nerve territory. Individual example of electromyographic (EMG) recordings from the biceps femoris for 500 ms after the stimulus (top) with amplification and time scales in the lower left-hand corner. Digitized EMGs were full-wave rectified and the C-fiber-evoked responses integrated within 100- to 450-ms poststimulus time window. Individual reflex responses were plotted against time (abscissa) and as % of mean control value (ordinate) calculated during the 2-min period preceding conditioning procedure. Before and after conditioning procedure the 2-ms stimulus was applied every 6 s (0.17 Hz) at an intensity of 1.5T. During conditioning procedure 20 stimuli were applied to the sural nerve territory at a frequency of 1 Hz with different intensities: 1.5 (A), 5 (B), and 10T (C). Right histograms: conditioning periods on expanded scales [abscissa, rank of order number (nb) of conditioning stimulus]. During 1st part of conditioning period the C-fiber reflex was facilitated in a stimulus-dependent fashion from 1 stimulus to the next, a phenomenon called wind-up by Mendell (1966) . During 2nd part of conditioning it decreased gradually from 1 stimulus to the next to reach intermediate value after 20th pulse at the end of conditioning. Left histograms: pre- and postconditioning periods (abscissa, minutes after end of conditioning period). Shaded areas correspond to conditioning period. C-fiber reflex was inhibited after conditioning period in a stimulus-dependent fashion. Note the maximum effect seen during the 2nd minute of postconditioning period and the stimulus-dependent duration of inhibition.
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FIG. 2.
Mean curves showing effects of temporal summations of C-fiber inputs on the C-fiber reflex during (A) and after (B) conditioning procedure; effects of stimulus intensity (see symbols). Conditioning stimuli applied to the sural nerve territory were 20 2-ms duration pulses delivered at 1 Hz and different intensities (1.5-10T). A: during conditioning procedure. Each individual C-fiber response (ordinate) was expressed as % of control responses recorded during 2-min period preceding conditioning procedure. Abscissa, rank order (nb) of 20 conditioning stimuli. Three different periods can be described over 20 stimuli: during 1st 7 conditioning stimuli, theC-fiber reflex progressively increased; it reached a plateau during the8th-13th stimuli and then slightly decreased. This effect was more pronounced at 10T. B: after conditioning procedure. In each individual case the C-fiber reflex was calculated as % of mean control value recorded during 2-min period preceding conditioning procedure and then results were expressed as means of 10 successive individual responses that corresponded to 1-min period. Abscissa, time (min) after conditioning procedure. Stippled bar, conditioning period. Postconditioning effects were dependent on the intensity of conditioning stimuli. In all cases, a maximal inhibition was observed at 2 min followed by a slight recovery after 20 min. Note that at 1.5T, the inhibitory period (during the 2nd-4th min) was followed by a facilitatory period (during the 6th-11th min that was significant at 9 min).
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FIG. 3.
Mean curves showing effects of temporal summation of C-fiber inputs on the C-fiber reflex during (A) and after (B) conditioning procedure; effects of frequency of stimulation (see symbols). Conditioning stimuli applied to sural nerve territory were 20 2-ms duration pulses delivered at 10T and different frequencies (1-0.17 Hz). A: during conditioning procedure. Each individual EMG response (ordinate) is expressed as % of the control responses recorded during 2-min period preceding conditioning procedure. Abscissa, rank order (nb) of 20 conditioning stimuli. C-fiber reflex was facilitated significantly at all frequencies, but the wind-up phenomenon was seen only at the higher frequencies of stimulation, namely 1 and 0.5 Hz. In this latter case, 3 different periods could be described on basis of their temporal evolution; during 1st 10 conditioning stimuli the C-fiber reflex increased progressively (wind-up phenomenon), reached a plateau during the 10th-16th stimuli, and decreased slightly at the end of conditioning period. B: after conditioning procedure. In each individual case theC-fiber reflex was calculated as % of mean control value recorded during 2-min period preceding conditioning procedure and results are expressed as mean of 10 successive individual responses, which corresponded to a1-min period. Abscissa, time (min) after conditioning procedure. Stippled bar, conditioning period. Postconditioning effects were similar for all frequencies of stimulation applied during conditioning period. In all cases a maximal inhibition was observed at 2 min followed by a slight recovery after 20 min.
