Physiological Properties of the Lamina I Spinoparabrachial Neurons in the Rat

Hervé Bester, Victoria Chapman, Jean-Marie Besson, and Jean-François Bernard

Institut National de la Santé et de la Recherche Médicale U.161, École Pratique des Hautes Études, F-75014 Paris, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bester, Hervé, Victoria Chapman, Jean-Marie Besson, and Jean-François Bernard. Physiological Properties of the Lamina I Spinoparabrachial Neurons in the Rat. J. Neurophysiol. 83: 2239-2259, 2000. Single-unit extracellular recordings of spino-parabrachial (spino-PB) neurons (n = 53) antidromically driven from the contralateral parabrachial (PB) area were performed in the lumbar cord in anesthetized rats. All the spino-PB neurons were located in the lamina I of the dorsal horn. Their axons exhibited conduction velocities between 2.8 and 27.8 m/s, in the thin myelinated fibers range. They had an extremely low spontaneous activity (median = 0.064 Hz) and a small excitatory receptive field (<= 2 toes or pads). They were all activated by both peripheral A (mainly Adelta ) and C fibers after intense transcutaneous electrical stimulation. Their discharge always increased in response to noxious natural stimuli of increasing intensities. The great majority (75%) of spino-PB neurons were nociceptive specific, i.e., they were excited only by noxious stimuli. The remaining (25%) still were excited primarily by noxious stimuli but also responded moderately to innocuous stimuli. Almost all spino-PB neurons (92%, 49/53) were activated by both mechanical and heat noxious stimuli. Among them, 35% were in addition moderately activated by noxious cold (thresholds between +20 and -10°C). Only (8%, 4/53) responded exclusively to noxious heat. Spino-PB neurons clearly encoded the intensity of mechanical (n = 39) and thermal (n = 38) stimuli in the noxious range, and most of the individual stimulus-response functions were monotonic and positive up to 40/60 N · cm-2 and 50°C, respectively. For the mechanical modality, the mean threshold was 11.5 ± 1.25 N · cm-2 (mean ± SE), the response increased almost linearly with the logarithm of the pressure between 10 and 60 N · cm-2, the mean p50 (pressure evoking 50% of the maximum response) and the maximum responsiveness were: 30 ± 2.4 N · cm-2 and 40.5 ± 5 Hz, respectively. For the thermal modality, the mean threshold was 43.6 ± 0.5°C, the mean curve had a general sigmoid aspect, the steepest portion being in the 46-48°C interval, the mean t50 and the maximum responsiveness were: 47.4 ± 0.3°C and 40 ± 4.4 Hz, respectively. Most of the spino-PB neurons tested (13/16) had their noxiously evoked responses clearly inhibited by heterotopic noxious stimuli. The mean response to noxious stimuli during heterotopic stimuli was 31.7 ± 6.1% of the control response. We conclude that the nociceptive properties of the lamina I spino-PB neurons are reflected largely by those of PB neurons that were suggested to be involved in autonomic and emotional/aversive aspects of pain.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous retrograde and anterograde anatomic data have clearly demonstrated that the parabrachial (PB) area---a group of neurons that surround the superior cerebellar peduncle at the ponto-mesencephalic junction---is one of the major projection targets for the neurons located in the most superficial laminae (I and II outer, or to simplify: lamina I) of the dorsal horn (Bernard et al. 1989, 1995; Blomqvist et al. 1989; Burton et al. 1979; Cechetto et al. 1985; Craig 1995; Feil and Herbert 1995; Hylden et al. 1989; Kitamura et al. 1993; Lima and Coimbra 1989; Menétrey and De Pommery 1991; Menétrey et al. 1992; Panneton and Burton 1985; Slugg and Light 1994; Wiberg and Blomqvist 1984; Wiberg et al. 1987; Yamada and Kitamura 1992; see also Menétrey et al. 1982; Saper 1995; Swett et al. 1985).

The superficial laminae of the spinal cord receive heavy and direct input from the thin primary afferents Adelta and C fibers and contain primarily nociceptive neurons some of which being identified as projecting to the thalamus (Besson and Chaouch 1987; Brown and Culberson 1981; Cervero and Connell 1984; Culberson and Brown 1984; Fitzgerald 1984; Light and Perl 1979; Mizumura et al. 1993; Sugiura et al. 1986, 1988, 1989, 1993; Woolf and Fitzgerald 1983, 1986). More recently, several studies also have demonstrated that a high number of nociceptive neurons in the superficial laminae project to the PB area (Hylden et al. 1985, 1986a,b; Light et al. 1987, 1993; see also McMahon and Wall 1983).

These studies, as a whole, strongly support a substantial noxious input from the spinal cord to the PB area. The effect of this noxious input was observed directly at the PB level. Several electrophysiological studies have recorded the unitary activity of PB neurons, a large number of which respond specifically to somatosensory stimuli in the noxious range irrespective of whether these neurons were antidromically driven from the central nucleus (Ce) of the amygdala or from the ventromedial (VMH) nucleus of the hypothalamus or whether their projecting target was not determined (Bernard and Besson 1988, 1990; Bernard et al. 1994; Bester et al. 1995b; Hayashi and Tabata 1990; Huang et al. 1993; Matsumoto et al. 1996; Menendez et al. 1996; Slugg and Light 1990). Further indirect evidence for the involvement of the PB area (Bellavance and Beitz 1996; Bullitt 1990; Clement et al. 1996; Hamba et al. 1994; Hermanson and Blomqvist 1996; Lantéri-Minet et al. 1993, 1994; Pertovaara et al. 1993) and spino-PB pathway (Bester et al. 1997b; Buritova et al. 1998) in nociception was obtained from c-Fos studies. Because the nociceptive region of the PB area is involved in numerous autonomic regulatory functions and densely projects to the Ce and the hypothalamus, the spino-PB pathway is thought to be involved in autonomic and emotional/aversive reactions to painful stimuli (see refs and discussion in Bernard and Besson 1990; Bester et al. 1995b).

Paradoxically, at least in the rat, the properties of PB neurons now are known more precisely than those of their main input, the spino-PB neurons. More specifically, their precise encoding properties to mechanical and thermal stimuli and the effect of stimuli applied outside the excitatory receptive field (heterotopic stimuli) of a consistent subgroup of PB neurons have been investigated in some detail (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b). To date, these properties have not been investigated for the spino-PB neurons. Although a study in the rat investigated the main characteristics of cells projecting through the dorsolateral funiculus (McMahon and Wall 1983), it did not give details concerning spino-PB neurons. This issue has been considered in the cat but neither the encoding properties nor the effect of heterotopic stimuli were determined (Hylden et al. 1985, 1986a,b; Light et al. 1987, 1993). Indeed, the encoding properties of superficial laminae neurons only have been investigated precisely for spino-thalamic neurons or for neurons without identified projection in the cat and monkey (Bushnell et al. 1984; Chung et al. 1979; Craig and Kniffki 1985; Craig and Serrano 1994; Ferrington et al. 1987; Hayes et al. 1981; Hoffman et al.,1981; Kenshalo et al. 1979; Light et al. 1993; McHaffie et al. 1994; Price et al. 1976, 1978). The aim of the present study was to provide a detailed electrophysiological characterization of the responses of the lamina I spinal neurons backfired antidromically from the contralateral PB area. Furthermore the present investigation should permit an accurate comparison between spino-PB and PB neurons because the studies were made under identical experimental conditions. Part of these results have been presented in abstract form (Bester et al. 1995a).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation

Experiments were performed on 53 Sprague-Dawley male rats weighing 250-275 g. In an anesthetizing box, animals were deeply anesthetized with 2% halothane (Belamont, France) in a nitrous oxide-oxygen mixture (2/3-1/3). This anesthesia regimen was maintained throughout the surgery procedures. A tracheal cannula was inserted, the jugular vein was cannulated, and then animals were paralyzed with a continuous intravenous injection of gallamine triethiodide (Flaxedil) and artificially ventilated using a Palmer pump. The expiratory CO2 was monitored continuously using a Capnomac (Datex Instruments) throughout the experiment, and the end-tidal CO2 was maintained at ~4%. Neither blood pressure nor electreoencephalogram (EEG) were monitored because these same conditions have been found to provide reproducibly stable and reliable anesthesia. Use of these conditions enables comparisons across all prior studies in this laboratory. The level of halothane, O2, and N2O also was checked systematically during the experimental procedure. The heart rate was monitored continuously, and the core temperature maintained at 37 ± 0.5°C using a homeothermic blanket system.

The animals were mounted in a stereotaxic frame. The head was fixed in a dorsiflexed position with the incisor bar elevated 10 mm above the standard position (Paxinos and Watson 1986). A craniotomy was performed at the parieto-occipital level on the right side for the passage of the stimulating electrodes targeting the PB area. A second small craniotomy was performed rostrally in the left frontal bone to implant the reference electrode for central stimulation. Vertebras L1-3 were exposed and fixed in "spinal hooks." A laminectomy then was performed to expose the L4-5 spinal segments. After surgery, the halothane level was reduced to 0.5%, and the gas mixture of nitrous oxide-oxygen was maintained at 2/3-1/3. This level of anesthesia did not excessively depress neuronal responses to noxious stimuli and was adequate in terms of ethical purposes (Benoist et al. 1984; Le Bars et al. 1980). These conditions were in agreement with the ethical guidelines of the International Association for the Study of Pain (IASP) for investigations of experimental animals (Zimmermann 1983) and received the approval from the local ethical committee.

Recordings

Extracellular unit recordings made with metal microelectrodes (9-12 MOmega ), tungsten or stainless steel (Frederic Haer). Single-unit activity was fed into a window discriminator, the output of which was stored on a digital audiotape (Biologic DTR 1200). A multichannel analyzer was used to perform further data processing.

