Brain Research, 124 (1977) 437-448 ,:~, Elsevier/North-Holland Biomedical Press, Amsterdam - Printed in The Netherlands

437

RESPONSES OF T H O R A C I C D O R S A L H O R N I N T E R N E U R O N S TO CUTANEOUS S T I M U L A T I O N A N D TO T H E A D M I N I S T R A T I O N OF A L G O G E N I C SUBSTANCES I N T O T H E M E S E N T E R I C A R T E R Y IN T H E S P I N A L CAT

G. GUILBAUD*,***, G. BENELLI** and J. M. BESSON*** Laboratoire de Physiologie des Centres Nerveux, UniversitF Pierre et Marie Curie, 4, avenue Gordon Bennett, 75016 Paris (France)

(Accepted July 28th, 1976)

SUMMARY The effects of the injection of algogenic substances (bradykinin, acetylcholine) into the inferior mesenteric artery were studied at the thoracic level on 47 dorsal horn interneurons responding to cutaneous stimulation. Each unit was characterized by its electrophysiological properties and carefully located within the cord by extracellular injection of pontamine sky blue. Twenty cells, driven only by non-noxious cutaneous stimulation and mainly located in lamina IV, were not affected by the administration of algogenic substances. The activity of 25/27 cells, excited by both non-noxious and noxious cutaneous stimulation and mainly located in lamina V, was strongly modified by nociceptive visceral stimulation, induced by bradykinin and acetylcholine: 8/27 cells were activated, 14/27 were inhibited, 3/27 had a mixed inhibitory-excitatory response. From our study it clearly appears that nociceptive visceral messages only project on dorsal horn cells receiving noxious cutaneous afferents. Thus viscerosomatic convergence seems only to concern nociceptive messages; the existence of this kind of convergence reinforces the hypothesis suggested by several authors to explain referred pain from a neurophysiological point of view.

INTRODUCTION Viscerosomatic convergences have been described at various levels of the CNS" spinal cord, reticular formation, thalamus and cortexl,Z,9,at,14Aa,17,z1,z2,e4,ao,32-a4,37, * Chercheur 1.N.S.E.R.M. Present address: Zambon Laboratory, Bresso, Milan, Italy. * ** Present address : Groupe de Recherches de Neurophysiologie Pharmacologique, U 161 1NSERM, 2, rue d'A16sia, 75014 Paris, France. **

438 as. in most cases the visceral inputs were of very brief duration as Ihe2~ ~.,c~c m;tip, iy induced by splanchnic nerve or sympathetic chain stimulation. In the [;i-e>cm stud, we will consider certain spinal dorsal horn mechanisms of visceral pare i~ the c~it: this will be done by producing experimental conditions which are knox~ io reduce intense nociceptive reactions of long duration in animals and to prow,ke durable painful sensations in man. For this purpose activities of dorsal horn i~terneuro~> were recorded in spinal preparations at the thoracic level during injectioll ,~i cdgogemc substances (brakykinin, acetylcholine) into the inferior mesenteric after 3 Several studies carried out at various levels of the CNS, using it~tra-.arterial injection of bradykinin into the limbs, have al,'eady been reported by our group :,:~.~', 19,29