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FIG. 4.
Example of temporal evolutions of C-fiber reflex recorded from biceps femoris after electrical stimulation of sural nerve territory in 3 different preparations, namely obex-transected, anesthetized (A), obex-transected, nonanesthetized (B), and spinal, nonanesthetized (C) animals. Presentation as in Fig. 1. Inserts: individual examples of EMG recordings from biceps femoris with amplification and time scales in lower left-hand corner. Digitized EMGs were full-wave rectified and the C-fiber-evoked responses integrated within a 100- to 450-ms poststimulus time window. Individual reflex responses were plotted against time (abscissa) and responses were expressed as % of mean control value (ordinate) calculated during the2-min period preceding conditioning procedure. Before and after conditioning procedure the stimulus was applied every 6 s (0.17 Hz). During conditioning procedure 20 stimuli were applied to the sural nerve territory at a frequency of 1 Hz and an intensity of 10 times threshold for the C-fiber reflex. Right histograms: conditioning periods on expanded scales (abscissa, rank of order (nb) of conditioning stimulus). C-fiber reflex increased continuously from 1 stimulus to the next during the whole conditioning period. Left histograms: pre- and postconditioning periods (abscissa, minutes after end of conditioning period). Shaded areas correspond to conditioning period. After conditioning period the C-fiber reflex was facilitated only in nonanesthetized rats. Note different time courses of postconditioning facilitations, depending on the level of anesthesia. After conditioning in obex-transected, anesthetized animals (A) a facilitation was seen only during 1st minute of postconditioning period with complete recovery occurring during the next minute. Conditioning in obex-transected, nonanesthetized rats (B) or in spinal rats (C) was followed by a facilitatory period lasting 15 and >20 min, respectively.
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FIG. 5.
Mean curves showing effects of temporal summation of C-fiber inputs on C-fiber reflex during (A) and after (B) conditioning procedure in 3 different preparations, namely obex-transected and anesthetized, obex-transected and nonanesthetized, and spinal and nonanesthetized animals (see symbols). Conditioning stimuli applied to sural nerve territory were 20 2-ms duration pulses delivered at 1 Hz and an intensity of 10 times threshold of C-fiber reflex. A: during conditioning procedure. Each individual C-fiber response (ordinate) was expressed as % of control responses recorded during 2-min period preceding the conditioning procedure. Abscissa, rank order (nb) of conditioning stimulus. Note that progressive increase in reflex from 1 stimulus to the next was greater in spinal rats than in anesthetized or nonanesthetized, obex-transected rats. B: after conditioning procedure. In each individual case the C-fiber reflex was calculated as a % of mean control value recorded during 2-min period preceding conditioning procedure and then results were expressed as means of 10 successive individual responses, which corresponded to 1-min period. Abscissa, time (min) after conditioning procedure. Stippled bar, conditioning period. Note that in obex-transected, anesthetized rats a slight facilitatory effect occurred only during the 1st min and that this was followed by a very quick recovery. In obex-transected, nonanesthetized rats, facilitatory postconditioning effect was long-lasting and significant during the 1st 8 min. In spinal, nonanesthetized rats the facilitatory postconditioning effects were clearly greater and lasted a longer period of time (significant for 11 min).
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Schouenborg and Dickenson 1985; Schouenborg and Kalliomäki 1990; Schouenborg and Sjölund 1983; Strimbu-Gozariu et al. 1993).
). One hour after transection and lowering of the anesthetic level to 0.9 or 0% halothane, there were no obvious signs of rigidity and aC-fiber reflex in the biceps femoris could be elicited easily. By comparison with what was seen in the intact animals, the EMG responses were generally weaker and not so clearly time locked to the stimuli. However, the C-fiber reflexes in anesthetized rats exhibited similar thresholds regardless of whether the animals were intact or transected at the level of the obex. As expected, the thresholds for the C-fiber reflexes were lower in nonanesthetized rats, regardless of whether these were transected at the level of the obex or spinal.
