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Depressive effects of morphine upon lamina V cells activities in the dorsal horn of the spinal cat
Brain Research, 98 (1975) 261-277 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands
261
DEPRESSIVE EFFECTS OF MORPHINE UPON LAMINA V CELLS ACTIVITIES IN THE DORSAL HORN OF THE SPINAL CAT
D. LE BARS, D. MENI~TREY, C. CONSEILLER AND J. M. BESSON Laboratoire de Physiologie des Centres Nerveux, Universit~ Pierre et Marie Curie, avenue GordonBennett, Paris 75016 (France)
(Accepted May 5th, 1975)
SUMMARY
The effects of morphine upon the transmission of nociceptive messages at the spinal level have been investigated in spinal cats by studying its effects on the activities of lamina V dorsal horn interneurons. Morphine (2 mg/kg i.v.) induced a direct depressive action at the spinal level, since it strongly reduced both spontaneous and evoked activities of lamina V cells. The spontaneous firing rate and the responses elicited by natural nociceptive stimulation were decreased by 50 %. The responses of these units evoked by supramaximal electrical stimulation were reduced to 67 % of their initial value; in this case, the depressive effect was much more prominent on the late component of the long duration responses. The observed depressive effects are specific since they are immediately reversed by administration of opiate antagonists (nalorphine or naloxone).
INTRODUCTION
Two hypotheses have been advanced to explain the depressive effects of morphine on various spinal activities: (1) a direct depressive action reported in the spinal animalS,20,24,~7,51,5~ and (2) an indirect effect by strengthening descending inhibitory controls of brain stem origin41,4a. Nevertheless, these studies were mainly concerned with motor reflexes and were not necessarily related to analgesia. The aim of the present study is to consider the first hypothesis by studying the effects of morphine on lamina V dorsal horn interneurons in the spinal cat. The role that lamina V dorsal horn cells play in the transmission of pain has been
262 emphasized by various authors: these cells are preferentially activated by small cutaneous afferent fibers and respond in a graded fashion to progressively more intense natural stimuli, including those at highly noxious levelslS,3~,49,50,.~)a; they receive visceral afferents4,a4, 42 and are strongly activated by intra-arterial injection of bradykinin 4,~. Concerning ascending projections of lamina V cells, several studies have shown that some lamina V cells are at the origin of the spino-cervico-thalamic tract 9-11,19,44 and of the spinothalamic tract 2,13,26,45,~3. From the available data, it can be concluded that lamina V cells are of special interest in the study of an eventual depressive effect of morphine on the transmission of nociceptive messages at the spinal level. Because of the particular behavioral reactivity of the cat to larger doses of morphine, we used a single dose of 2 mg/kg. With such a dose this drug has essentially analgesic properties. It is only at larger doses (4-6 mg/kg and above) that a 'bizarre motor behavior becomes dominant' (see ref. 28). METHODS
Twenty-seven adult cats of both sexes, each weighing 2-3.2 kg were used. After halothane anesthesia, they were immobilized by gallamine triethiodide (Flaxedil), artificially ventilated and placed in a Horsley-Clarke stereotaxic apparatus. A spinal cord section was performed at C1 and the carotid arteries were ligatured; anesthesia was then withdrawn and recordings were begun more than 4 h later. Arterial blood pressure, end-tidal CO2 and rectal temperature were continuously monitored; special care was taken to keep these physiological parameters under control: blood pressure always being above 80 mm Hg, central temperature being maintained at around 38 °C and end-tidal CO2 being adjusted to between 4 and 4.5 ~. Alter laminectomy, the dura mater was opened and the cord exposed from L4 to SI and covered with paraffin oil. The dura was retracted with 4 stitches to form a sling and used to lift the cord slightly in order to reduce the effects of respiratory movements. Under microscopic control small openings were made in the pia using fine forceps, to allow microelectrode penetration. Extracellular unitary recordings were made in the L6 and L7 segments using 3 M KCI and Pontamine blue filled glass microelectrodes, the resistance of which remained between 2 and 5 MfL The only cells considered in this analysis were those presenting no major alterations in spike amplitude or waveform during a period of study which lasted at least one hour before and one hour after morphine injection. Potentials were amplified, displayed on an oscilloscope and recorded on magnetic tape; firing frequency was continuously recorded through a spike integrator. Analysis of recorded activities were performed on an intertechnique apparatus (interspike histogram, post-stimuli histogram). Characterization of cells was made by using electrophysiological criteria described by Wall 5° and Hillman and WalP 8. These criteria allow a relatively precise differentiation between the units of laminae type IV, V and VI. Lamina IV type cells
263
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Fig. 1. Characterization of a lamina V type cell. A: location (see arrow) within the dorsal horn by extracellular injection of Pontamine blue (the subdivision in 10 layers has been added according to Rexed48). B: cutaneous excitatory receptive field (E.F.) of this unit. C: cell response to a pinch of long duration (time base 500 msec).
