Involvement of solitary tract nucleus in control of nociceptive transmission in cat spinal cord neurons

323

Pain, 40 (1990) 323-331

Elsevier

PAIN 01545

Involvement

of solitary tract nucleus in control of nociceptive transmission in cat spinal cord neurons Huan-Ji

Shanghai Brain Research Instiiute, Academia Shanghai Medical

(Received

Du and Shi-Yi Zhou

*

Sinica, Shanghai (People’s Rep. of China), and * Department

of Physiology,

University, Shanghai (People’s Rep. of China)

19 April 1989, revision received 11 July 1989, accepted

2 October

1989)

Summary

In cats anesthetized with Nembutal and immobilized with Flaxedil, extracellular recordings were made from dorsal horn neurons and lamina X neurons in the lumbar spinal cord. The nociceptive responses of these neurons elicited by peripheral nerve stimulation were significantly inhibited by stimulation of the nucleus tractus solitarius (NTS) at low intensity without any noticeable cardiovascular reaction. As usual, the late response or C-response was found to be preferentially inhibited by NT’S stimulation as compared with the early response or A-response. The effective current intensity for NTS stimulation-produced inhibition ranged from 80 gA to 200 pA. Stronger inhibition was induced when the stimulating site was within or in the immediate vicinity of the NTS. There was no significant difference in the efficacy of the NTS stimulation-produced inhibition of nociceptive response between dorsal horn neurons and lamina X neurons. A similar inhibitory effect was elicited by microinjection of monosodium glutamate into the NTS area. The results demonstrate that the NTS may be involved in the control of nociceptive transmission at the spinal cord level. Key words:

Dorsal horn neurons; Pain modulation

Lamina

X neurons;

Nucleus

Introduction It has been well established that a number of medullary nuclei play important roles in the modulation of nociceptive information at the spinal cord level [4,7,21,22,25&t]. Of the medullary regions involved in this modulation, at least 4 nuclei have been extensively examined and recognized. They are the nucleus raphe magnus (NRM) [3,7,8,10,12-16,20,24], the nucleus reticularis magnocellularis (in cat) or the nucleus reticularis paragigantocellularis (in rat) [3,4,13,22,25], the

Correspondence to: H.-J. Du, Shanghai Brain Research Institute, Academia Sinica, 319 Yue-Yang Road, 200031 Shanghai, People’s Rep. of China. 0304-3959/90/.$03.50

0 1990 Elsevier

Science Publishers

tractus

solitarius;

Nociceptive

transmission;

Descending

inhibition;

rostra1 ventrolateral medulla including the nucleus reticularis paragigantocellularis lateralis [30,31] and the nucleus reticularis lateralis [23,35,41]. However, all of these nuclei are positioned in the ventral half of the medulla, and little is known about the role played by the dorsal medulla in pain modulation. Within this region, the nucleus tractus solitarius (NTS) may be related to antinociception for the following reasons: (i) histochemical studies have indicated that the NTS area is rich in opiate receptors [l] and all 3 members of the opioid peptide families, i.e., endorphins [27,39], enkephalins [40] and dynorphins [43]; (ii) the NTS has connections with NRM and the periaqueductal gray (PAG) [2,3,5,29] which play important roles in the control of nociception. The purpose of the present study was to define

B.V. (Biomedical

Division)

324

the neurophysiological characteristics of descending control of the NTS on nociceptive transmission of the cat’s spinal dorsal horn neurons and Rexed’s lamina X neurons. Lamina X neurons have been implicated in nociception [9,26,36,45] and they are known to be modulated supraspinally [45,46]. Abstracts of some portions of this study have already appeared [10,17,18].

Methods General procedures Twenty-three cats (2.0-3.5 kg) of either sex were used for the experiments. The animal was anesthetized with sodium pentobarbital (initial dose of 42 mg/kg, i.p.) and maintained during surgery and the experiment on supplemental doses (4-6 mg/kg, i.v.) when necessary. The anesthesia was considered to be deep enough if the pupils were maximally constricted and no changes in pupil diameter or blood pressure occurred during strong (30 V) peripheral nerve stimulation. Mean arterial blood pressure, end-tidal CO, and rectal temperature were monitored and kept within physiological limits. The lumbar spinal cord was exposed by laminectomy (L4-Sl). The medulla oblongata was approached by removing the occipital bone and by partial ablation of the cerebellum. The left superficial peroneal (SP), posterial tibia1 (PT), and sural or gastrocnemius nerves were dissected for stimulation. The exposed nerves were immersed in a warm paraffin oil pool. The temperature of the paraffin oil pool covering the exposed lumbar cord was maintained at 37 + 1” C by a temperature controller. Bilateral pneumothoracotomies were carried out to reduce the influence of respiratory movement on recording. The cat was then immobilized with gallamine triethiodide (Flaxedil) and ventilated artificially. Recording and data analyses Extracellular recordings were obtained from neurons in the dorsal horn (usually in laminae IV-VI) and central canal-surrounding gray (lamina X) at the L7 or L6 level, using glass micropipettes filled with 1 M sodium acetate and