; Schouenborg and Kalliomäki 1990; Schouenborg and Sjölund 1983). Several studies have shown that the effects of halothane on the activities of dorsal horn convergent neurons are minimal. Le Bars and Chitour (1983) demonstrated that the responses of these neurons to either radiant heat or repetitive innocuous mechanical stimulation were identical in intact, halothane-anesthetized and spinal, nonanesthetized rats. Several other pieces of evidence suggest that gaseous anesthesia does not act merely on the afferent part of the reflex arc (Go and Yaksh 1987; Puil et al. 1990; Sorkin et al. 1992). Nicoll and Madison (1982) showed that the potency of halothane to hyperpolarize motoneurons was strongly correlated with its anesthetic potency. Recording of the spinal C-fiber reflex was not feasible in our experiments with spinal rats anesthetized with 0.9% halothane, although dorsal horn convergent neurons, which presumably belong to the circuitry of the reflex, can be recorded under the same experimental conditions. It is tempting to speculate that a depressive effect of halothane, acting mainly on the motor part of the C-fiber reflex pathway, was added to the reflex depression due to spinal shock. This assertion is strongly supported by the fact that EMG responses were easily recorded in spinal, nonanesthetized animals.
; Wiesenfeld-Hallin 1985; Wiesenfeld-Hallin et al. 1990, 1991; Woolf and Wall 1986; Woolf and Wiesenfeld-Hallin 1986). However, it is worth pointing out that the excitability of spinal reflexes is not stable after a spinal section. In the rat spinal shock lasts ~10-20 min and is followed by a gradual increase in excitability of withdrawal reflexes during the following 5-8 h (Schouenborg et al. 1992). The EMG responses recorded after spinal section in the present experiments were weaker than those in intact, anesthetized animals and, to some extent, were desynchronized. Interestingly, the thresholds, latencies, and durations of the C-fiber reflex were similar to those recorded in obex-transected, nonanesthetized rats.
during recordings from dorsal horn neurons in spinal cats. However, beyond the 7th to 10th conditioning stimuli the increasing responses reached a plateau and then decreased. A completely different evolution was seen in spinal rats where the wind-up phenomenon occurred as a monotonic accelerating function, which was obvious during the whole conditioning period from the first to the last (20th) stimulus. An intermediate situation was observed in the obex-transected rat, with clear differences in this preparation between anesthetized and nonanesthetized animals.
0.5 Hz. If conditioning was applied every 4 or 6 s (0.25 or 0.17 Hz), the increase in stimulus intensity elicited an increase in the response to the first conditioning stimulus with no further significant modifications of the responses to the following stimuli.
; Price 1972; Schouenborg and Sjölund 1983). With regard to spinal neurons, a progressive increase in their discharges to repeated electrical stimulation at a level sufficient to recruit high-threshold unmyelinated fibers is observed from one stimulus to the next after relatively low (>0.3 Hz) frequencies of stimulation (Mendell 1966; Mendell and Wall 1965; Woolf 1983a; Woolf and Swett 1984). Price and colleagues pointed out that the frequency should be >0.3 Hz for an effective wind-up phenomenon to be seen during recordings from dorsal horn neurons in the cat and the monkey (Price and Wagman 1970; Price et al. 1971; Wagman and Price 1969). Again, wind-up occurred only if the stimulus was intense enough to activate peripheral C-fibers.
; Sivilotti et al. 1993; Woolf and King 1987; Yoshimura and Jessel 1989). A role in wind-up for N-methyl-D-aspartate (NMDA) and neurokinin receptors is well established in mammals (Davies and Lodge 1987; De Koninck and Henry 1991; Dickenson and Sullivan 1987, 1990; Thompson et al. 1990, 1993a; Xu et al. 1992). It has been found that, in turtle dorsal horn neurons, wind-up elicited either by repetitive stimulation of the dorsal roots or by repetitive depolarizing current pulses is mediated by a depolarizing potential produced by increasing activation of postsynaptic l-type Ca2+ channels that can be blocked by nifedipine (Russo and Hounsgaard 1994).
; Price et al. 1977, 1989, 1994). At lower frequencies the sensation of second pain was perceived as constant from one stimulus to the next over a series of 6-8 stimuli. Our observations during the first seven conditioning stimuli in both intact and spinal animals are consistent with these observations. To the best of our knowledge there are no published observations in humans with longer periods of repetitive electrical stimulation.
during recordings of dorsal horn convergent neurons and motoneurons in a preparation very similar to that used in the present study (see also Herrero and Cervero 1996). Wagman and Price (1969) also noted in the intact monkey that an initial wind-up was often followed by a decrease in the output of action potentials by dorsal horn neurons.