METHODS

This study was carried out on 17 cats weighing 1.8--3.2 kg. Animals were prepared under deep halothane anesthesia. They were immobilized by gallamme triethiodide (Flaxedil) and artificially ventilated. A spinal cord section was performed at the ('~ level, carotid and vertebral arteries being previously ligated or clamped. Arterial blood pressure, rectal temperature and end-tidal COe were continuously monitored. Femperature was maintained at about 38 '-'C, end-tidal CO,, was kept between 4 and 4.5 "~,. and arterial pressure was always higher than 80 mm Hg. A laparotomy was performed to take out a section of small intestine with the corresponding portion of mesentery. A small catheter was introduced upstream into a collateral of the inferior mesenteric artery. The permeability of the catheter was maintained by a continuous and ver\, slow infusion of physiological fluid (2-4 ml/h). Then the intestine was replaced and the peritoneum and skin were sewn. The cat was placed in a stereotaxic apparatus and a laminectomy was performed from T6 to TIi. The dura mater was opened and the exposed cord was covered with warm mineral oil. The troublesome effects of respiratory movements, very numerous at this level, were reduced in two ways. (a) A 'tank' was realized by the dura mater, folded back and maintained with threads: and (b) a double pneumothorax was systematically created at the beginning of the e.xperiment Recordings were performed 3 or 4 h after elimination of the volatile anesthetic. Extracellular unitary recordings were made using glass micropipettes filled with KCi and a solution of pontamine sky blue; electrode resistance was of 3- 6 M t2. Cells were characterized according to the electrophysiological criteria described by WaltZ% Their location within the dorsal horn was established by extracellular injection of pontamine blue (Fig. 1). In order to carefully specify the size of the cutaneous receptive fields. abdominal and thoracic hairs were shaved. The discharge frequency of units wa~ recorded by means of a spike integrator (time base, 2 sec). Units, the amplitude or' which changed during bradykinin injection, were eliminated. Synthetic bradykinin(Sandoz: 5-20 7) or acetylcholine (5-10 7) was injected into the inferior mesenteric artery. through a 3-way tap in 1 ml solution. Control injections were made with an identical volume of physiological fluid. Injection of methylene blue solution at the end of the experiment permitted a check on the site of injection and the satisfactory irrigation

439

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Fig. 1. Histological section of the thoracic spinal cord showing an extracellular coloration by pontamine blue.

of the intestine; cats were then anesthetized with pentobarbital sodium (40 mg/kg i.v.), the spinal cord was removed and placed in 10~ formol-saline. Serial 100#m sections and staining by cresyl violet or safranin was made for verification of recording sites. RESULTS

General Jindings Sixty-eight cells were recorded at the thoracic dorsal horn level; only 47 had sufficiently long and steady records to test the effects of the administration of algogenic substances. Twenty units classified as non-noxious presented the characteristics of lamina IV type cells. They were only activated by light cutaneous stimulation such as touch, tap, brushing or air jet. Their peripheral receptive fields were of very limited size (less than 1 sq. cm) and very often included few hairs. These fields were located on the thorax (65/o) o/ Stimulation gave short duration bursts of °~ or on the abdomen (35 /o)activity which were rapidly adapting.

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\ Fig. 2. Schematic reconstruction of recording sites, tn A, cells are classified according to the properties of their peripheral cutaneous excitatory receptive fields; in B, according to the effects induced by administration of bradykinin into the inferior mesenteric artery.

Twenty-seven units excited by both non-noxious and noxious stimuli presented the characteristics of lamina V type cells. They were driven by light cutaneous stimuli and their discharges were increased when the stimulus became noxious (intense pinches). These cells were driven from wide receptive fields: 32 ?;, were ol 3 4 sq. cm and 68 i!~i were larger than 4 sq. cm (maximal 27 sq. cm). These fields were located on the thorax (37 ",,) or on the abdomen (63",,). For this group 16/27 units presented an inhibitory cutaneous receptive field which surrounded or was adjacent to the excitatory one. As shown in Fig. 2, the cells of the first group were essentially located in lamina IV while the cells of the second group were deeper, located mainly in lamina V.

TABLE 1 E ~ ' c t s o / bradykinin injection into the inlerior mesenteric artery upon 47 dorsal hort~ ce/A

The numbers indicate the number of cells in each category. Non-noxious and noxious toll/.,,

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Fig. 3. The activity of a lamina IV type cell only driven by light touch is not affected by the administration of algogenic substances.

Effects of bradykinin and acetyleholine injections into the inferior mesenteric artery As shown in Table I, the effects induced by the administration of algogenic substances depended upon the nature of the cutaneous stimulation which was able to drive the cells.

Non-noxious units The 20 units of this group were not affected by bradykinin. Acetylcholine injection tested on 6 of these 20 units was also ineffective. (Fig. 3).