2% Pontamine Sky Blue. The action potentials ol the neuron were amplified by a preamplifier (MEZ-8201) displayed on an oscilloscope (VC-10, Nihon Kohden), and stored in a TEAC data recorder. In the meantime, the signals were processed by a window discriminator and then fed into a microcomputer, which counted the total number of impulses over a given period of time and produced averaged rate histograms of the neuronal activity. The data were printed out for on-line evaluation. Peripheral stimulation Two or 3 hind limb nerves (SP, PT, sural or gastrocnemius) were separately or concomitantly stimulated with a train of electrical pulses (30-40 V, 0.2-0.5 msec, 3-5 pulses at 200 Hz) through platinum electrodes. The stimulus was delivered 5 times with intervals of 5 sec. The evoked compound action potentials from the nerves were monitored and compared with the discharge of the dorsal horn neuron from time to time. The stimulus intensity was supramaximal for excitation of unmyelinated fibers as estimated by the full-sized C-component of the evoked action potential. In the present study, the C-response (or slow A-response plus C-response, see below) of the spinal neurons elicited by nerve stimulation was used as a major indicator of nociceptive response. Sometimes, natural noxious stimuli, such as pinch (with forceps) or radiant heat (48-55” C), were also applied to the skin in the receptive field of the neuron tested. However. these data were not used in the evaluation of the change in the nociceptive response induced by brain stimulation because of the possible complications described by Duggan and Morton [19]. Indeed, NTS stimulation may cause particularly larger changes in peripheral sympathetic reactions (such as local microcirculation in the skin) than those produced by PAG stimulation [19]. These reactions might affect the excitability of cutaneous multimodal nociceptors. Medullary stimulation and microinjection According to the stereotaxic coordinates for cat brain-stem [6], 2 guided cannulae (0.9 mm OD) were placed such that the opening of each cannula was just touching the surface of the medulla above

the NTS and NRM areas, respectively. Bipolar stainless steel electrodes, insulated except for the tip and contained within a metal cannula (0.5 mm OD), were inserted via the guided cannulae and lowered to their target nuclei. Through these electrodes, repetitive constant current negative pulses (SO-450 PA, 0.2 msec, 100 Hz) were applied to the NTS or NRM area. The medullary stimulation was usually started 15 set before the nerve stimulation and lasted for 40 sec. The interval between 2 successive brain stimulations was not less than 5 min. For microinjection of monosodium glutamate (250 mM in 2 pl), the stimulating electrodes were replaced by an injection tube which had the same outer diameter and length as the stimulating electrodes. Histology The locations of the units tested were marked during the experiment by injecting negative current (10 PA, 5-10 min) through the dye-filled recording micropipette. At the conclusion of the experiment, positive current (1 mA, 5-10 set) was passed through the stimulating electrodes for the Prussian Blue reaction. The animal was routinely perfused and the medulla and lumbar cord were dissected out and cut into coronal sections (40 pm thick). All the sites of single unit recording and medullary stimulation or microinjection were determined histologically after staining the sections with neutral red (for spinal cord) or cresyl violet (for medulla). Details of the methodology have been described elsewhere [15,16,45,46].

Results Sample and characteristics of neurons Data were obtained from 63 neurons, 37 in the dorsal horn and 26 in lamina X. The neurons responded to the C-volley or to the A- and C-volleys from more than one of the hind limb nerves stimulated. A great majority of the spinal neurons were of the wide-dynamic-range (multireceptive) type. They could be excited by non-noxious stimuli (light touch of the glabrous skin and air puff

