Temporal Summation of C-Fiber Afferent Inputs: Competition Between Facilitatory and Inhibitory Effects on C-Fiber Reflex in the Rat

Manuela Gozariu1, Dominique Bragard2, Jean-Claude Willer1, and Daniel Le Bars1

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

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

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.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

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; 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.

In this context the phenomenon of "wind-up" consists of a progressive increase in neuronal or reflex responses to a consistent nociceptive stimulus applied repetitively to the same area of the body. Mendell and Wall (1965) 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).

A related phenomenon was observed in spinal animals when a test response is conditioned by strong, repeated, short-lasting conditioning stimuli. Woolf and Wall (1986) 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.

However, the transmission of nociceptive signals at the level of the spinal cord is subject to various other influences. Modulations with segmental, propriospinal, and supraspinal origins are essentially inhibitory and can profoundly modify the original peripheral input (see Besson and Chaouch 1987; Willis and Coggeshall 1991; 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).

It could be that in experimental situations where central sensitization occurs at the spinal level, descending inhibitory controls with supraspinal origins are triggered in parallel. In the case of inflammation, there is evidence that the development of hyperalgesia or hyperexcitability of spinal neurons is counteracted by an enhancement of descending inhibitions (Cervero et al. 1991; 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.

The results were very much dependent on the type of preparation. In intact, anesthetized rats the reflex was facilitated during the conditioning period, but then inhibited in a stimulus-dependent manner for a long period of time. In spinal, unanesthetized rats such inhibitions were replaced by long-lasting facilitations. Inhibitions were never observed in animals whose brains were transected at the level of the obex, but postconditioning facilitations were seen only in the absence of anesthesia.

These results have been presented previously in abstract form (Gozariu et al. 1995).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

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.

The procedure of transection at the level of the obex consisted of fixing the rat in a stereotaxic frame with the head ventroflexed by using a metal bar and exposing the brain stem by a slit into the dura overlying the cisterna magna. Transection at the level of the obex was then made by electrocoagulation. Spinalization was performed at the T8-T10 level after exposure of the cord by laminectomy.

After surgery the concentration of halothane was lowered to 0.9% in 100% oxygen or completely abolished in spinal rats. In rats whose brains were transected at the level of the obex, the effects of two levels of anesthesia (0 and 0.9% halothane) were tested.

The heart rate and the parameters of ventilation and anesthesia were monitored continuously throughout the experiment. The respiratory rate (50 counts/min) and levels of O2, end-tidal CO2(3.2-3.5%), and halothane (0.9%) were monitored continuously by using a capnometer (Capnomac II, Datex Instruments, Helsinki, Finland) and each was under the control of an alarm. Body temperature was maintained at 37.5 ± 0.5°C (SE) by means of a homeothermic blanket system.

Electrophysiological recordings

This method was described previously (Falinower et al. 1994; 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.

The conditioning stimuli (trains of 20 2-ms pulses) were delivered to the same area but at variable intensities (1.5-10 times threshold for the C-fiber reflex) and frequencies (1-0.17 Hz). The stimulus intensities and the EMG responses were fed to an oscilloscope for continuous monitoring and to a computerized system for on-line digitization. The digitized EMG responses were full-wave rectified and the C-fiber-evoked responses integrated within a time window from 100 to 450 ms after the stimulus. The individual reflex responses were plotted against time to allow the study of their temporal evolution.

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.

In intact, anesthetized (0.9% halothane) rats, after a control period of 10 min (with 1.5 T test stimuli being applied at 0.17 Hz), 20 conditioning stimuli were delivered. In one group the conditioning stimuli were single 2-ms square-wave shocks with a constant frequency of stimulation and a variable intensity of stimulation of the sural nerve (1.5, 2, 3, 5, and 10T). In a second group the conditioning stimuli were also single 2-ms square-wave shocks but with a constant intensity of stimulation (10T) and a variable frequency (1, 0.5, 0.25, and 0.17 Hz). After the conditioning period the reflex evoked by the test stimulus was recorded continuously for 20 min.

In spinal, nonanesthetized rats, after a control period of 10 min (with 1.5 T test stimuli being applied at 0.17 Hz), 20 conditioning stimuli were delivered (2 ms, 1 Hz, and 10T) and the postconditioning effects were recorded for 20 min.