Non-noxious and noxious units The activity of 25/27 cells of this group was affected by bradykinin. Three kinds of efl~cts were observed: excitatory, inhibitory and mixed. For these cells the injection of the same volume of physiological fluid was ineffective. Excitatory effects. Eight out of 27 cells were excited. As shown in Fig. 4, they were also strongly excited by natural nociceptive stimulation applied within their large peripheral cutaneous fields; they never presented inhibitory fields. The latency and the duration of these effects were variable from one cell to another. The latencies were between 11 and 20 sec; the durations of the increase in firing rate were between 40 and 60 sec. The degree of activation was also variable between cells, generally between 50 and 150%. As shown in Fig. 4 for the same unit the activation due to bradykinin was easily reproducible. However, in some cases, the degree of the increase of the firing rate presented small fluctuations between injections without clear tachyphylaxis. Due to these fluctuations and to the difficulty in maintaining long duration recordings during repeated administration of bradykinin, no systematic investigations were performed to consider the dose relationship effect. Nevertheless large doses of bradykinin seem to be more effective (see Fig. 4). Inhibitory effects. Fourteen of 27 cells were inhibited after bradykinin administration (Fig. 5). As in the previous group, large variations in duration of the effects

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Fig. 4. Excitatory effects induced by successive administration of bradykinin and acetylcholine on the activity of a lamina V type cell. Note that this cell was strongly excited by pinches (1 and 2) applied within the excitatory field. were found between cells. The latencies were between 10 and 14 sec; these inhibitory effects were always well marked (Fig. 5) and in several cases the spontaneous firing rate was even totally abolished. These cells were strongly excited by noxious stimuli applied within their excitatory receptive fields; they had similar properties to those described for excited cells but in the majority o f cases (12/14 cells) these units presen ted a clear inhibitory cutaneous receptive field. Inhibitory fields were large; they surrounded the excitatory fields or were adjacent to them but in several cases they were more removed. Excitatory and inhibitory fields belong to the same dermatomes but in most cases the inhibitory areas spread to several dermatomes and can even reach the

443

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Fig. 5. Inhibitory effects of bradykinin and acetylcholine upon the spontaneous activity of a lamina V type ceil. Note that this cell was strongly activated by pinches applied in the excitatory field (E.F.) and inhibited by cutaneous stimulation applied within the inhibitory field (I.F.).

444 hip. Light cutaneous repetitive stimulation was generally sufficient to induce a clear inhibition of the spontaneous firing rate; more intense stimulation was only necessary in a few cases. Mixed effects. Three ceils presented a mixed inhibitory-excitatory response to bradykinin administration. The depression of spontaneous activity always preceded the excitatory effect (Fig. 6). These three cells presented a wide inhibitory cutaneous receptive field. Effects of acetylcholine. The modifications induced by acetylcholine were considered for 17 of the 27 cells of this group; 8 cells were excited and 9 inhibited, in all cases the effects of acetylcholine were of the same type as those induced by bradykinin (Figs. 4 and 5). Generally the modifications induced by acetylcholine were instantaneous and sometimes occurred during the injection itself; the importance of these effects was of the same order as that observed with bradykinin but their" duration was usually shorter.

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Fig. 6. Mixed inhibitory-excitatory effects induced by bradykinin on the activity of a lamina V type cell.