directed to the hair) and noxious cutaneous stimuli (skin pinching or heating) in the receptive fields. The lamina X neurons mostly had extensive receptive fields as characterized previously [9,45]. A small population of spinal neurons (n = 3) which responded only to noxious stimuli were also seen in this study. All of the spinal neurons responded to hind limb nerve stimulation, generally with a dramatic increase in the discharge rate; however, a remarkable population of neurons, especially in lamina X, reduced spontaneous firing in response to natural stimuli applied to the skin and to C-volleys from the somatic nerves. For easy evaluation of the effect of NTS stimulation, it was decided to test only those neurons with an increased discharge rate during nociceptive stimulation. As has been shown in previous studies [15,44], the nociceptive response in a number of spinal neurons is differentiated into two components (see Figs. lD-F and 3B). A fast short-lasting component (latency was shorter than 4 msec), which was maximally activated by stimulation of the nerve at an intensity of 2-3 V and lasted for lo-20 msec, was associated with the A-volleys, perhaps originating mainly from activation of the lowthreshold Aq /3 and part of the fast-conducting AS fibers. This component was termed early response or just A-response for easy comparison with those of previous studies. A late long-lasting component (with latency longer than 100 msec), which consistently occurred with the C-volleys (due to the C-fiber activation) and was sensitive to morphine or y receptor agonist (unpublished observation), was termed late response or C-response. The total number of spikes of the clear-cut C-response was counted 100 msec after the stimulus artifact for 0.5 set or 1 sec. More often (in 40/63 neurons studied), however, an intermediate component, i.e., slow short-lasting component, was interposed between the two components mentioned above, which might be associated with the excitation of high-threshold and slow-conducting A6 fibers. This component would mix the fast A-response with the C-response (as seen in Figs. lA-C, 2A and 5), especially when a longer bin width was used. In this circumstance, we counted

326

l-A--l-kPT

Set ’

Set 2

PT

G Pinch

Puff

-

-

NTS

NTS

Fig 1. Effects of stimulation of NTS on the responses of spinal cord neurons. Each peristimulus time histogram was constructed from 5 superimposed records. A-C: the A- and Cresponses from a dorsal horn neuron before (A), during (B) and after (C) NTS stimulation. D-F: responses from a lamina X neuron. G: a pen-recorder record of neuronal firing (after passing a window discriminator). Left, response of a dorsal horn neuron to persistent pinching of skin, applied to the toe pad. Right, response of the same neuron to continuing puff movement of the hair around the toe pad. NTS stimulation (200 PA) was delivered as indicated by the bar underneath. Marking points at the recording and stimulating sites are illustrated in the insets in A and B, respectively. Abbreviations: PT. posterior tibia1 nerve stimulation (30 V); X, lamina X.

the total number msec, depending

of spikes with a delay of 20-50 upon the bin width used.

Inhibition of activity of spinal cord neurons by NTS stimulation The effect of NTS stimulation was estimated in 37 dorsal horn neurons and 26 lamina X neurons after their nociceptive responses (i.e., C-response or slow A- and C-responses) had been stable in 2 or 3 successive trials (each trial was composed of 5 repeated stimuli to the nerve). The effect induced by brain stimulation was defined as inhibitory only when the total number of the spikes of the nociceptive responses was reduced by an average of 20% or more as compared with the control. It was found that repetitive NTS stimulation showed

a marked inhibitory effect in 31 out of 37 (83.8%) dorsal horn neurons. According to 25 units whose total spike number was counted throughout the test. the nociceptive responses were inhibited to 46.5 1_ 21.6% (mean + S.D.) of the control. A typical example is shown in Fig. IA---C. No significant inhibition was seen in other 61’37 neurons (16.2%). In some instances. the late response or C-response was nearly completely inhibited (Figs. 1B and 3C). The inhibitory effect was usually maintained for a few minutes after the NTS stimulation. In addition. the high-rate spontaneous discharge of the neuron was often depressed by the brain stimulation. The NTS stimulation was also shown to produce an inhibition of nociceptive response to 54.7 ) 21.4% of the control in 1 X/26 (69.2%) lamina X neurons tested (Fig. 1D-F). There was no change of the nociceptive response in 6 and increased nociceptive response (i.e., facilitation) in 2 neurons. The efficacy in producing inhibition by NTS stimulation in these neurons appeared to be relatively weak as compared with that in the dorsal horn neurons. The data are summarized in Table 1. Selective inhibition of nociceptive responses In general, the C-responses of both the dorsal horn and the lamina X neurons were predominantly depressed by the NTS stimulation, whereas the A-response, especially its fast component, was less affected (see Figs. 1, 2, 3 and 5). The selective inhibition of NTS stimulation on the nociceptive response produced by nerve stimulation was further confirmed in the experiments in which innocuous and noxious cutaneous stimuli were em-

TABLE

I

INHIBITION OF NOCICEPTIVE RESPONSES OF SPINAL CORD NEURONS INDUCED BY NTS STIMULATION -. Location of neuron

Number

NTS stimulation Inhibition

Facilitation

Dorsal horn Lamina X

37 26

31 (83.8%) 18 (69.2%)

0 (0%) 2 (7.7%)

6 (16.2%) 6 (23.1%)

Total

63

49 (77.8%)

2 (3.2%)

12 (19.0%)

No effect

-_

-

321

sity was used in order to avoid the blood response.