The electrode was inserted vertically in the left spinal cord close to the surface. From this zero point, the search of a neuron was started by driving the electrode through the dorsal horn. Each time only one spike or any change in the background activity was observed, the response to electrical stimuli delivered in the PB area was tested. If no response was detected, the effects of high-intensity current delivered on different distal sites of the hindpaw were tried rapidly. If the presence of a unit was confirmed, antidromic identification was tested by applying electrical stimuli in the PB area at different depths from each of the three stimulating electrodes (see following text). When antidromic activation was obtained, the cell responses to different cutaneous stimuli (electrical, mechanical, and thermal) were tested. Only one neuron was studied per animal to avoid sensitization.

Response to PB area stimulation

Stimulations of the PB area were applied with a linear array of three concentric monopolar electrodes (model No. SNE-300 modified; insulated contact protrudes from shaft, 120-µm diam, 5-mm length; exposed contact, 100-µm diam, 150-µm length; Rhodes Medical Instruments) separated by 600 µm from each another. The set of three electrodes was inserted into the right PB area with the following coordinates for the middle electrode: 1.9 mm rostral to the lambda and 1.8 mm lateral to the midline. The depth was 7,000 µm from the brain surface. The three center contacts could be independently stimulated (1-mA square wave negative current, 0.2-ms duration).

When, in the spinal cord, a unit was backfired from the PB area, the stimulation thresholds were measured for each electrode. The site of minimum threshold was determined by repeating this measure at different depths (above and below). Finally, the set of electrodes was positioned at the depth where the lowest threshold had been observed, and we used the electrode from which this lowest threshold was obtained. Then the intensity of the central stimulation was set at twice the threshold to test the criteria of antidromic activation: 1) the lack of variance (<100 µs) of the spike latency (t), 2) the ability of the evoked response to follow a train of high-frequency stimulation (>300 Hz, for 5 stimuli). This test was coupled with the measure of the refractory period (R), determined as the shortest interval between two stimuli required to produce two antidromic spikes. The refractory period always corresponded to a higher frequency (1/r > 500 Hz). And 3) the observation of a systematic collision between an orthodromic spike (spontaneous or electrically evoked from the receptive field) and the antidromically evoked response in the 2t + R period.

Response to transcutaneous electrical stimulation

Electrical square-wave stimuli (2-ms duration) were delivered through pairs of stainless steel stimulating electrodes inserted subcutaneously in the toes. We usually observed that a high-intensity stimulation evoked two periods of activation. The threshold of each peak of activation was determined by a progressive increase in the intensity of stimulation from a 0 starting point. The effects of repetitive electrical stimuli (16 trials at a frequency of 0.5 Hz) at an intensity corresponding to threefold the highest threshold (i.e., that evoking the late peak of activation) were analyzed by building poststimulus time histograms (PSTHs) off-line on a MacIntosh 7100, with the Spike2 software and CED 1401 hardware (CED, Cambridge England). The length of the sciatic nerve was measured at 15 cm from the tip of the toes (III-V) to the dorsal horn entry zone of the L4-5 lumbar segments. The conduction velocity of the peripheral fibers then was calculated as follows: 150 mm/[observed latency of the first spike in each individual peak - 1 ms]. The subtracted 1 ms corresponds to an estimate of the synaptic delay, assuming that spino-PB neurons were monosynaptically activated. All in all, our results happened to be in good agreement, a posteriori, with data obtained directly from peripheral fibers in the rat foot (Leem et al. 1993a,b), validating thus the assumptions and the methods.

Response to natural cutaneous stimulation

INNOCUOUS STIMULI. Innocuous stimuli were applied to the receptive field of the recorded neuron. They consisted of mechanical light touch and brush (paintbrush Gerand Séries SD 1, No. 6) or thermal stimuli applied with a thermostatically controlled water jet (20-40°C). To apply thermal stimuli, a 50-ml syringe was filled with water just above the desired temperature. When ejected through a 12-gauge needle, the stimulating water jet maintained a temperature (measured from a thermocouple placed next to the receptive field) at the desired level over the 20 s of stimulation. From 20 to 30°C, 5°C steps were used, then 2°C steps were used from 30 up to 50°C. In the absence of an evoked response, intervals between the discrete stimuli were of 2 min. All stimuli were applied during a 20-s period.

NOXIOUS MECHANICAL STIMULI. With a calibrated forceps (strain gauge on the arm connected to an amplifier: Hottinger, Baldwin Messtechnick, Darmstadt, Germany), we manually applied serially increasing pressures of 20-s duration (5, 10, 20, 40, 60, and 80 N · cm-2; 1 N · cm-2 = 10 kPa approx  100 g · cm-2) on the receptive field of the spino-PB neurons. The stimulating area was 10 mm2. The level (intensity) of the applied pressure was read, on-line, on the digital screen (4 digits) of the apparatus, allowing the trained experimenter to maintain, with a feedback reaction, an almost constant pressure (variation <10%) over the stimulating period. Between two stimulations, an interval of 5 min was observed, to avoid fatigue and/or sensitization of peripheral receptors.

NOXIOUS THERMAL STIMULI. Both noxious heat and noxious cold stimuli were applied using a thermostatically controlled water jet, directly on the receptive field, for 20 s periods. With the approach used for innocuous thermal stimuli, scanning the effect of heat stimuli toward the noxious range consisted of applications of discrete water jets of increasing temperatures from innocuous 30°C up to highly noxious 52°C in 2°C steps. Similarly, cold stimuli consisted of applications of discrete water jets of decreasing temperatures from innocuous 30°C down to highly noxious -15°C, with a 5°C step. In this case, the stimulating fluid was an alcohol-water mixture that was set just below the desired temperature. This procedure allowed the desired temperature to be achieved at the level of the receptive field (measured with a thermocouple) for the 20-s stimulation period. For both heat and cold stimuli, a minimum interval of 5 min (and 10 min for higher intensity stimuli) was observed, to avoid fatigue and/or sensitization of peripheral receptors.

Effects of heterotopic noxious stimulation on a noxiously evoked response

On 16 spino-PB neurons, we have tested the effects of a noxious stimulus applied far outside the receptive field (heterotopic stimuli) on the noxiously evoked responses of the spino-PB neurons. Briefly, on a standard response to noxious stimuli (50°C, or 60 N · cm-2) applied on the receptive field of a spino-PB neuron (toe or pad), we have tested the effects of a noxious stimuli (50°C, or 60 N · cm-2) applied far outside the receptive field, basically, a forepaw (and in some cases the tongue). The heterotopic stimulation started 10 s before the noxious stimulation of the receptive field, continued throughout this stimulation (20 s), and was prolonged 10 s after the end of the noxious stimulation of the receptive field (total duration of the heterotopic stimulus: 40 s).

Histological controls

For all the experiments, the location of the PB electrode tip that provided the lowest threshold to obtain an antidromic spike was marked by passing a 10 µA DC for 30 s (positivity to the electrode). This procedure allowed a small electrophoretic iron deposit at the tip of the stimulating electrode.

In a first series of 28 experiments, we used stainless steel recording electrodes. At the end of each experiment, the depth of the electrode was noted, and a small deposit of iron was made at the tip of the electrode by passing a 10 µA DC current (positivity to the electrode) during 30 s.

After the iron deposits, animals were killed by an overdose of intravenous sodium pentobarbitone. The brain and lumbar spinal cord were removed and fixed for 5 days in a mixture that would label the iron with a Prussian blue color (1 volume of 1% potassium ferrocyanide in 10% formalin solution added to 2 volumes of 2% acetic acid in 95% alcohol solution). The tissues then were cut into 100-µm thick sections, mounted on slides, Nissl stained with safranin, and coverslipped. Because the depth of electrode was related very closely to a Prussian blue point in the most superficial laminae of the dorsal horn (lamina I), it appeared reasonable to use only the depth measurement to locate the recordings when we used tungsten microelectrodes, given that all the experiments were performed with identical paradigms.

Both the recording and the stimulating sites were drawn individually through an optical microscope, using an attached camera lucida. The stimulation sites were plotted on a series of 12 camera lucida drawings of the PB area (150 µm apart). The spinal recording sites were plotted on camera lucida drawings of the L4-5 levels because no recordings were made outside these lumbar segments. For illustration purposes, the stimulation sites in the PB area were collated on four representative sections (2 pontine, 2 mesencephalic), and the recording sites on a representative section passing through the L4 segment.

Data analysis

The magnitude (or intensity) of the responses was defined as the mean spike frequency increase during the whole period of stimulation (20 s), i.e., mean spike frequency during the stimulation minus mean spike frequency during the 20 s preceding the stimulation. The responsiveness of a neuron was defined as the magnitude of the highest response observed for the considered modality.

In the case of heterotopic stimulation, the effect (inhibition) was defined as the percentage of the residual response as compared with the control test value. The results therefore were expressed as percentages of control responses.

Unless noted otherwise, the results were expressed as means ± SE. Statistical ANOVA were made using the Fisher's PLSD (protected least significant difference) tests. However, in some cases, we used the 10th, median, and 90th percentile because the distribution of the data were obviously not normal.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fifty-three dorsal horn neurons were recorded extracellularly and unitarily at the lumbar segment level of 53 rats. These neurons were selected by the following criteria: antidromically backfired from the contralateral PB area and located in the most superficial laminae of the dorsal horn.

General findings

The spontaneous activity of the spino-PB neurons was extremely low (10th percentile < median <90th percentile): 0.001 < 0.064 < 1.88 Hz (n = 53), being often very close to silent. All of these neurons were clearly nociceptive, i.e., responses were markedly increased to stimuli in the noxious range, irrespective of whether or not they responded to innocuous stimuli. Most of them (75%, see following text) were nociceptive specific (NS), responding only to stimuli in the noxious range. Almost all of them (92%, 49/53) responded to two modalities of noxious stimuli (mechanical and thermal). Generally, they had small receptive fields including a restricted portion of the paw. All of the spino-PB neurons responded to transcutaneous electrical stimulation applied to the receptive field with, at least, two distinct early and late periods of activity.