; Groves et al. 1969; Mendell 1984; Spencer et al. 1966a-c; Wickelgren 1967). Habituation was observed after trains of high-frequency stimulation of both cutaneous and muscular afferents and varied with the frequency and intensity of stimulation: the faster the stimulation rate, the more rapidly the response declined; the stronger the stimulus, the less habituation there was and the more slowly the response declined. Although lamina V dorsal horn neurons in the spinal cord seem to play a role in habituation (Groves and Thompson 1970), we do not believe that our results are related to such a phenomenon for two reasons. First, we studied the late component of a flexor reflex elicited by stimulation of C-fibers whereas habituating reflexes are triggered by myelinated fibers. Second, the decrease in the magnitude of the reflex during the last part of the conditioning procedure in intact rats disappeared after spinalization, whereas habituation is a purely spinal phenomenon. In this respect Dimitrijevic and Nathan (1970, 1971) showed that habituation also occurred in human spinal cords that were disconnected from supraspinal control. It therefore appears that the decelerating function that followed the wind-up phenomenon in our experiments was the result of an active process that involved supraspinal structures and counteracted the spinal accelerating function. Such an interpretation is further supported by the observations made during the postconditioning period.
also reported minimal effects of halothane on the wind-up phenomena seen during recordings of dorsal horn convergent neurons. As already mentioned, a preferential depressive effect of halothane on the motor part of the C-fiber reflex arc could explain the slight decrease in the wind-up phenomena.
View this table:
TABLE 1.
Control experiment: effects of the experimental protocol on blood pressure and heart rate in the intact anesthetized animal
reported that in the spinal, nonanesthetized rat an identical conditioning procedure resulted in a marked increase in the excitability of biceps femoris-semitendinosus motoneurons for 10 min. Only electrical conditioning stimuli at intensities that recruited afferent C-fibers were effective in producing a prolonged facilitation of the reflex. This observation was confirmed repeatedly in the same preparation with nearly identical experimental protocols (Wiesenfeld-Hallin 1985; Wiesenfeld-Hallin et al. 1990, 1991; Woolf and Wall 1986; Woolf and Wiesenfeld-Hallin 1986). It was also seen in spinal rabbits during recordings of responses elicited by stimulation of the sural nerve in the nerves to the semitendinosus or gastrocnemius muscles; in this case the facilitation was seen after noxious stimulation of the heel or application of a train of electrical stimuli to the sural nerve at an intensity that recruited A
- and C-fibers (Catley et al. 1984; Clarke et al. 1992). Finally, long-lasting postconditioning depolarizations have been observed during recordings from motoneurons in the neonatal rat spinal cord after homosynaptic or heterosynaptic repetitive stimulation of high-threshold afferent fibers (Sivilotti et al. 1993; Thompson et al. 1993b).
). However, a decrease in the tail flick latency can be seen after noxious thermal conditioning of the tail in the intact rats lightly anesthetized with pentobarbital-chloral (Cridland and Henry 1988; Yashpal et al. 1991, 1993). In the present study 0.9% halothane completely blocked the postconditioning facilitations seen in the obex-transected rat, suggesting a susceptibility of segmental effects to volatile anesthetics in our preparation. Interestingly, volatile anesthetics, but not barbiturates, have been reported to attenuate significantly spinal sensitization assessed with the paw formalin test (Abram and Yaksh 1993; O'Connor and Abram 1995). Central sensitization therefore appears to be very dependent on both the type of preparation and the anesthetic regime that is used. In keeping with our results in the intact rat, Fleischman and Urca (1988) described an increase in the tail flick latency after noxious pinching of the tail in intact, but not in spinal, nonanesthetized mice.