In rats whose brains were transected at the level of the obex, two levels of anesthesia were tested: 0.9 and 0% of halothane. The same conditioning procedure was applied; after a control period of 10 min (2-ms test stimuli, 0.17 Hz, and 1.5 T), 20 conditioning stimuli were applied (2 ms, 1 Hz, and 10T) and the postconditioning effects were recorded for 20 min.

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.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

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). 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.

The C-fiber reflex elicited by electrical stimuli of 1.5 times threshold (1.5T) was the test response. Twenty conditioning pulses were applied at various intensities (1.5-10T) and frequencies (1-0.17 Hz) through the same electrodes. In each case the results were expressed as percentages of the mean control value recorded during the 2-min control period preceding the conditioning procedure, which consisted of 20 successive C-fiber reflex responses.

The temporal summation of C-fiber inputs was studied during and after the conditioning procedure. The main series of experiments were performed in intact, anesthetized rats. Additional experiments were made in several other preparations, namely animals transected at the level of the obex (anesthetized or nonanesthetized) and spinal, nonanesthetized animals.

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.


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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.

These results were consistent observations as shown by the cumulative results in Fig. 2. Analysis of the mean curves for the conditioning period in Fig. 2A shows that the C-fiber reflex was significantly facilitated in a stimulus-dependent fashion. Three different periods can be described on the basis of the temporal evolution of the responses. During the first seven conditioning stimuli the C-fiber reflex progressively increased (wind-up phenomenon); it reached a plateau between the 8th and 13th stimuli and then decreased slightly between the 14th and 20th stimuli. This final decrease was particularly obvious during conditioning at 10T, when the response reached 742.4 ± 144.9% of the control level after the 10th conditioned stimulus and was only 575.6 ± 138% after the 20th stimulus.


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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).

A completely different picture was seen during the postconditioning period (Fig. 2B) where the reflex was inhibited in a stimulus-dependent manner. At 1.5T a significant inhibition was seen during the period 2-3 min after conditioning (72.7 ± 8.3% of control values after 2 min) and this was followed by a slight facilitation in the 6- to 11-min postconditioning period (significant at 9 min; 119.1 ± 10.8% of control values). Although increasing the intensity of the conditioning stimuli reduced this latter effect, the former effect increased. At 2T a significant inhibition was observed during the 2- to 4-min postconditioning period (52.2 ± 7.2% of the controls at 2 min) with a slight recovery of activity during the 6- to 11-min postconditioning period (101.4 ± 9.6% of control values at 8 min). At 3T a significant inhibition was observed during the first 11-min postconditioning (maximum at 2 min; 41.1 ± 7.9% of the controls) with a progressive recovery toward baseline responses occurring within 15 min. The postconditioning time course at 5T was almost exactly the same as that for 3T: a significant inhibition occurred in the 2- to 10-min period with a maximum at 2 min (33.7 ± 7.3% of the controls) and this was followed by some recovery. At 10T, a strong inhibition with a maximum at 2 min (20.1 ± 5.1% of controls) followed by a complete recovery within 20 min was observed. In the latter cases no rebound facilitation was seen.

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%.


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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.

During the postconditioning period (Fig. 3B) the reflex was inhibited in a similar way regardless of which frequency of stimulation had been applied during the conditioning period. At 1 Hz a strong inhibition (significant for 13 min) with a maximum at 2 min (28.1 ± 5.1% of the control level) and a complete recovery within 16 min was seen. At 0.5 Hz a significant inhibition occurred only during the first 4 min postconditioning (maximum at 2 min; 34.5 ± 5.8% of the control level), probably because a slight recovery of activity took place during the 6- to 11-min postconditioning period. At 0.25 Hz a significant inhibition was observed during the first 12-min postconditioning (maximum at 2 min; 39.3 ± 5.8% of the control level) and this was followed by a complete recovery within 20 min. At 0.17 Hz the inhibition was significant only during the first 9 min of the postconditioning period (maximum at 2 min; 34.6 ± 12.1% of the control level) and again a complete recovery occurred within20 min.

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.


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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.

In spinal (T8-T10) animals anesthetized with 0.9% halothane, no clear EMG responses could be recorded. In nonanesthetized rats recordings were started one hour after the surgical procedure, including recording of the spinal section. Again the EMG signal was weak compared with that in intact animals and was desynchronized (see individual example as an insert in Fig. 4C). The values of the threshold (1.6 ± 0.5 mA), latency (118.3 ± 7.5 ms), and duration (281.7 ± 24.8 ms) of the C-fiber reflex were similar to those recorded in obex-transected, nonanesthetized rats.