Antidromic activation of the spino-PB neurons

All of the spino-PB neurons were driven antidromically from the contralateral PB area and satisfied the antidromic criteria (Fig. 1): the latency of the antidromic spike was stable (Fig. 1B), i.e., with a variation <100 µs, or <1% of the latency; it was always possible to obtain a collision between the antidromic spike and a spontaneous or electrically evoked orthodromic spike (Fig. 1, C and C'); the antidromic spike could follow a high-frequency stimulation (>500 Hz; Fig. 1, D and D'). The refractory period of the spino-PB neurons was short with a mean of 0.7 ± 0.2 ms (mean ± SD, n = 53).



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Fig. 1. Criteria for antidromicity. A: single antidromic spike. B: superimposition of 15 sweeps showing the stability of the antidromic spike. C and C': collision test. C: antidromic spike (star ); C': orthodromic spikes (), one of which have collided with the antidromic spike that does not appear where it should occur (). D and D': high-frequency testing. D: antidromic spike was evoked 5 times by 5 electrical stimuli delivered with a frequency of 714 Hz; D': antidromic spike was evoked only 4 times by 5 electrical stimuli delivered with a frequency of 770 Hz. up-arrow , electrical stimuli (delivered in the parabrachial area). E: drawing of a coronal section of the parabrachial (PB) area passing close to the junction between its pontine and mesencephalic divisions. This also shows an electrode tract and the different intensities required along the tract to elicit an antidromic spike in the lamina I neuron.

The mean latency of the antidromic spikes was 9 ± 6.1 ms (mean ± SD, n = 53). The mean of the calculated conduction velocities for the spino-PB neurons (~100 mm distance between the L4 segment and the contralateral PB area divided by the spike latency) was 15.3 ± 7.3 m/s (mean ± SD, n = 53). The lowest conduction velocity was 2.8 m/s (Fig. 2), indicating that all of the spino-PB neurons recorded here had a thin myelinated axon.



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Fig. 2. Histogram of conduction velocities of spino-parabrachial axons. Abcissa: conduction velocities range of each class in m/s, left and right brackets indicate inclusion and exclusion of the value, respectively. Ordinate: number of neurons in each class.

The stimulation site was always located in or in the close vicinity of the PB area (Fig. 3, A-E). The thresholds needed to evoke an antidromic spike were often low (150-300 µA) or very low (5-150 µA): 26% (14/53) and 57% (30/53), respectively (Fig. 3, A-E). A few thresholds (17%, 9/53) were higher (300-500 µA) when a systematic search for the lowest threshold was not achieved. In numerous experiments, a low threshold could be obtained from only one of the three stimulating electrodes, whereas from the other two, even a current >1 mA was not always sufficient.



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Fig. 3. Parabrachial stimulation sites. A-D: caudal to rostral schematic drawing of the parabrachial are in coronal sections. Antidromic stimulation site with threshold in the range of 5-150 µA (odot ), 150-300 µA () and >300 µA (black-triangle). E: photomicrograph of a coronal section of the right pontine parabrachial area. Arrow indicates the Prussian blue point located between the pPBdl and pPBlcr subnuclei of the parabrachial area. Scale bars = 1 mm. bc, Brachium conjunctivum; Cnf, Cuneiformis nucleus; IC, inferior colliculus; KF, Kölliker-Fuse nucleus; LC, Locus coeruleus; mPBcl, mPBel, mPBem, mPBil, mPBm, mPBsl, central lateral, external lateral, external medial, internal lateral, medial, and superior lateral subnuclei of the mesencephalic parabrachial area; pPBcl, pPBdl, pPBel, pPBem, pPBil, pPBm, pPBvl, central lateral, dorsal lateral, external lateral, external medial, internal lateral, medial and ventral lateral subnuclei of the pontine parabrachial area; sct, Spino-cerebellar tract.

Location of the spino-PB neurons

The depths of the recording sites of the 53 spino-PB neurons were in the 118- to 363-µm range with a mean of 240 ± 12 µm (n = 53), i.e., corresponding to the superficial dorsal horn. The relevance of this measurement was confirmed further in a subgroup of 28 spino-PB neurons for which the recording sites were labeled with a Prussian blue point (Fig. 4A) in the superficial laminae (Fig. 4B). The mean depth of the recordings corresponding to these labeled sites was 260 ± 23 µm (n = 28). The mean depth of the 25 neurons recorded with tungsten microelectrodes was similar 233 ± 14 µm (n = 25), indicating that sites without histological confirmation were similarly located close to the lamina I. 



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Fig. 4. Spinal cord recording sites. A: photomicrograph of the left dorsal horn in coronal section at L4 lumbar enlargement. Arrow indicates the Prussian blue point. B: schematic representation of the L4 dorsal horn. : Prussian blue points corresponding to the location of recorded spino-PB neurons. Scale bars = 0.5 mm.

Receptive field

Overall the spino-PB neurons had small receptive fields. In most cases (72%, 38/53), the receptive field was extremely small (Fig. 5), including only one toe (n = 33) or one pad (n = 5). In some cases (28%, 15/53), the receptive field extended to two toes (n = 10), two pads (n = 2), or one toe and one pad (n = 3).



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Fig. 5. A: receptive field and responses of a spino-parabrachial neuron. Toes in dark and light gray, parts of the receptive field in which noxious stimuli evoked a heavy and lower responses, respectively. Histogram in black: firing rate; the mechanical (40 N · cm-2) and heat (50°C) noxious stimuli were applied, between the 2 arrows, in the corresponding toe. B and C: examples of the responsiveness reproducibility of 2 different spino-PB neurons. Two 48°C, as well as the 2 50°C stimuli were applied with a 5-min interval. Note the clear reproducibility of the responsiveness. Bin width of histograms = 2 s.

Characterization of spino-PB neurons by electrical stimuli

All the spino-PB neurons responded to suprathreshold transcutaneous electrical stimulation of the receptive field with at least two periods of activation (Fig. 6). The mean intensity threshold of these early and late periods of activation were 0.4 ± 0.1 and 3.1 ± 0.5 mA, respectively (n = 53). The mean latency (±SD) of the early and late periods were 7.7 ± 3 ms (range 3.9-20 ms, n = 53) and 162 ± 54 ms (range 51-290 ms, n = 53), respectively. These latencies correspond to calculated mean conduction velocities (±SD) of 26 ± 9 m/s (range 7.9-52 m/s, n = 53) and 1.1 ± 0.6 m/s (range 0.5-2.9 m/s, n = 53), respectively. These ranges of conduction velocities correspond with those of A and C fibers, respectively. Intense electrical stimulation (three times the C-fibers threshold) evoked 3.7 ± 0.4 and 14 ± 1.4 spikes (n = 53), in the early (A fibers) and late (C fibers) periods of activation, respectively.



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Fig. 6. Response of a spino-parabrachial neuron to transcutaneous electrical stimuli. A: single sweep recording showing 3 (1 early and 2 late) peaks of firing evoked by the stimulation of 1 toe. B: poststimuli time histogram (PSTH) made from responses to repetitive electrical stimuli (16 trials, 0.5 Hz). Note 1 early and 2 late components of response well individualized. Bin size is 0.5 ms.

The distribution of the A-fiber conduction velocities, calculated from the latency of the earliest spike in the early peak of activation for each single spino-PB neuron appeared normal (Fig. 7A). The majority of the conduction velocities (58%, 31/53) were in the 8- to 26-m/s range, i.e., clearly in the range of conduction velocities of Adelta fibers. The remaining 42% (22/53) corresponded to conduction velocities between either 26-38 or 38-60 m/s (Fig. 7A), i.e., to conduction velocities of peripheral fibers that were either at the border between Adelta and Abeta fibers (n = 18) or within Abeta -fiber (n = 4) ranges, respectively. Even in neurons with a clear Abeta -fiber activation, the duration of the first period of activation was such that most of the evoked spikes, and more specifically the relatively late ones, were likely to be triggered by Adelta fibers. Furthermore in 12 cases, two peaks of activation, with mean conduction velocities of 28 ± 6 and 13.6 ± 2 m/s, respectively (mean ± SD, n = 12), were identified. These clearly correspond to Adelta /Abeta -fiber and Adelta -fiber conduction velocity ranges, respectively.



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Fig. 7. Histogram of the peripheral conduction velocities. A: conduction velocities of peripheral fibers triggering the early period of activation. B: conduction velocities of the peripheral fibers triggering the late period of activation. Abcissa: conduction velocities range of each class in m/s; left and right brackets indicate inclusion and exclusion of the value, respectively. Ordinate: number of neurons in each class.

The distribution of the C-fiber conduction velocities, calculated from the latency of the earliest spike in the late peak of activation for each single spino-PB neuron (Fig. 7B) was also normal and clearly separated from the A-fiber group (Fig. 7A). The great majority of the C fibers (87%, 47/53) activating the spino-PB neurons had very slow conduction velocities, between 0.3 and 1.4 m/s, the few remaining cases corresponded to intermediate weaker peaks (Fig. 7A). Furthermore, as clearly illustrated in the single sweep of Fig. 6, numerous spino-PB neurons responded to suprathreshold electrical stimuli with two peaks in the late period of activation. In 26 cases where the two late peaks were clearly separated, their mean conduction velocities were 1.2 ± 0.6 and 0.6 ± 1 m/s, respectively (mean ± SD, n = 26).

Figure 8 shows that in the late period corresponding to C-fiber activation, for 36 representative spino-PB neurons stimulated over 16 trials at 0.5 Hz with the same intensity (3 times the threshold), the number of spikes evoked by the first stimulus increased slightly after a few stimulations and remained stable thereafter. Thus the wind-up was relatively weak in the present population of superficial spino-PB neurons.



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Fig. 8. Number of spikes in the late component (C fibers) during repetitive transcutaneous electrical stimuli. Mean C-fiber component increased moderately during the 1st 8 stimuli (0.5 Hz) before plateauing. open circle  and error bars: mean ± SE (n = 36).