445 DISCUSSION The results of this study, which considered the modifications of activity of dorsal horn interneurons during intense nociceptive stimulation, are in good agreement with previous results using splanchnic and sympathetic chain stimulation 17,zl, 22,34,37,38. Indeed from the present work, it clearly appears that the interneurons characterized as lamina IV type cells and which were only driven by light cutaneous stimulation from small receptive fields were never affected by intense visceral stimulation. Similar findings have been reported at the thoracic level with splanchnic nerve stimulation 17,34 and at the lumbar level by stimulation of the sympathetic chain 37. Activation of a small number of lamina IV type cells by splanchnic volleys was only reported by Hancock et al. 22. In contrast the great majority of cells 25,27 characterized as lamina V type cells, and which were driven from larger receptive fields by both noxious and non-noxious cutaneous stimuli, were affected by the intense visceral stimulation. The difference in the effects of algogenic substances on the two types of units can be explained by the fact that lamina IV type cells do not receive visceral afferents; indeed this was clearly demonstrated by stimulating the splanchnic nerve and the sympathetic chain17,34,37, 38. These results are in good agreement with previous studies which indicate that the large splanchnic afferents innervating mesenteric Pacinian corpuscules project directly without relays along the dorsal column pathways l°,11,16. By contrast lamina V type ceils which are also unaffected by large splanchnic afferents, receive A(5 and C volleys 17,34,37,3s. Thus in our experimental conditions the modifications induced by algogenic substances most likely result from the activation of thin afferent fibers belonging to the A5 and C groups which are located in the walls of the small intestine or in its mesentery, and which only respond to pinches or prolonged and strong mechanical stimuli 16,31. As suggested by Lira et al. 27 it may well be that these sensory endings in visceral are the free branching nonmyelinated terminals of the perivascular nerves running in the adventitia of all blood vessels 2~ (or any rate derived from them), and which are very sensitive to chemical substances 26. Although it is hazardous to make a comparison between interneuronal discharge variations and pseudo-affective reactions of painful sensation induced by algogenic substances, from the psychophysiological point of view, it is interesting to underline the similarity of their time courses. Indeed the latencies and durations of the excitatory effects are comparable to those of the pain obtained in man by Burch and De Pasquale s by bradykinin administration into the brachial artery or by the intraperitoneal route2S; similarly the time course of the pseudo-affective manifestations of pain evoked by bradykinin injection into the splenic artery in dogs 2° was also of the same order. The shorter latency and duration of the modifications induced by acetylcholine correspond to the observations of others particularly to those of Keele and Armstrong 23: these authors, by blister area application or intradermal injection, obtained cutaneous pain in man, ('stinging' or 'smarting') of short duration with a 'typical sharp

446 peak.' Using bradykinin in the same way, they induced, after a latent period of 15-45 sec, a sensation of pain which ~rose to a peak in stages and was maintained at a high level for 1-2 min and then declined at a variable rate'. The observations of numerous viscerosomatic convergences on lamimi V cells confirm previous studies 17,21,e2,34,37,3s. Similar convergent properties have also been reported for cells located in laminae 1-Ill, VII and VIII 17,~1,22. Hancock et ~1.~1 found that some of these cells were at the origin of spinothalamic fibers and several studies 14,15 pointed out that a certain number of fibers themselves, located in the ventral quadrant of the white matter of the cord also present convergent properties. From all these studies it appears that this intermingling of visceral with somatic afferents is largely distributed within the cord. From our study, which was essentially devoted to laminae IV and V type cells, and in which each unit was located by extracellular injection of pontamine blue, a clear distribution appeared between these two groups. This distribution corresponds to Rexed's lamination 35 and seems, from our study, much more pronounced at the thoracic than at the lumbar level. Indeed a large number of studies have mentioned that the electrophysiological properties ot ° dorsal horn interneurons at the lumbar level were not always strictly correlated with Rexed's laminae. A great proportion of the considered cells have been inhibited by the ~tdministration of bradykinin into the mesenteric artery and it must be pointed out that most of these cells (12/14) presented an inhibitory cutaneous field. Similar inhibitory effects have already been described at the lumbar level for lamina V type cells, which besides their cutaneous excitatory fields also presented a large cutaneous inhibitory field4,L Thus in the present study it can be hypothesized that the inhibition induced by bradykinin could be due to the existence of visceral inhibitory fields. Our experimental conditions did not enable us to search for these visceral inhibitory fields, but Fields et al. a5 have shown that mechanical stimulation of hollow organs such as the distension of the gallblader or urinary bladder could inhibit fiber activity recorded in the ventral quadrant. It is also of interest to mention that several authors observed the appearance of IPSP of EPSP-IPSP sequences induced on various spinal cells by splanchnic stimulationlV,~L However these inhibitory phenomena, which are of postsynaptic nature 3, are difficult to explain and certainly result from complex interactions. This is well illustrated by the fact that mixed inhibitory-excitatory effects have also been observed in the present work and in previous studies4,5~ As it was suggested by several authors 21,a4,a7,z~s the existence of excitatory viscerosomatic convergence on lamina V interneurons may be used as a ncurophysiological basis to explain referred pain and supports Ruch's convergence projection theory a6. Indeed, it is probable that certain of these interneurons are involved in viscerosomatic reflexes -t2,1a, but it is also possible to suppose that a number of them are at the origin of ascending fibers included in the spinocervicothalamic tract 6,7 or the spinothalamic tracCL This assertion seems to be reinforced by the fact that several authors have also described convergence of somatic and visceral input into supraspinal structures t,2,9,24,3°,

447 ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by the C . N . R . S . ( E . R . A . 237). T h e s y n t h e t i c b r a d y k i n i n u s e d in this s t u d y was k i n d l y s u p p l i e d by S a n d o z L a b o r a t o r i e s (Basel, S w i t z e r l a n d ) .