; Cavallari and Pettersson 1989; Fitzgerald 1982; Gerhart et al. 1981; Hobbs et al. 1992; Nagasaka et al. 1993; Pitcher et al. 1995; Sandkühler et al. 1993; Schomburg et al. 1986; Sherrington and Sowton 1915; Wall et al. 1993). When applied homotopically these stimuli appear to be either facilitatory, as mentioned previously, or inhibitory. Indeed, strong segmental inhibitory effects have been reported after high-intensity conditioning applied ipsilaterally on or near to the same dermatome (Catley et al. 1983; Chung et al. 1983, 1984a,b; Clarke et al. 1988, 1989; Hentall and Fields 1980; Shin et al. 1986; Taylor et al. 1989-1991; Woolf 1983b, 1984). The complexity of interactions in the dorsal horn was stressed by Hentall and Fields (1980) who recorded from convergent neurons in the unanesthetized, spinal cat and observed that the late response to sural nerve stimulation, presumably elicited by the activation of C-fibers, was either potentiated or depressed after homotopic, high-frequency (20-50 Hz) conditioning; by contrast they were always depressed after heterotopic conditioning of split sections or natural branches of the sural nerve. Analogous observations were made in the unanesthetized, spinal rat by Woolf (1983b, 1984); prolonged homotopic stimulation at low frequencies (0.2-0.5 Hz) produced inconsistent changes in the C-fiber responses of convergent neurons to sural nerve stimulation whereas higher frequencies (1-2 Hz) elicited profound inhibitions of these responses. Heterosynaptic inhibitions elicited from the common peroneal nerve were also reported by the same author.
; Collingridge and Singer 1990; Ito 1989; Linden and Connor 1995; Malenka 1994). Little is known about synaptic plasticity at primary afferent synapses with dorsal horn neurons. Randic et al. (1993) defined LTP as 30% increase in amplitude of the synaptic response that occurred during brief high-frequency stimulation (300 pulses at 100 Hz) and was maintained for 20 min thereafter. They showed in a spinal cord slice preparation that brief, high-frequency stimulation of primary afferent fibers produced LTP of mono- and polysynaptic EPSPs recorded in dorsal horn neurons and that the induction of the potentiation required the activation of NMDA receptor-gated conductances. They also demonstrated that the same tetanic stimulation can induce either LTP or LTD of the synaptic response, depending on the level of membrane potential of the postsynaptic neuron. If the depolarization is strong enough to reach the activation threshold for NMDA receptor-gated channels, the tetanus causes LTP; if the depolarization remains below this level, an LTD 20% is induced. The mechanisms downstream from intracellular Ca2+ increases may be of critical importance in determining whether synaptic potentiation or depression occurs. It is difficult to conclude as to whether LTP or LTD were involved in our experimental paradigm.
). These observations are in keeping with several other reports in intact, anesthetized animals (Banks et al. 1992; Cadden 1985; Calvino et al. 1984; Chapman and Way 1982; Clarke and Matthews 1985; Clarke et al. 1988; 1989; Fleischman and Urca 1988, 1993; Hayes et al. 1978; Kalliomäki et al. 1992; Kawakita and Funakoshi 1982; Morgan and Whitney 1996; Morgan et al. 1994; Pitcher et al. 1995; Schouenborg and Dickenson 1985; Tal et al. 1981; Taylor et al. 1991; Yashpal et al. 1995).
, 1989). Such inhibitions of the RIII reflex were not observed in tetraplegic patients suffering from clinically complete spinal cord transections of traumatic origin (Roby-Brami et al. 1987). Similarly, a jaw reflex evoked by perioral stimuli in humans was reported to be strongly inhibited by heterotopic noxious stimuli (Cadden and Newton 1994).
; Cadden et al. 1983; Cadden and Morrison 1991; Calvino et al. 1984; Dallel et al. 1990; Dickenson and Le Bars 1983; Dickenson et al. 1980; Fleischman and Urca 1989; Gerhart et al. 1981; Hu 1990; Le Bars et al. 1979a; Morgan et al. 1994; Morton et al. 1987; Ness and Gebhart 1991a,b; Schouenborg and Dickenson 1985; Sher and Mitchell 1990; Tomlinson et al. 1983). DNIC are not observed in 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 mechanisms underlying DNIC are not confined to the spinal cord and that supraspinal structures must be involved. The supraspinal loop sustaining DNIC is confined to the most caudal part of the medulla (Bouhassira et al. 1995a) including the subnucleus reticularis dorsalis (Bouhassira et al. 1992). This conclusion from animal studies is in agreement with data obtained in patients with unilateral caudal medullary lesions (De Broucker et al. 1990). In any case, it is of particular interest that lesions of the rostral ventromedial medulla (RVM) did not modify DNIC (Bouhassira et al. 1993). Because DNIC were found to be triggered by activities in A- and C-peripheral fibers (Bouhassira et al. 1987) and to be closely related to the intensity of the noxious conditioning stimulus (Le Bars et al. 1981; Villanueva and Le Bars 1985), such diffuse inhibitory controls could have contributed to the postconditioning effects reported herein.