Characterization of the spino-PB neurons by natural stimuli

RESPONSIVENESS. All the spino-PB neurons responded with a strong and sustained discharge to thermal and/or mechanical noxious stimuli. From a very low or absent spontaneous activity (see preceding text), the responses of the spino-PB neurons to noxious stimuli had a rapid onset that could reach values ~100 Hz (Figs. 5, 10, and 13), giving to the initial part of the response a phasic aspect. The response then decreased but remained strong and sustained throughout the period of stimulation, giving to the second part a tonic pattern. The majority of neurons had an afterdischarge, especially for the highest noxious intensities of stimulation (Figs. 5, 10, and 13). Overall, the spino-PB neurons had a high responsiveness, the mean of the maximum response (whatever the modality) was 47 ± 5 Hz (n = 53). Furthermore the response to noxious stimuli was clearly reproducible, the variation of responsiveness to two noxious heat stimulation of the skin (5-20 min apart) being in the 0.5-25% range (n = 29). Figure 5, B and C, shows two individual examples of the reproducibility of responsiveness to heat stimuli.

Most of the spino-PB neurons (75%, 40/53) were clearly NS, i.e., they were excited only by stimuli in the noxious range. The remaining spino-PB neurons were not so easy to classify. They could be activated by innocuous stimuli with a low responsiveness (mean response to brush: 4.6 ± 1.7 Hz, n = 13). When noxious stimuli were applied, these neurons had a much higher responsiveness, which, in fact, did not differ from that of the main group (see, however, encoding properties to thermal stimuli). They were then considered as nociceptive nonspecific neurons or wide dynamic range (WDR) to match the nomenclature in the current literature. The spontaneous activity of this population of WDR neurons is 0.08 < 0.36 < 3.25 Hz (10th percentile < median <90th percentile), i.e., there was a tendency to be moderately greater than that of the general population.

Interestingly, consistent with the lack of substantial "wind-up" in response to electrical stimuli, we observed that repeated stimuli of same intensity after 5-min intervals did not lead to increased responsiveness (Fig. 5, B and C).

Almost all the spino-PB neurons (92%, 49/53) were activated by both intense noxious thermal (50°C) and noxious mechanical (60 N · cm-2) stimuli. These stimuli gave rise to similar responsiveness of the spino-PB neurons, i.e., 40 ± 4.4 Hz (n = 45) and 40.5 ± 5 Hz (n = 51), respectively. In the group of neurons excited by both thermal and mechanical noxious stimuli, about a third (35%, 17/49) also responded to noxious cold stimuli (response >= 1 Hz). Responses to cold were much lower (7.6 ± 2.5 Hz; range, 1-43 Hz, n = 17) than those to noxious heat or mechanical stimuli. Examples of the encoding properties to cold stimuli of four of them are shown in Fig. 9, demonstrating that these neurons only responded to and encoded the intensity of noxious cold. Most (14/17) of the neurons responding to noxious cold stimuli did not respond to brush stimulation, thus were clearly of NS type.



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Fig. 9. Stimulus-response curves of some spino-parabrachial neurons to cold stimuli. Decreasing temperatures (30 to -20°C in 5°C step) were applied on the receptive field, each of them during 20 s with delays without stimuli >3 min between each temperature. Abcissa: stimulus temperature. Ordinate: mean frequency of response (see METHODS).

Only 8% (4/53) of spino-PB neurons responded exclusively to heat stimuli (heat specific). These neurons had a lower heat-evoked response (28 ± 8 Hz; n = 4) as compared with that of modality nonspecific neurons. Mechanical-specific, cold-specific, and innocuous cooling spino-PB neurons were not observed.

ENCODING PROPERTIES TO MECHANICAL STIMULI. The spino-PB neurons increased their firing rate when the pressure stimulus increased in the noxious range as shown in Fig. 10, A-C, for three neurons having different thresholds (5, 10, and 20 N · cm-2) close to and in the noxious range. The progressive increase in neuronal firing paralleled the increase in intensity of the noxious pressure, this was true for the whole tonic excitation throughout the stimulation period and the afterdischarge where present (Fig. 10, B and C).



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Fig. 10. Mechanical encoding properties of spino-parabrachial neurons. A-C: examples of encoding properties of 3 spino-parabrachial neurons having different thresholds (5, 10, and 20 N · cm-2, respectively). Graded mechanical stimuli were applied on the receptive field during the 20 s between the arrows; binwidth of histograms = 2 s. D: histogram of mechanical thresholds. Mean threshold was 11.5 ± 1.5 N · cm-2 (n = 39).

Pressure thresholds (n = 39) were distributed between 5 N · cm-2 (the lowest controlled pressure possible with our equipment) and 40 N · cm-2, with a mean of 11.5 ± 1.25 N · cm-2 (n = 39) clearly in the noxious range (Fig. 10D). Thus the majority (62%, 24/39) of the thresholds was clearly in the noxious range (>= 10 N · cm-2) the remainder (38%, 15/39) even if lower (<= 5 N · cm-2) were still close to the noxious range. Most of the neurons responding to brush stimuli (WDR) had a pressure threshold <= 5 N · cm-2, thus being clearly part of the low-threshold class discussed in the following text. A few of them, however, had higher pressure thresholds, ~10 N · cm-2.

Neuronal-encoding properties related to thresholds were analyzed by grouping individual stimulus-response curves by neuronal thresholds (4 groups with thresholds being: <= 5, 10, 20, and 40 N · cm-2, respectively). As shown in Fig. 11, all of the spino-PB neurons encoded pressure in the noxious range. In a few cases, this characteristic was relatively poor. Most of the individual curves were monotonic and positive <= 40/60 N · cm-2. The pattern of the stimulus-response curves was relatively homogeneous for the four groups. However, neurons in the lowest threshold group often exhibited a greater responsiveness (Fig. 11A) as compared with those in the higher threshold groups (Fig. 11, B-D). Furthermore in all groups the mean intensities of neuronal responses increased progressively and almost linearly with the logarithm of the pressure.



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Fig. 11. Stimulus response curves to mechanical stimuli gouped in accordance to the thresholds. A-D: stimulus response curves of spino-PB neurons with a low (5 N · cm-2), medium (10 N · cm-2), high (20 N · cm-2), and very high (40 N · cm-2) mechanical threshold, respectively. Insets: histogram of the thresholds, the class of threshold the curves presented being in black for each group. Thin lines: curves of individual neurons; thick line mean curve of the corresponding group.

The mean stimulus-response curve of the whole population (Fig. 12) demonstrated mean response intensities of 1.75 ± 0.5, 4.8 ± 1, 14.4 ± 2.6, 25.8 ± 3.8, 33.1 ± 3.9, and 37.1 ± 4.3 Hz ( n = 39), for pressures of 5, 10, 20, 40, 60, and 80 N · cm-2, respectively. The slope of the mean curve was low from 5 to 10 N · cm-2, then steeper and relatively constant from 10 to 60 N · cm-2 (with a weak maximum between 40 and 60 N · cm-2). Finally, the slope became a little shallower between 60 and 80 N · cm-2. Thus as well as the mean curves of the subgroups (Fig. 11, A-C), the mean curve of the whole population (Fig. 12) increased almost linearly between 10 and 60 N · cm-2 with the logarithm of the pressure. The mean p50 (pressure evoking 50% of the maximum response) was 30 ± 2.4 N · cm-2 (n = 39), thus emphasizing the involvement of these neurons in the processing of noxious mechanical information.



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Fig. 12. Mean stimulus-response curve to mechanical stimuli. Increasing mechanical stimuli were applied on the receptive field. open circle : mean response, error bars: SE. Abcissa: pressure in N · cm-2. Ordinate: mean frequency of response (see METHODS).

ENCODING PROPERTIES TO HEAT STIMULI. The firing rate of spino-PB neurons increased with increasing temperature of stimulation in the noxious range as shown for three individual examples (Fig. 13, A-C) of neurons with different noxious thresholds (44, 46, and 48°C, respectively). The response increase, primarily in the 46-50°C range, was maintained over the whole period of stimulation in addition to the afterdischarge in some cases (Fig. 13B).



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Fig. 13. Thermal encoding properties of spino-parabrachial neurons. A-C: examples of encoding properties of 3 spino-PB neurons having different thresholds (44, 46, and 48°C, respectively). Graded temperatures were applied on the receptive field during 20 s between the arrows; binwidth of histograms = 2 s. D: histogram of heat thresholds. Mean threshold was 43.6 ± 0.5°C (n = 38).

The mean heat threshold was 43.6 ± 0.5°C (n = 38). The thresholds of the individual spino-PB neurons were widely distributed (range: 38-48°C; Fig. 13D), the majority of the thresholds (60%, 23/38) were high (>= 44°C), i.e., in the noxious range. The remaining neurons (40%, 15/38) had lower thresholds, between 38 and 42°C.

Neuronal-encoding properties related to thresholds were analyzed further by grouping the neurons (n = 38) according to their thresholds and studying the individual stimulus-response curves (Fig. 14). All the neurons encoded the stimulating temperature in the noxious range. Most of the individual curves were monotonic positive up to 50°C; however, there was a tendency toward decreased responses at the highest temperature (52°C); the pattern of the individual stimulus-response curves were relatively homogeneous. As was the case for the mechanical stimulation, the group of neurons with the lowest thresholds (38-40°C, n = 7; Fig. 14A), although small in number, were the most responsive. The steepest portion of the mean curve of this group was in the 46-48°C interval. The midthreshold group of neurons (42-44°C, n = 17; Fig. 14B) showed homogeneous curves with minimally scattered responses. Finally, the highest threshold group (46-48°C, n = 14; Fig. 14C) was more heterogeneous and included numerous neurons with a broader range of responses for a narrow range of temperatures (mainly 46-50°C).



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Fig. 14. Stimulus response-curves to heat stimuli gouped in accordance to the heat thresholds. A-C: stimulus response curves of spino-PB neurons with low (38-40°C), medium (42-44°C), and high (46-48°C) thermal thresholds, respectively. Responses of individual neurons are presented with thin lines, the average stimulus-response curve is the thick line. In each case, the insert represents the histogram of the thresholds distribution, with in black the thresholds concerned by the curves.