Set D

NTSIBOuA)

$50

g 50I

IIIIIIII

PT

5

Fig. 2. Inhibition of nociceptive neuronal response by low-intensity NTS stimulation. Repetitive NTS stimulation at 80 PA caused a 36.7% decrease (B) in the total number of spikes of a lamina X neuron in response to C-volleys produced by F’T stimulation. The intensity for causing a detectable inhibition was about 60 PA in this neuron. D: a simultaneous blood pressure record, showing that the NTS stimulation which induced inhibition did not change the mean blood pressure.

ployed in the same neuron (n = 3). The neuronal responses caused by hair deflection or light touch of the skin were not changed during the NTS stimulation. By contrast, the responses elicited by skin pinching or heating of the toe pad were significantly reduced (Fig. 1G). Change in blood pressure produced by NTS stimulation As usual, repetitive NTS stimulation with a current lower than or just at 200 PA would not cause any remarkable fluctuation in the mean blood pressure level (as shown in Fig. 2D). When the intensity of NTS stimulation was over 300 PA, the blood pressure sometimes decreased or increased by 15-20 mm Hg or more. However, this fluctuation did not seem to be related to the occurrence or the degree of the NTS stimulationproduced inhibition. Even so, anesthetics were routinely supplemented or a lower stimulus inten-

pressure

Effect of parameters of NTS stimulation The threshold intensity of NTS stimulation for inducing inhibition was examined in some experiments. In general, 60-70 PA was found to be the intensity to elicit a detectable inhibition of nociceptive response in the spinal neuron. Fig. 2 demonstrates that NTS stimulation of 80 PA could reduce the PT response (B) to 63.3% of its control without any change in the blood pressure (D). Stronger stimulation caused stronger inhibition as illustrated in Fig. 4A. At a proper intensity (e.g., 200 PA), even a single train (100 msec in duration) of pulses was enough to induce a significantly inhibitory effect (Fig. 3B), and a longer train (500 msec) resulted in a complete inhibition of the C-response in this neuron (Fig. 3C).

20

1

A

:1-L1

B NTS(200uA,lOOms)

-

Fig. 3. Comparison of inhibition induced by NTS stimulation with different pulse train duration. A short tram of pulses (100 msec, 100 Hz, 200 PA) applied to the NTS produced apparent inhibition of nociceptive response of a lamina X neuron (B) as compared with the control (A and D), and a longer tram (500 msec) caused complete inhibition of the C-response (C). Note that the C-responses were selectively depressed. Five records superimposed.

32X

Specificity of stimulating sites In some experiments, the stimulating electrodes were inserted, step by step, from the surface of the medulla to cover the NTS and its adjacent neurol structures, as depicted in Fig. 4A. The effective sites for producing a marked inhibition were located within the NTS area. This was also confirmed by the distribution of the stimulating intensities at different depths (Fig. 4A) necessary for inducing a significant inhibition. Stimulation of the NTS, especially its medial half, was most effective, as indicated in Fig. 4B. Effect of microinjection of monosodium glutamate (Na-Glu) into NTS In 3 spinal neurons, after an NTS stimulationproduced inhibition had been observed the stimulating electrode was substituted by an injecting

AlT

G\

PT

Fig. 5. Effect of monosodium glutamate (Na-Glu) on nociceptive response of a dorsal horn neuron. A-C: inhibitory effect of NTS stimulation as a control. D-E: inhibitory effect of Na-Glu microinjection in NTS. For details see text.

tube, and Na-Glu (250 mM) was then microinjetted into two loci (1 ~1 each) of the NTS, separated vertically by 1 mm. The application of Na-Glu induced a marked inhibition with a 67.9% decrease in the total spike number of nociceptive response (Fig. 5E), which was comparable with that of electrical stimulation (75.0% decrease, Fig. 5B). However, microinjection of NaCl solution (250 mM, 2 x 1 ~1) had no effect (not shown).