; Willis and Coggeshall 1991; Zieglgänsberger 1986) and it is impossible to conclude which may be involved on the basis of the present results. There is however at least one other system that could potentially be triggered by noxious peripheral inputs.
-c; Lundberg 1982) clearly showed that limb flexor reflexes, elicited by stimulation of thin myelinated or unmyelinated muscular and cutaneous afferents (the flexor reflex afferents), are subject to inhibitory controls originating from the RVM. The RVM also appeared to be of particular interest with respect to pain modulation, notably because its activation triggers descending inhibitory controls that block the spinal transmission of nociceptive signals in the dorsal horn (see Basbaum and Fields 1978; Fields and Basbaum 1994; Oliveras and Besson 1988; Willis and Coggeshall 1991). Because the RVM receives afferents from the adjacent reticular formation and the periaqueductal gray matter, the physiological roles of these modulatory systems acting on the spinal transmission of nociceptive signals were interpreted in terms of spino-bulbo-spinal regulatory mechanisms (Fields 1992; Fields and Basbaum 1989; Fields et al. 1991). They could therefore be involved in the postconditioning inhibitory effects described in intact animals in the present study. Interestingly, preliminary experiments have shown that lesions of the RVM reduce such inhibitory processes to a great extent.
; Dubner and Ren 1995; Ren and Dubner 1995; Schaible and Grubb 1993; Schaible et al. 1991). Because the postconditioning increase in excitability of the flexor reflex was very sensitive to halothane anesthesia in our preparation, it is clear that the postconditioning supraspinally mediated inhibitory effects were maximized in the present experiments in intact, anesthetized rats. On the other hand, in both the present and previous studies (Cervero et al. 1991; Dubner and Ren 1995; Ren and Dubner 1995; Schaible and Grubb 1993; Schaible et al. 1991), the postconditioning spinally mediated facilitatory effects were maximized when the animals were spinal and nonanesthetized. Identical experiments are not feasible in nonanesthetized animals both for ethical and for scientific reasons, notably the inevitable biasing interference of stress. We are thus dealing with two opposing phenomena; both are powerful, both are long-lasting, but evidence for each can be obtained only in the absence of the other. The net global effect cannot be assessed with the present experimental paradigm in animals. We are presently designing a similar experiment in human volunteers by using the recording of the biceps femoris RIII reflex elicited by stimulation of the sural nerve. Pilot studies indicate that inhibitory processes are indeed triggered in nonanesthetized human volunteers after homotopic high-intensity electrical stimulation. It therefore appears that supraspinally mediated inhibitions cannot be neglected when one is referring to long-term changes in spinal cord excitability after noxious stimulation. It is not our purpose here to deny a role for central sensitization in clinical situations involving hyperalgesia and allodynia, but the overestimation of this factor along with others (e.g., Herrero and Headley 1995) may have contributed to the rather disappointing results reported in most clinical trials of "preemptive analgesia" (Dahl and Kehlet 1993; McQuay 1995), which is based mainly on the assumption of a prevalent role of central sensitization.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. B. Bussel, S. W. Cadden, and L. Villanueva for advice in the preparation of the manuscript.
This work was supported by Institut National de la Santé et de la Recherche Médicale (Clinical Research network).
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FOOTNOTES |
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Address for reprint requests: D. Le Bars, INSERM U-161, 2, rue d'Alésia, 75014 Paris, France.
Received 18 November 1996; accepted in final form 14 July 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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- and C-fibres triggers inhibition of trigeminal and spinal convergent neurones in the rat.
J. Physiol. (Lond.)
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