Overall, the thermal stimulus-response function of the global neuronal population was smooth (Fig. 15). The mean stimulus-response curve of the whole population demonstrated mean response intensities of: 0.14 ± 0.07; 0.8 ± 0.4, 1.9 ± 0.7, 5.4 ± 1.7, 12 ± 3, 26 ± 4, 37 ± 5, and 43 ± 6 Hz (n = 38), in response to 38, 40, 42, 44, 46, 48, 50, and 52°C, respectively. This mean curve had a general sigmoid aspect; its slope increased progressively from 38 to 46°C, reached the steepest portion in the 46-48°C interval, then decreased weakly between 48 and 50°C and more clearly 50°C. The slopes of the different portions of this mean curve were significantly different (ANOVA test F7,3 = 9.5, P < 0.0001). The maximum value observed between 46 and 48°C (16 ± 2%/°C; n = 38) was significantly (P < 0.0001) greater than the slopes between 42 and 44°C (4 ± 1.5%/°C) and between 50 and 52°C (4.8 ± 3.3%/°C). The mean t50 (temperature evoking 50% of the maximum response) was of 47.4 ± 0.3°C (n = 38).



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Fig. 15. Mean stimulus-response curve to heat stimuli. Increasing temperatures were applied on the receptive field. open circle : mean response, error bars: SE. Abcissa: temperature in °C. Ordinate: mean frequency of response (see METHODS).

RELATIONSHIPS BETWEEN ENCODING PROPERTIES OF NEURONS TO NOXIOUS HEAT AND MECHANICAL STIMULI. Neurons were divided into two groups with respect to mechanical thresholds, low (<= 5 N · cm-2, n = 8) and high (>= 20 N · cm-2, n = 9), thus corresponding to the commonly adopted classification of WDR and NS cells. The encoding properties of these two neuronal groups to heat stimuli were compared. Low mechanical threshold neurons had a tendency to lower heat thresholds (42 ± 1.3°C; n = 8), whereas high mechanical heat threshold neurons had higher heat thresholds (45 ± 0.8°C; n = 9). In addition, the mean stimulus-response curves of the two groups to heat stimuli were significantly different (Fig. 16; ANOVA, F1,151 = 5, P = 0.027). Thus the low mechanical threshold neurons (WDR) had a higher responsiveness than the high mechanical threshold neurons (NS) for all the graded temperatures in the noxious range (>= 44°C).



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Fig. 16. Mean stimulus-response curve to heat stimuli of subgroups of wide dynamic range (WDR) and nociceptive specific (NS) neurons. open circle  and  correspond to WDR (low mechanical threshold <= 5 N · cm-2, n = 8) and to NS (mechanical threshold >= 20 N · cm-2, n = 9) neurons, respectively. Error bars: SE. Abcissa: temperature in °C. Ordinate: mean frequency of response (see METHODS).

Inhibition of spino-PB neurons by concomitant heterotopic noxious stimuli

Noxious heat stimulation (50°C) applied to the receptive field of the recorded lumbar neuron could be considerably inhibited by a noxious mechanical stimuli (60 N · cm-2) of the forepaw (Fig. 17A, 1 and 2). Heterotopic noxious stimuli (stimulation applied outside the receptive field of the recorded lumbar neuron) clearly inhibited the response to the control noxious stimulation of the great majority of the spino-PB neurons (13/16) studied. The percentage of inhibition was clearly different between neurons (Fig. 17B), but in most cases (9/13) the neuronal response was reduced to <50% of the control value. The mean intensity of the responses during heterotopic noxious stimulation was 31.7 ± 6.1% of control responses (n = 13).



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Fig. 17. Effects of heterotopic noxious stimuli on nociceptive responses of spino-PB neurons. A1: control response of a spino-PB neuron to noxious heat (50°C) applied during 20 s (between arrows) on the receptive field (1 toe). A2: response of the same neuron to the same stimuli when a 2nd mechanical noxious stimuli (60 N · cm-2) was applied simultaneously in another part of the body (the forepaw). Mechanical stimuli, started 10 s before, and finished 10 s after the heat stimuli. In this case (A2) we observed a marked diminishing of the response to heat stimuli; binwidth of histograms = 2 s. B: histogram of the effect of heterotopic noxious stimuli. Abcissa: remaining response in percent of the control. Ordinate: number of neurons in each class.

This phenomenon of heterotopic inhibition was maximum during concomitant stimulation, but decreased rapidly thereafter, rarely lasting more than a couple of minutes after cessation of the heterotopic stimulation. The level of inhibition did not seem to be dependent on the intensity of the control response, which indicates that whatever the amplitude of the initial responsiveness, the neurons seemed to have the same susceptibility to inhibition by heterotopic noxious stimuli.

We did not find any evidence for a difference between the different categories of spino-PB neurons (low or high mechanical threshold) when considering their susceptibility to inhibition by heterotopic stimuli.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Results were obtained from 53 neurons, all recorded in or close to the lamina I at the lumbar level of the spinal cord, and backfired antidromically from the contralateral PB area. All spino-PB neurons were excited by and encoded the intensity of noxious mechanical and/or thermal stimuli applied to their small receptive field. None of the spino-PB neurons were activated by innocuous stimuli only. However, some neurons were lightly activated by innocuous mechanical stimuli, as well as responding to noxious stimuli. A distinction between NS (75%) and WDR (25%) spino-PB neurons was made. All neurons had a very low spontaneous activity, close to silent, and exhibited two distinct periods of activation, corresponding to A- and C-fiber-evoked activity, respectively, in response to suprathreshold transcutaneous electrical stimuli.

Methodological considerations

ANESTHETIC REGIMEN AND IMMOBILIZING DRUGS. The anesthetic conditions used in the present study were the same as those used in all of the previous electrophysiological studies of the PB area in our laboratory (e.g., Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b). This point is of critical importance to ensure accurate comparison between responses of spino-PB and PB neurons. It must be emphasized that the precise measurement of the halothane concentration together with the endtidal PCO2 allowed good control of the stability and reproducibility of the anesthesia depth. Furthermore in our hands, this anesthetic regimen did not seem too depressive in terms of neuronal responses. This indeed allowed us to observe clear, and often strong, neuronal responses to light and/or noxious stimuli in different spinal laminae and several supraspinal nuclei (present results; see refs in Bernard et al. 1996; Besson and Chaouch 1987). Despite numerous advantages, our anesthesia regimen, like any other, inevitably induces some changes in comparison to the awake state. This issue is probably even more critical with other types of anesthesia regimen where the physiological assessments of the anesthesia quality and levels of drug delivery cannot be constantly monitored and accurately adapted.

Even if, in our daily experience, the main parameter of the anesthetic depth is the level of halothane (see also Kitahata et al. 1975), the opiate-related analgesic effect of nitrous oxide gas (Gillman 1986; see, however, Willer et al. 1985) could specifically affect the responses to noxious stimuli (Sonner et al. 1998). In our hands, considering the great responsiveness of numerous neurons to noxious stimuli, the analgesic effects of nitrous oxide did not seem to be very important. This could be due to the fact that the analgesic effect of nitrous oxide was counteracted, at least in part, by halothane as recently suggested (Goto et al. 1994, 1996). In addition, nitrous oxide as well as halothane could separately interfere with spinal sensitization (Goto et al. 1994; O'Connor et al. 1995) and thus diminish the number of sensitized NS neurons that consequently appeared as WDR-like. However, here again the N2O and halothane in association do not seem to interfere with the sensitization (O'Connor et al. 1995). In fact, the relatively low level of sensitization observed in the present study is probably more a credit to our paradigms where we only studied one neuron per animal each identified by antidromic activation first and where we avoided strong and repetitive stimuli.

Although very widely used as an immobilizing drug in electrophysiological experiments, it appears to some that gallamine triethiodide (flaxedil) could be responsible for important side effects because it is a potent blocker of potassium conductance (Smith and Schauf 1981). However, in vivo, the central effect of gallamine is limited considerably by the fact that it does not cross the blood brain barrier. Some authors evidenced however an excitatory effect of gallamine at the cortex and cuneatus nucleus levels in vivo (Galindo et al. 1968; Halpern and Black 1967). On the other hand, others showed that gallamine had no effect at the spinal cord, or cortical levels (Guilbaud et al. 1992; Jong et al. 1968). It appears, that under our experimental conditions---constant injection with doses just above the immobilizing concentration---gallamine has probably only limited excitatory effects. Indeed, the main putative side effect that could be attributed to gallamine---important and long afterdischarge, bursting and lengthening of spikes---were not especially noticed in the present study.

ANTIDROMIC ACTIVATION AND LOCATION OF SPINO-PB NEURONS. The PB area has been shown to be the densest field of projection of contralateral spinal lamina I neurons in rats, as demonstrated recently with the Phaseolus vulgaris leucoagglutinin (Bernard et al. 1995; Feil and Herbert 1995; Slugg and Light 1994). The selectivity of the projection origin from lamina I is supported further by studies using retrograde tracers (Bernard et al. 1989; Cechetto et al. 1985; Hylden et al. 1989; Kitamura et al. 1993; Lima and Coimbra 1989; Menétrey and De Pommery 1991; Menétrey et al. 1982, 1992; Swett et al. 1985; Yamada and Kitamura 1992). In the present study, most of the thresholds (83%) needed to evoke an antidromic spike were low (<= 300 µA) or very low (<150 µA), and all the stimulating sites were clearly located in the PB area, it is therefore highly probable that spino-PB neurons terminals were primarily stimulated. This confidence in the specificity of the antidromic stimulation for spino-PB neurons is reinforced by the personal observation that in some cases a current approx 1,000 µA did not spread up to 600 µm (the distance between 2 monopolar electrodes). This short distance of current spread is a little lower than indicated by Ranck (1975). Furthermore identifying well-isolated spikes that lasted long enough (>= 1 ms) and had a high enough amplitude (>= 2 mV) supports the somatic origin of the recordings (Lipski 1981). Consequently, as validated by the location of the Prussian blue points in the lamina I and sometimes in the Lissauer's tract, we can state that our recordings of neurons driven antidromically from the contralateral PB area were those of lamina I spino-PB neurons. However, because the axonal trajectory of spinothalamic neurons could be in the vicinity of the PB area (unpublished anatomic data from PHA-L injection centered in lamina I) (see also Mehler et al. 1960), one cannot exclude the possibility that the higher currents have stimulated spinothalamic neurons.