Discussion

Fig. 4. Comparison of inhibitory effects of varied stimulating loci and different current intensities. A: stimulating electrode was inserted downward with 0.5 mm steps (on the left), and the inhibitory effects produced by stimulation of these loci (200 CA) were expressed as a percentage of the control (on the right). B: effects of stimulating intensities. The current intensities, at which the NTS stimulation induced a significant inhibition (i.e., a more than 30% decrease in nociceptive response), are depicted by different symbols: filled circle, not more than 200 PA; half-filled circle, 250-450 PA; open circle, more than 450 p A. Abbreviations: AP, area postrema; G, nucleus gracilis; X, dorsal motor nucleus of vagus; XII, hypoglossal nucleus.

The present study reveals that the activity of spinal neurons, including dorsal horn and lamina X neurons, can be inhibited by electrical stimulation of the NTS area. It is unlikely that this inhibition is due to excitation of the fibers running through the NTS or to the current spread to nearby structures because chemical stimulation (Na-Glu microinjection) causes a similar inhibition. Also, the possibility that changes in the central blood pressure and/or peripheral blood flow might be related to the inhibitory effect [19] could be ruled out, as described above. An inhibition, predominantly on the responses elicited by both C-input and noxious cutaneous stimuli, was seen following the NTS stimulation.

329

This differential effect is in agreement with those observed in experiments with PAG and NRM stimulation [4,22,24,44]. Despite the great discrepancy among the results from different laboratories, comparison of stimulation-produced inhibitory effects on noxious- and non-noxious-evoked spinal neuronal responses indicates that descending influences on nociceptive responses of spinal neurons are more marked than those on nonnociceptive responses [for review see 221. It is possible that the partial inhibition of the A-reobserved in our experimental circumsponse, stances, may have been due to a selective depression of the slow component of the A-response elicited by high-threshold A6 inputs. It has long been believed that the NTS participates only in a variety of autonomic functions. In the early 1980s this conception was first challenged by Oley et al. [37]. They presented evidence that administration of morphine into the NTS region produced behavioral analgesia in the rat, suggesting that the NTS is involved in the morphine-sensitive pain-suppressing system. However, this result was recently questioned by Morgan et al. [34] who failed to produce analgesia following morphine microinjection into the NTS, and thus attributed Oley’s result to an overdose of opiate or the diffusion of drug into the ventricular system. On the other hand, the latest evidence showed that electrical stimulation of the NTS produced analgesia [28,34]. Furthermore, Ran et al. [38] reported that stimulation of the unmyelinated fibers of the rat vagal nerve caused a behavioral analgesia; Menetrey and Basbaum [33] revealed the projections from the spinal lamina X area and the superficial laminae of the trigeminal nucleus caudalis to the NTS, which are thought to be responsible for the nociceptive transmission. These data imply that the NTS might play a role in nociceptive modulation. In the present study, we have provided electrophysiological evidence to support the argument that the NTS may take an active part in not only the regulation of autonomic functions, but also in the modulation of nociceptive transmission at the spinal cord level. It seems to us that nociceptive responses of the spinal relay cells, including dorsal horn and lamina X neurons, are controlled concomitantly, although to a differ-

ent degree, by the descending activities originating in a number of medullary nuclei. With regard to the mechanisms underlying the NTS stimulation-produced antinociceptive effects, little is known at the present time. Pharmacological studies conducted in our laboratory, which will be reported in detail elsewhere [47], demonstrated that the inhibition of spinal nociceptive response induced by NTS stimulation is naloxone reversible but not yohimbine sensitive, suggesting that this effect is mediated by opioid receptors but not a,-adrenoceptors. The exact site of the action of these antagonists was not determined. The NTS is known to be one of the medullary regions which have complicated connections with many parts of the central nervous system [29,42]. However, there is no evidence for a direct NTS projection to the spinal dorsal horn and lamina X regions [11,29]. On the other hand, it is interesting that almost all of the regions which have been shown to be involved in the control of nociceptive transmission in the spinal cord have 2-way connections with the NTS. Among these are two major neural structures, the PAG [2,29] and NRM [3,5,29], which take an active part in the descending modulation of spinal nociceptive transmission [4,7,12-15,21, 22,24,25,44]. Thus, the possibility that the NTSmediated inhibition is relayed in the PAG or NRM must be considered. The NRM was tested by microinjection of novocaine, and a partial and reversible blockade of NTS-mediated inhibition was observed [47]. Based on these studies, it appears reasonable to suggest that the inhibition induced by NTS stimulation is mediated by opiate receptors, and that its efferent pathway is partly relayed in the NRM and/or the adjacent reticular formation.

Acknowledgements

We are grateful to Mrs. Y.F. Zhao for histological assistance, and Mr. Q. Lan for photography. The study was supported by Grant No. 85030520 from the National Natural Science Foundation of China.

330

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