The experimental protocol employed under these differing experimental conditions is illustrated in Fig. 4 by the individual histograms of the temporal evolution of the C-fiber reflex (histograms to the left show the pre- and postconditioning periods; histograms to the right show the conditioning periods). The C-fiber reflex increased continuously from one stimulus to the next during the whole conditioning period. The amplitude of the reflex increased to a maximum of 354, 482, and 534% of the control values at the end of conditioning in obex-transected and anesthetized, obex-transected and nonanesthetized, and spinal animals, respectively.

Figure 4, left, also shows that after the conditioning period the C-fiber reflex was facilitated only in nonanesthetized rats. Note the different time courses of the postconditioning facilitations, depending on the level of anesthesia. After conditioning in obex-transected, anesthetized animals a facilitation was seen only during the first minute of the postconditioning period (180.6% of the control level), and this was followed by a complete recovery during the following minute. The conditioning in obex-transected, nonanesthetized rats was followed by a facilitatory period lasting for 15 min (to a maximum of 225% of the control level after 1 min and in the 140-160% range during the 1st 10 min). Similarly, in spinal rats a facilitation of the response occurred during the postconditioning period (to a maximum of 247% of the control level at 1 min and in the 125-140% range during the first 20 min).

These were consistent observations as is illustrated by the cumulative results in Fig. 5. The mean curves related to the conditioning period in Fig. 5A show that the C-fiber reflex was facilitated significantly in all the experimental conditions. The progressive increase of the reflex from one stimulus to the next, the wind-up phenomenon, was more marked in spinal rats than in anesthetized or in nonanesthetized, obex-transected rats. This is exemplified by the values for the maximum facilitation seen after the 20th stimulationat the end of the conditioning period: 780.8 ± 168.2%,273.2 ± 54.9%, and 458.9 ± 79.2% of the control levels for the spinal, the anesthetized, and the nonanesthetized and obex-transected rats, 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).

As shown by the cumulative results in Fig. 5B, the postconditioning effects were also dependent on the level of anesthesia and the surgical preparation. In obex-transected, anesthetized rats, the inhibition seen during the postconditioning period in intact animals was blocked completely. Note that a small facilitatory effect occurred only during the first minute (to 156.1 ± 20.8% of the control level) and was followed by a very quick recovery. In obex-transected, nonanesthetized rats, the facilitatory postconditioning effect was significant throughout the first 8 min (208.2 ± 28.0% of the control level at 1 min and in the 160-120% range between the 2nd and 10th minutes). In spinal, nonanesthetized rats, the facilitatory postconditioning effects were clearly higher. They were significant during the first 11 min (259.4 ± 53.0% of the control level at 1 min and in the 185-130% range from the 2nd to the 20th minute).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

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.

The discussion will be organized into several sections as follows: 1) EMG signals and the preparations, 2) wind-up phenomenon observed during conditioning, 3) postconditioning effects including facilitatory mechanisms involving the spinal cord and inhibitory mechanisms involving the brain, and 4) conclusions and functional implications.

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; Schouenborg and Dickenson 1985; Schouenborg and Kalliomäki 1990; Schouenborg and Sjölund 1983; Strimbu-Gozariu et al. 1993).

In some rats transections were performed at the level of the obex, which is caudal to the vestibular nuclei that are probably the main sources of the muscular rigidity usually elicited by decerebration (Lundberg 1982). 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.

No clear EMG responses could be recorded from the biceps femoris muscle in anesthetized, spinal rats. As discussed elsewhere, convergent neurons in the dorsal horn of the lumbar spinal cord are part of the circuitry involved in the polysynaptic C-fiber-evoked reflex (Falinower 1994; Schouenborg and Dickenson 1985; 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.

In nonanesthetized rats recordings were started 1 h after the surgical procedure including the spinal section. This experimental procedure was chosen after reference to previous studies (Wall and Woolf 1984; 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.

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) 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.

Interestingly, wind-up phenomenon was seen only at frequencies of stimulation >= 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.

The present results in spinal and intact animals (at least for the 1st responses in the latter case) are very much in keeping with earlier studies of polysynaptic reflexes and of dorsal or ventral horn neurons. In our experimental paradigm we are dealing with a global response, presumably involving both a pool of interneurons and a corresponding pool of motoneurons. Such a wind-up phenomenon was described previously for C-fiber reflexes in the spinal cat or the intact rat, when the iterative electrical shocks were applied at a frequency >0.3 Hz (Laird et al. 1995; 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.