SELECTION CRITERIA. Neurons were selected primarily on the basis of the fulfillment of antidromic activation from the PB area. Consequently we have investigated the physiological characteristics of all of the recorded spino-PB neurons, and we were able to determine, with minimal bias, that all lamina I spino-PB neurons studied were excited by noxious stimuli. With the aim of avoiding central and peripheral sensitization, we have described only one neuron per animal, thus allowing us to report a homogenous neuronal population with homogenous properties.

Physiological properties of spino-PB neurons

CENTRAL CONDUCTION VELOCITIES. A wide range of central conduction velocities was observed. Because the slowest fiber we recorded had a conduction velocity of 2.8 m/s, we can assume that all the axons of the spino-PB neuronal population were myelinated. Previous studies have also described a wide range of central conduction velocities of spino-PB axons; however, a higher representation of low to very low conduction velocities were then reported (Hayashi and Tabata 1989; Hylden et al. 1985, 1986a,b; Light et al. 1993; McMahon and Wall 1985). Furthermore one study (Light et al. 1993), suggested that a substantial proportion (~40%) of axons of NS spino-PB neurons had very low conduction velocities in the unmyelinated range. The basis for these discrepancies may be due to species differences (Hayashi and Tabata 1989; Hylden et al. 1985, 1986a,b; Light et al. 1993). Furthermore in our experimental condition the few spikes evoked with very long latencies were not sufficiently stable to be recognized as antidromic. Consequently it is not possible to exclude the possibility that we missed a few antidromic spikes carried by very thin unmyelinated fibers. Interestingly, others have reported antidromic activation of unmyelinated lamina I axons with the use of long (2 ms) stimulus pulses (Craig and Kniffki 1985). Although, Light et al. (1993) have suggested that multireceptive (WDR) neurons have the highest conduction velocities, we did not find any evidence for a relationship between the central conduction velocity and the physiological properties of the spino-PB neurons.

RECEPTIVE FIELD. Spino-PB neurons responding from the distal territory of the sciatic or saphenous nerves had small receptive fields. This observation is consistent with earlier studies of spinal neurons of the superficial and deep dorsal horn, projecting either to the PB area, the thalamus or other undefined projecting targets in the rat, cat, and monkey (Christensen and Perl 1970; Hylden et al. 1985, 1986a,b; Kumazawa et al. 1975; Light et al. 1993; McMahon and Wall 1983; Woolf and Fitzgerald 1983).

SPONTANEOUS ACTIVITY. In our hands, spino-PB neurons had very low spontaneous activity, being close to silent. This physiological parameter of the lamina I neurons has rarely been investigated systematically but has been reported to be low (<2 Hz) in several species (rat, cat, and monkey), irrespective of the projection site of the neurons (Christensen and Perl 1970; Ferrington et al. 1987; Hayashi and Tabata 1989; Hylden et al. 1986a,b; Light et al. 1993; McHaffie et al. 1994; McMahon and Wall 1983; Meng et al. 1997; Woolf and Fitzgerald 1983).

PERIPHERAL ELECTRICAL STIMULATION. After intense electrical stimulation (3.1 ± 0.5 mA; n = 53) of the receptive field of all of the spino-PB neurons, at least two periods of activation, corresponding to peripheral A and C fibers, were observed. This is in agreement with earlier anatomic and electrophysiological work, showing that superficial laminae neurons are contacted monosynaptically by A and C fibers (Craig and Kniffki 1985; Fitzgerald and Wall 1980; Gobel et al. 1981). The constant coinnervation by A- and C-fiber inputs found in the present study was not consistently observed in earlier studies of spino-PB neurons that reported a dissociated innervation by either A- or C-fibers inputs (Hylden et al. 1986a,b; Light et al. 1993; McMahon and Wall 1983; Meng et al. 1997).

Considering the conduction velocities of Abeta , Adelta , and C fibers being >24 m/s, in the 2.5-24 m/s range, and <2.5 m/s, respectively (Handwerker et al. 1991; Leem et al. 1993a; Sanders and Zimmermann 1986), we have determined that the A-fiber group was made of a higher proportion of Adelta fibers, as observed in other studies (Light et al. 1993; McMahon and Wall 1983). In our study, all of the spino-PB neurons were activated by C fibers. In previous studies, a wide range of proportions (30-71%) of spino-PB neurons responding to only C-fiber stimulation have been reported (Hylden et al. 1986a,b; Light et al. 1993; McMahon and Wall 1983). These differences may result from the varying depths of anesthesia, which could, when deep, mask the weaker C components. Interestingly, among NS spino-thalamic units in the monkey, none were found to be activated exclusively by either A- or C-fiber volleys (Chung et al. 1979; Kenshalo et al. 1979; Price et al. 1978). In contrast, a dissociated innervation by Adelta or C fibers has been described in the cat and in the rat (Craig and Kniffki 1985; Woolf and Fitzgerald 1983).

RESPONSIVENESS TO NATURAL STIMULI. We have shown that all of the spino-PB neurons were nociceptive, the large majority of them (75%) being NS and the remainder WDR. None of the lamina I spino-PB neurons responded exclusively to innocuous stimuli; this is in agreement with some previous studies (Hayashi and Tabata 1989; Hylden et al. 1986a,b; Meng et al. 1997). However, the literature describes spino-PB neurons responding to innocuous stimuli only (Light et al. 1993; McMahon and Wall 1983). In the latter studies, the percentage of innocuous spino-PB neurons was low (3 and 12%). The 12% of innocuous spino-PB neurons found in the cat corresponded to neurons responding to innocuous cooling (Light et al. 1993).

The relative proportion of NS and WDR spino-PB neurons remains controversial because both high (Hayashi and Tabata 1989; Hylden et al. 1986a,b; Light et al. 1987, 1993) and low (McMahon and Wall 1983) proportions of NS neurons have been described. However, it appears that these discrepancies are probably more a semantic question than a physiological problem. For example, McMahon and Wall (1983) state that 51% of lamina I neurons responded to low level of stimulation, increasing their discharge in noxious range. However, two-thirds of these "WDR" neurons did not respond to the lightest form of stimulation and therefore correspond probably to the NS group in the present study. Thus it appears that, in previous studies as well as here, the majority of the spino-PB neurons are NS, or close to the NS group. The proportion of NS neurons within the lamina I spinothalamic group was found to be even higher in the cat because Craig and Kniffki (1985) reported only NS lamina I cells projecting to the thalamus, the WDR lamina I cells not being antidromically driven from the thalamus. On the other hand, in the monkey, the proportion of NS neurons within the lamina I spinothalamic group was found clearly lower (24-43%) the remainder being of WDR types (Ferrington et al. 1987; Kenshalo et al. 1979; Price et al. 1978).

In the present study, almost all spino-PB neurons (92%) responded to both noxious mechanical and noxious thermal stimuli and thus were bimodal. These findings are in agreement with some of the earliest studies of lamina I neurons (Christensen and Perl 1970; Dostrovsky and Hellon 1978) as well as with studies of lamina I spinothalamic neurons in the monkey (Ferrington et al. 1987; Kenshalo et al. 1979). However, in the cat, Craig and Kniffki (1985) reported a lower proportion (approx 65%) of neurons responding, at least, to both pinch and heat noxious stimuli, the other lamina I spinothalamic neurons responded to only one modality (pinch, noxious heat, or innocuous cold).

The few remaining spino-PB neurons (8%) responded to noxious heat only and had similar characteristics to those of the bimodal neurons. Similar noxious heat specific nociceptive neurons also were encountered, but in larger proportion (approx 20%) in the spino-thalamic tract of the cat (Craig and Kniffki 1985).

It would be tempting to suggest that the difference in proportion of NS versus WDR neurons is due to the projection of lamina I neurons either to the parabrachial area or to the thalamus. Unfortunately, it seems more likely that most discrepancies could be due to changes in mechanical testing (difficulties in reproducibility and calibration) and criterion used to define WDR neurons in different laboratories. Furthermore it seems clear that differences in anesthetic levels could be important at least on this criterion. The sensitization of NS cells or cells that respond only to noxious heat, pinch, and cold stimuli (HPC cells) that could become sensitive to innocuous mechanical stimuli after noxious stimulation is another factor of discrepancy that cannot be excluded (Craig and Kniffki 1985; Han et al. 1998; Woolf et al. 1994; see, however, Ferrington et al. 1987).

ENCODING PROPERTIES TO THERMAL STIMULI. This is the first study to systematically analyze the encoding properties of a well identified population of lamina I spino-PB neurons. The capacity of lamina I neurons of the dorsal horn to increase firing rates with noxious temperature was demonstrated in early studies, in the cat (Christensen and Perl 1970), monkey (Kumazawa and Perl 1978; Price et al. 1976, 1978), and rat (Menétrey et al. 1977, 1979). However, systematic studies of the thermal encoding properties of neurons were concentrated on lamina I neurons projecting to the thalamus in the monkey (Bushnell et al. 1984; Ferrington et al. 1987; Hayes et al. 1981; Hoffman et al. 1981; Kenshalo et al. 1979; Price et al. 1976, 1978) and the cat (Craig and Kniffki 1985; Craig and Serrano 1994) and neurons with unidentified projection in the rat (McHaffie et al. 1994). These previous studies classified nociceptive neurons into two main groups (NS and WDR) according to responses to mechanical stimuli. Therefore we first used the same classification (Fig. 16) to make an accurate comparison. In our study, the mean firing rate of WDR neurons was higher than that of NS neurons, which is in agreement with previous systematic studies of lamina I spino/trigeminothalamic neurons (Bushnell et al. 1984; Ferrington et al. 1987; Hayes et al. 1981; Hoffman et al. 1981). Furthermore in both WDR and NS neurons, the mean discharge increased progressively with temperatures from ~40 to 52°C.