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448 22 Hancock, M. B., Rigamonti, D. D. and Bryan, R. N., Convergence in the lumbar spinal cord of pathways activated by splanchnic nerve and hind limb cutaneous nerve stimulation, Exp. Neurol., 38 (1973) 337-348. 23 Keele, C. A. and Armstrong, D., Substances Producing Pain and Itch, Aznold, London, 1964, 399 pp. 24 Langenstein, H. and Rubia, F. J., Representation of splanchnic afferents in the nucleus ventropostero-lateralis of the cat, Pfliigers Arch. ges. Physiol., 331 (1972) 279-282. 25 Lira, R. K. S., Visceral receptors and visceral pain, Ann. N. Y. Acad. Sci., 86 (1960) 73 89. 26 Lira, R. K. S., Neuropharmacology of pain and analgesia. In R. K. S. Lirn, D. Armstrong and E. G. Pardo (Eds.), Pharmacology of Pain, Pergamon Press, Oxford, 1968, pp. 69 216. 27 Lira, R. K. S., Guzman, F. and Rodgers, D. W., Note on the muscle receptors concerned with pain. In D. Barker (Ed.), Symposium in Muscle Receptors, University of Hong Kong Gotd~,n Jubilee Congress, Hong Kong University Press, Hong Kong, 1961, pp. 215--219. 28 Lira, R. K. S., Miller, D. G., Guzman, F., Rodgers, D. W., Rogers, R. W., Wang, S. K., Chao. P. Y. and Shih, T. Y., Pain and analgesia evaluated by the intraperitoneal bradykinin evoked pain method in man, Clin. Pharmacol. Ther., 8 (1967) 52l 542. 29 Lombard, M. C., Guilbaud, G. and Besson, J. M., Effects of the intra-arterial injection of bradykinin into the limbs upon the activity of mesencephalic reticular units, Europ. J. Pharmacol., 30 (1975) 298-308. 30 McLeod, J. G., The representation of the splanchnic afferent pathways in the thalamus of the cat, J. Physiol. (Lond.), 140 (1958) 462-478. 31 Morrison, J. F. B., Mechanoreceptors in the region of the mesentery with A-delta and C fibres in the splanchnic nerves of cats, .I. Physiol. (Lond.), 226 (1972) 100-101. 32 Newman, P. P., Interaction in the visceral sensory areas of the cerebral cortex, J. Phy~dol. {Lomk ). 157 (1961) 29P. 33 Pavlasek, F. and Duda, P., The relation of activity of postsynaptic elements in the spinal cord to propriospinal and spinobulbospinal reflexes, Physiol. bohemoslov., 20 (1971) 398. 34 Pomeranz, B., Wall P. D. and Weber, W. V., Cord cells responding to fine myelinaled afferents from viscera, muscle and skin, J. Physiol. (Lond.), t99 (1968) 511-532. 35 Rexed, B., The cytoarchitectonic organization of spinal cord in cat, J. comp. Neto'o[., 96 (1952) 415-456. 36 Ruch, T. C., Pathophysiology of pain. In F. Fulton (Ed.), A Textbook of Physiology. 17th ed., Saunders, Philadelphia, Pa., 1955, pp. 358-376. 37 Seizer, M. and Spencer, W. A., Convergence of visceral and cutaneous afferent pathways in the lumbar spinal cord, Brain Research, 14 (1969) 331-348. 38 Seizer, M. and Spencer, W. A., Interactions between visceral and cutaneous afferents in the spinal cord: reciprocal primary afferent fiber depolarization, Brain Research, 14 (1969) 349-366. 39 Wall, P. D., The laminar organization of dorsal horn and effects of descending impulses, d. PhysioL (Lond.), 188 (1967)403-423.