Incrementing cumulative depolarizations of spinal neurons have been observed after repetitive stimulation of high-threshold unmyelinated primary afferent fibers (Price et al. 1971; 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).

The fact that wind-up phenomenon at the spinal level has physiological consequences in terms of pain has been strongly suggested by experiments in humans. Price and coworkers reported that the sensation of "second pain" elicited by percutaneous electrical stimulation or noxious heat pulses increased with each successive shock when the stimulus was repeated at least once every 3 s (Price 1972; 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.

In the present experiments the monotonic accelerating function was seen over the 20 consecutive stimuli only in the spinal or obex-transected preparations. In the intact rats the wind-up phenomenon was obvious for seven stimuli, then plateaued and decreased. An identical observation was made by Schouenborg and Sjölund (1983) 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.

In animals and humans a decrease in the magnitude of spinal reflexes with repetitive suprathreshold cutaneous stimuli occurs after an initial increase in these responses and is termed habituation (Egger 1978; 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.

Another observation deserves comment: the wind-up observed in the present experiments in obex-transected preparations was slightly diminished by halothane. Dickenson and Sullivan (1987) 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.

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.

The possibility that there was a direct relationship between the inhibitory effects described herein in intact animals and neurovegetative reactions appeared very unlikely. Indeed, although high-intensity stimuli (5 and 10T) elicited increases in blood pressure in control experiments (see Table 1), the time courses of such increases did not match those of the inhibitory effects of the EMG signal. In addition there were cases (e.g., conditioning at 3T) of strong inhibitory postconditioning effects without any change in blood pressure. Finally, heart rate was unchanged by the conditioning procedures. It therefore appears that we are not dealing with a phenomenon generated by a global neurovegetative reaction to a noxious stimulus but rather with a phenomenon triggered by specific control mechanisms.

 
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TABLE 1. Control experiment: effects of the experimental protocol on blood pressure and heart rate in the intact anesthetized animal

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) 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 Adelta - 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).

Similarly, it was reported that in the spinal, nonanesthetized rat there is a decrease in the latency for hindpaw withdrawal from noxious heat after electrical conditioning stimuli similar to ours applied to the sciatic nerve; interestingly, this latter effect disappeared after pentobarbital sodium administration (Cleland et al. 1994). 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.

It is interesting to note that signs of inhibitory effects were seen neither in spinal animals nor in anesthetized or nonanesthetized animals that had been transected at the level of the obex. This lack of effect was particularly striking in the anesthetized, obex-transected preparation where theC-fiber responses were remarkably stable and identical to the controls after high-intensity conditioning. This observation suggests that intrinsic spinal mechanisms that were reported to decrease the spinal transmission of nociceptive signals were not involved in our experimental paradigm.

In fact, studies on single dorsal horn neurons and on nociceptive reflexes have shown that intersegmental inhibitory influences elicited by noxious stimuli are generally triggered from remote segments or the contralateral side of the same segment (Cadden et al. 1983; 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.

In many brain structures repetitive activation of synaptic connections can lead to long-term potentiation (LTP) or long-term depression (LTD) of synaptic transmission (Bliss and Lomo 1973; 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.

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.

When the C-fiber reflex was recorded in intact, anesthetized rats in conditions similar to those used in the present experiments, it was reported to be strongly inhibited by noxious heterotopic stimuli; such effects disappeared when the reflex was recorded either in spinal animals or ipsilaterally to a rostral unilateral lesion of the dorso-lateral funiculus (Falinower et al. 1994). 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).

In humans, analogous results have been obtained by means of EMG recordings of a nociceptive flexion reflex in the biceps femoris muscle elicited by electrical stimulation of a cutaneous nerve, the sural nerve (the RIII reflex); painful heterotopic conditioning stimuli depress such a reflex in an intensity-dependent manner (Willer et al. 1984, 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).

Such inhibitory processes acting on reflex activities are probably related to diffuse noxious inhibitory controls (DNIC) observed on dorsal horn neurons in various species (Brennan et al. 1989; 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 Adelta - 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.

However, many other brain regions are sources of descending inhibitions to the dorsal and/or the ventral horns of the spinal cord (Besson and Chaouch 1987; Fields and Besson 1988; 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.

Lundberg and colleagues (Engberg et al. 1968a-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.

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; 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.

    ACKNOWLEDGEMENTS

  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).

    FOOTNOTES

  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.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society