The preceding analysis appeared at first sight a little paradoxical because individual responses of lamina I NS spino/trigeminothalamic neurons were sometimes described as shallow and/or not monotonically increasing with the temperature (Bushnell et al. 1984; Hayes et al. 1981; Hoffman et al. 1981). In fact, even in the latter studies, the mean curve (pooling all the individual discrepancies) increased clearly with the temperature in the noxious range. Thus Hoffman et al. (1981) in the awake monkey mentioned that some NS neurons (most recorded in superficial laminae) lacked monotonic stimulus-response functions to noxious-heat, whereas the mean stimulus-response function was found to be monotonic between 37 and 49°C. Bushnell et al. (1984) also presented some NS neurons with poor encoding properties between 45 and 49°C, nevertheless their mean stimulus-response curve was clearly monotonic in the investigated range (43-49°C). Thus the latter authors concluded that there was a positive relationship between thermal stimulus intensity and response frequency for both WDR and NS neurons, although the stimulus response curves were generally steeper and the response rate higher for the WDR than for the NS neurons. Interestingly, the curves we presented in Fig. 16 for WDR and NS neurons in anesthetized rats were very similar to those presented in Bushnell et al. (1984, Fig. 5-means curves) in awake monkeys and to those in Ferrington et al. (1987, Fig. 10B) in anesthetized monkeys. Although, precise comparison was a little more difficult, our results were also in agreement with those of Craig and Serrano (1994) in the spino-thalamic tract of cat. Finally, our mean results differ slightly from those of Kenshalo et al. (1979) in the monkey and of McHaffie et al. (1994) in the rat because these authors did not find differences between NS and WDR neurons.

Although for the purpose of comparative analysis we have separated NS and WDR spino-PB neurons, it is probable that this classification, based on their mechanical sensitivity, might not be the most appropriate for two reasons. First, the mechanical properties of the lamina I WDR found in the present study are close to those of lamina I NS neurons and probably not equivalent to those of the "lamina V WDR" neurons: in our study, the lamina I WDR responded weakly to innocuous stimuli and not at all to very weak stimuli. Interestingly, it was not possible to obtain a high response with a repeated vigorous innocuous stimulus, whereas this was observed for "lamina V WDR" neurons (Le Bars and Chitour 1983; Price et al. 1976, 1978; Wall 1967). Furthermore instead of a clear separation between NS and WDR neurons, we observed a continuum between neurons responding clearly, although weakly, to innocuous stimulus and those that did not fire at all even for a vigorous innocuous or weak noxious stimulus. Second, because very dense projections from lamina I spino-PB neurons converge from all of spinal cord segments onto PB neurons (Bernard et al. 1995; Craig 1995; Feil and Herbert 1995), it is very likely that a single PB neuron receives information from both WDR and NS lamina I neurons. The striking point is that all PB neurons are NS under normal conditions (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b). Thus to analyze the thermal properties of the lamina I spino-PB neurons, we have considered this neuronal population collectively. It is remarkable that the mean curve of the present study (Fig. 15) resembles very closely the equivalent curve obtained from PB neurons (see Fig. 10 in Matsumoto et al. 1996).

The comparison of the encoding properties of individual lamina I neurons (Fig. 14) with those of previous studies is much more difficult because of the strong interneuronal variability and the relatively small sample sizes. Nevertheless we observed a large variability of individual responses and a significant number (around one-third in our study) of neurons poorly encoded to temperature in the noxious range as described in several previous studies (Bushnell et al. 1984; Ferrington et al. 1987; Hoffman et al. 1981; Price et al. 1976, 1978). However, unlike these previous authors, it must be emphasized that we found a large majority (around two-thirds) of lamina I spino-PB neurons (with a high maximal responsiveness rate >30 Hz) clearly encoding the thermal stimuli in the noxious range, including numerous NS neurons (Fig. 14C). Our individual results, even if less homogenous, resemble those of the recent study of McHaffie et al. (1994) in the rat, which analyzed a large population of NS neurons mainly located in lamina I. We have shown that heat thresholds of the lamina I spino-PB neurons were scattered from 38 to 48°C and that even neurons with high-thresholds (46 and 48°C; Fig. 14C) have a high responsiveness and a monotonic stimulus-response curve up to 52°C. These data show that lamina I spino-PB neurons could transmit noxious thermal information not only through their individual encoding properties but also by the means of a progressive recruitment of the whole population. Indeed, from studies using noxiously evoked c-Fos expression at the spinal level, it is clear that the number of Fos-immunoreactive (Fos-ir) neurons in the laminae I and II of the dorsal horn increases in parallel with the intensity of the heat stimuli (Abbadie et al. 1994; Bester et al. 1997b; Hunt et al. 1987). Whereas electrophysiological techniques analyze the increase in firing rates of a single neuron to graded stimuli, the c-Fos technique describes the location and the increasing size of a neuronal population. In this regard, the anatomo-functional technique is in agreement with our results. It is likely that the number of Fos-ir neurons observed in the superficial laminae after a heat stimulation will depend on the number of cells having a heat threshold below the stimulating temperature. Overall, combining the benefits of these two approaches might further our understanding of the physiology of the laminae I neurons.

Previous studies in the awake monkey (Dubner et al. 1989; Maixner et al. 1986, 1989) demonstrated that WDR neurons (especially the subgroup of WDR1) have a greater capacity to discriminate differences between innocuous and noxious temperatures and to differentiate small temperature variations in the noxious range, as compared with NS neurons. We think this analysis is not necessarily contradictory to our demonstration of a large population of lamina I NS neurons providing a reliable information about thermal noxious events to the PB area level. Indeed it is tempting to hypothesize that the WDR1 could send, mainly to the sensory thalamus, very discriminative information. The ambiguity of this message (the WDR1 neurons also respond to innocuous mechanical stimuli) could be lifted collectively, in the thalamus, by the information arising from NS neurons. Considering this hypothesis, it is not surprising that the lamina I spino-PB neurons are essentially of an NS nature because the PB area is probably not involved in the most discriminative aspects of pain. Furthermore it appears logical that these lamina I NS neurons are able to collectively provide a reliable thermal noxious message to the PB area that could in turn induce appropriate autonomic and emotional responses to pain events.

ENCODING PROPERTIES TO MECHANICAL STIMULI. The present study is the first to give a detailed analysis of the mechanical encoding properties of lamina I spino-PB neurons. We have demonstrated using a semilogarithmic scale that spino-PB neurons increase their firing rate linearly in response to increasing mechanical stimuli over a very large noxious range (10-80 N · cm-2). Even though most earlier studies used brush, pinch, and squeeze as mechanical stimuli without building stimulus-response curves, they showed, as does the present study, that spino-PB, and spino(trigemino)-thalamic neurons increased their firing rate with increasing intensities of mechanical stimuli in the noxious range (Chung et al. 1979; Craig and Serrano 1994; Light et al. 1993; McHaffie et al. 1994; Price et al. 1976). The mechanical encoding properties of NS neurons located in the sacral dorsal horn was systematically studied in the rat by Cervero et al. (1988). The encoding properties of the spino-PB neurons we describe here resemble those of NS neurons previously described (Cervero et al. 1988). The mean mechanical threshold (11.5 N · cm-2) we observed in the present study using a stimulation area of 10 mm2 is comparable with the mean threshold (16.7 N · cm-2) observed in spino-thalamic neurons recorded in cats with a 3 mm2 probe (Craig and Serrano 1994).

COLD INFORMATION PROCESSED BY SPINO-PB NEURONS. In our hands, some spino-PB neurons responded moderately to cold stimuli, with thresholds on the border (20-15°C) or frankly in the noxious ranges (<0°C). None of the spino-PB neurons responded only to cooling in the innocuous range, whereas they increased firing rates when the temperature was lowered within the noxious range (down to -5 and -10°C). All of the cold activated neurons described in the present study also responded to mechanical noxious stimuli. These spino-PB neurons sensitive to noxious cold resembled the HPC neurons described by Craig and Kniffki (1985; see also Craig and Serrano 1994). These results contrast a previous study describing cooling specific spino-PB neurons in cat (Light et al. 1993) and spino-thalamic neurons in cat and monkey (Craig and Hunsley 1991; Craig and Kniffki 1985; Craig and Serrano 1994; Dostrovsky and Craig 1996). In these latter species, the innocuous cooling neurons are mostly of pyramidal shape, whereas the HPC and NS neurons are mostly of multipolar and fusiform shape, respectively (Han et al. 1998; Zhang and Craig 1997; Zhang et al. 1996). If we admit similar morpho-functional relationships in the rat, it is tempting to explain this discrepancy by the fact that pyramidal cells (potentially innocuous cooling neurons) project only weakly to the PB area in the rat (Lima and Coimbra 1989). Interestingly, the stimulus-response curves for cold stimuli of these spino-PB neurons are relatively close to those of PB neurons. However, it cannot be excluded that our sample missed these innocuous cold responsive neurons because of a relatively restricted searching window in the dorsal horn. Indeed although a particular distribution of innocuous cooling neurons was not demonstrated in the rat, some data in the medullary dorsal horn of the monkey were in favor of special location of this type of neurons (Craig et al. 1999). Finally, one cannot exclude a specific depressant effect of our anesthetic regimen on responses to innocuous cold, thus explaining the failure to observe neurons responding to that type of stimuli in our study. Indeed, N2O, used as a sole anesthetic drug, has been shown to change responses to cold in humans (Cheung and Mekjavik 1995). Nevertheless, this question of responses to cold stimulation would need further investigation devoted specifically to this particular but important point.

INHIBITION OF SPINO-PB NEURONS BY A CONCURRENT HETEROTOPIC NOXIOUS STIMULI. Our results showed that a large proportion of spino-PB lamina I neurons were subject to the effects of inhibitory controls generated from noxious stimuli applied far outside their receptive field. This phenomenon was very strong, virtually abolishing responses in some cases. Inhibition was obtained with different types of heterotopic noxious stimuli (thermal or mechanical) and was effective on the different modalities that activated the spino-PB neurons. Because it was possible to obtain inhibition with heterotopic stimuli delivered to all sites of the rat's body, it is probable that this inhibition corresponds to the diffuse noxious inhibitory controls (DNIC) (Dickenson et al. 1980; Le Bars et al. 1979a,b, 1981; Villanueva and Le Bars 1986; Villanueva et al. 1984). Previously, DNIC was described predominantly for WDR neurons located around the lamina V, whereas DNIC exerted considerably less effect on lamina I NS neurons. Our findings seem to extend the concept of DNIC, showing that lamina I neurons projecting to the PB area are inhibited greatly by heterotopic stimuli. These results are in keeping with a recent study showing WDR, as well as NS, neurons (in the spinal trigeminal nucleus) are sensitive to DNIC (Meng et al. 1997). These studies are supported indirectly by a study of c-Fos expression in the lumbar spinal cord level that showed a significant decrease in the number of noxious-pinch evoked Fos-ir neurons in lamina I by immersion of the tail in 50°C (Morgan et al. 1994). Thus the present results and previous literature suggest that the concept of DNIC preferentially acting on WDR neurons in the deep dorsal horn should be questioned, and this phenomenon extended to NS neurons of the superficial dorsal horn.

Relationship between spino-PB and primary afferent fibers

The electrophysiological data presented in the preceding text taken together with earlier studies of spino-PB neurons (Hylden et al. 1986a,b; Light et al. 1993; McMahon and Wall 1983; Meng et al. 1997) and data obtained from the rat foot (Leem et al. 1993a) suggest that lamina I spino-PB neurons primarily were innervated by Adelta and C fibers. This is in keeping with anatomic data showing that these fibers densely arborize in the superficial spinal laminae (Brown and Culberson 1981; Cervero and Connell 1984; Cervero et al. 1988; Culberson and Brown 1984; Gobel et al. 1981; Mizumura et al. 1993; Sugiura et al. 1986, 1988, 1989, 1993; Swett and Woolf 1985). In fact, Adelta and C fibers are involved in detection of noxious heat and mechanical and cold stimuli and also encode the intensity of the stimulation.

Responses of spino-PB neurons to noxious heat may essentially be driven by C-polymodal receptors the properties of which were shown to be similar to those of spino-PB neurons. Indeed, in the rat hindpaw, Leem et al. (1993a,b) have shown that the C-polymodal receptors are numerous, that their heat thresholds were in the 37-47°C range (many being in 43-47°C range), and that they encoded thermal stimuli in the noxious range (see also Long 1977).

Responses of spino-PB neurons to noxious mechanical stimuli chiefly would be driven by Adelta -mechanical and C-polymodal receptors. Indeed, Leem et al. (1993b), using a 20 mm2 probe (see their Fig. 5) have shown that Adelta - and C-mechanical cutaneous nociceptors are numerous and have clear encoding properties at least in the 1-6 N range (5-30 N · cm-2 range). These data support the mean stimulus-response curve obtained from spino-PB neurons, even if spino-PB neurons increased their firing rate over a larger range (5-60 N · cm-2). However, considering the difficulty of applying adequately calibrated stimuli to the paw, or toe of the rat, it cannot be excluded that the moderate discrepancies observed may mainly be due to methodological differences. The weak response of some spino-PB neurons to innocuous stimuli may result from activation of C mechanoreceptors (which are numerous and respond to innocuous stimuli) rather than from activation of Abeta fibers, which did not seem to substantially innervate these neurons.

Finally in our study, responses of spino-PB neurons to noxious cold may be driven by Adelta -mechano-cold, Adelta -cold, C-mechano-heatcold, and C-mechano-cold fibers as observed in the rat foot (Leem et al. 1993a). These receptors were shown to respond and encode the intensity of cold stimuli in the noxious range, with a wide range of thresholds (22 to -14°C). Furthermore, none of these peripheral fibers responded exclusively to noxious cold stimuli in the rat (Fleischer et al. 1983; Handwerker et al. 1991; Kajander et al. 1994; Kress et al. 1992; Leem et al. 1993a,b; Lynn and Carpenter 1982). These properties are reminiscent of those of cold spino-PB neurons, which all responded also to mechanical and/or heat noxious stimuli. On the other hand, as discussed in the preceding text, we have probably not encountered the specific cooling neurons which are probably driven by "cold responsive C fibers" with a maximum sensitivity in the innocuous range (27-17°C).

Spino-PB and PB neurons: functional considerations

Comparison of the physiological properties of spino-PB and PB neurons is a relevant question to our further understanding of the manner in which noxious information is processed along the dense pathways relaying in the PB area and terminating in the Ce, the VMH, and the bed nucleus of the stria terminalis (Aldén et al. 1994; Bernard et al. 1993, 1995; Bester et al. 1997a; Feil and Herbert 1995; Slugg and Light 1994). Anatomic studies emphasized that spinal lamina I neurons send a dense projection to the PB area (Bernard et al. 1995; Craig 1995; Feil and Herbert 1995; Slugg and Light 1994). Consequently, it is not surprising that the properties of spino-PB neurons were in many respects very similar to those already observed in the target PB area (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b; Huang et al. 1993; Matsumoto et al. 1996; Menendez et al. 1996). Thus at both levels of the spino-PB pathway, neurons had a very low spontaneous activity, responded only, or preferentially to noxious stimuli, encoded the stimuli intensities in the noxious range, and were activated by Adelta and C fibers at the periphery. At both levels, neurons had similar thermal thresholds: 43.6°C for the spino-PB neurons, and 44-44.3°C for PB neurons (Bernard and Besson 1990; Bester et al. 1995b; Matsumoto et al. 1996). Similarly, the mean stimulus/response curve has a maximal slope between 46 and 50°C for both the spino-PB and the PB neurons (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b; Matsumoto et al. 1996). In addition, the t50 were similar: 47.4°C for the spino-PB neurons, and 47.5-47.7°C for PB neurons (Bester et al. 1995b; Matsumoto et al. 1996). The similar encoding properties to heat stimuli at both levels of the spino-PB pathway also has been demonstrated by studies of the expression of the c-Fos protein (Bester et al. 1997b). Similarly, the mean mechanical thresholds at the spinal and PB levels were close, 11.5 and 15.8 N · cm-2, respectively (Matsumoto et al. 1996), with a comparable encoding of mechanical noxious stimuli over the 10-60 N · cm-2 range.

In contrast to this similarity in neuronal response profiles, the receptive fields of spino-PB neurons were very small cutaneous areas, whereas PB neurons had rather large receptive fields, often including some parts of the body, and in some cases the body in its whole (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b; Huang et al. 1993; Matsumoto et al. 1996). This observation suggests a huge convergence of noxious information arising from lamina I neurons all along the neuro-axis toward the PB neurons. Considering that some PB neurons have a complex receptive field---the simultaneous stimulation of two different parts of the body can diminish the response evoked from only one these two parts (Bernard et al. 1994; Bester et al. 1995b; Huang et al. 1993)---one can assume that this characteristic of these PB neurons could be linked, at least in part, to the inhibition of spino-PB neurons by heterotopic noxious stimuli (DNIC phenomenon).

The latter considerations suggest that the PB area is a region for integration of noxious information. Interestingly, a substantial number of PB neurons are modality specific. Thus for example 78% of the PB-VMH neurons responded exclusively or very preferentially either to mechanical or to heat noxious stimuli (Bester et al. 1995b) and about half of the PB cold neurons were excited only by cold (Menendez et al. 1996). In contrast, a huge majority of spino-PB neurons responded to at least two modalities of noxious stimulation (mechanical and/or heat and/or cold) with a less marked preference for one modality and only very few (8%) of them responded specifically to noxious heat. It appears clearly that the nociceptive region of the PB area can be considered as a center where noxious information originating in lamina I is extracted, modality-wise, and where further links with autonomic controls are engaged.

In conclusion, considering all the similarities between spino-PB and PB neurons, it is suggested that the nociceptive neurons in the PB area behave more or less like an extension of lamina I neurons of the spinal cord. However, the remaining discrepancies can reflect the fact that noxious information is not only transmitted along that pathway, but also processed, integrated, and modified before reaching higher centers. In the rat, lamina I neurons send their densest projections to the contralateral PB area (Bernard et al. 1995; Craig 1995; Feil and Herbert 1995; Slugg and Light 1994) and in turn the PB area further projects very densely to the amygdala (Bernard et al. 1993), hypothalamus (Bester et al. 1997a), and bed nucleus of the stria terminalis (Aldén et al. 1994). It has been shown that PB-Ce and PB-VMH neurons are NS with large receptive fields (Bernard and Besson 1990; Bester et al. 1995b), and their general physiological properties resemble those of the lamina I spino-PB neurons. Taking into account both the roles of the brain targets (amygdala and hypothalamus) and the sizes of the receptive fields of the PB neurons, these pathways relaying in the PB area are unlikely to be involved in the sensory discriminative aspects of pain that usually are thought to be dependent on the classically studied spino-thalamic tract (see references in Besson and Chaouch 1987). In contrast, according to earlier studies (Bernard and Besson 1990; Bernard et al. 1994; Bester et al. 1995b, 1997a,b; Huang et al. 1993; Matsumoto et al. 1996; Menendez et al. 1996), it is suggested that the nociceptive spino-PB pathway, which further relays noxious inputs to the amygdala, hypothalamus and bed nucleus of the stria terminalis, would be a major entry point for the integration of the autonomic and emotional/aversive aspects of pain.


    ACKNOWLEDGMENTS

We thank J. Martin for technical support and R. Rambur for photographs.

This work was supported by the Institut National de la Santé et de la Recherche Médicale and by the Institut UPSA de la Douleur.

Present address of V. Chapman: School of Biomedical Sciences, E Floor Medical School, Queen's Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK.


    FOOTNOTES

Address for reprint requests: H. Bester, INSERM U-161, 2 rue d'Alésia, F-75014 Paris, France.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 January 1999; accepted in final form 21 December 1999.


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