have a narrow peripheral field and are only driven by low intensity stimuli (touch, hair movements, airjet); their responses adapt rapidly to such natural stimulation. Lamina V type cells have a much larger receptive field (Fig. 1), respond to various stimuli (touch, pressure, pinch), increase their discharge in relation to the stimulus intensity and have a threshold gradient between the center and the periphery of the receptive field: low intensity stimuli are effective in the center, while more intense stimuli are generally necessary at the periphery. A prolonged natural stimulation of
264 the receptive field of these cells induces a tonic response (Fig. l). Lamina VI type cells present characteristics of lamina V type cells, since they are polysynaptically activated from the latter cells by cutaneous afferents, but they are also driven by muscular and articular afferents and generally present a more regular spontaneous firing rate '~0. In addition to the electrophysiological criteria, we used anatomical techniques in order to localize the recorded cells : at the end of most of the experiments, extracellular injections of Pontamine blue were performed (Fig. 1). Although the great majority of lamina V type cells, characterized by physiological criteria, are located in lamina V, as described by Rexed as, we noticed that some of them were in the ventral part of lamina IV and the dorsal part of lamina VI. The effects of morphine were studied on various types of activities: spontaneous activity, responses to natural cutaneous stimulus of strong intensity (intense pinching of the skin with a clamp placed in the peripheral excitatory receptive field), responses to supramaximal (10-25 V intensity; 0.3 or I msec duration) and to threshold electrical stimulation (1-3 V intensity; 0.3 or I msec duration) applied in the center of the peripheral receptive field. Student t-tests were used for statistical analysis. Morphine sulfate was slowly injected (3 min) in single doses of 2 mg/kg and attempts to reverse its action were made in some experiments by i.v. injections of nalorphine (1 mg/kg) (Clin-Comar Laboratories, Paris, France) or naloxone (0.05-0.2 mg/kg) (Endo Laboratories, Garden City, U.S.A.). RESULTS
(1) Effects of morphine on spontaneous activity The spontaneous activity of lamina V cells shows great variability between cells since the mean frequency ranged in these experiments from 0 to more than 40 spikes/ sec.
For the 19 cells studied, the mean value of the initial spontaneous firing rate was 19.6 ~ 3.2 spikes/sec. Among these 19 cells, 16 had a spontaneous activity high enough to evaluate an eventual depressive effect. For 15 of these 16 cells, injection of morphine sulfate induced a marked decrease of the spontaneous activity. It is of interest to note that the unaffected cell presented the lowest initial firing rate (3.1 spikes/ see).
For the 16 cells which initially showed spontaneous activity, the mean value of the firing rate was 22.3 + 2.9 spikes/sec before, and 11.4 -k 2.0 spikes/sec after morphine administration. This difference was highly significant (t15 ..... 6.92; P <./ 0.001); thus, the spontaneous firing rate of lamina V cells was reduced to 49 ~ of its initial value after morphine administration. No relationship between the level of initial spontaneous activity and the degree of the observed depressive effect had been found. The depression occurred rapidly, reaching its maximum less than 5 min after beginning the injection (Fig. 2A). Recovery was never observed before 50 min. However, as shown in Fig. 2B, the time course of the depressive effect showed large variations from one cell to another. In two cases, strong depressive effects were still present 2
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(2) Effects of morphine on activities evoked by natural stimulation In the cells studied, the excitatory receptive fields were located on the distal part o f the hindlimb. The size o f these fields was never restricted to less than two toes, Natural nociceptive stimulation, strong pinches applied within the center o f the re-
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ceptive field, induced an intense increase of the firing rate. From a methodological point of view, it is of interest to emphasize the fact that the responses are easily reproduced by successive pinches (Fig. 3). In this study, we used stimulations of 5, 10 and 20 sec duration. For the 10 cells studied with this type of stimulation, the mean frequency of the responses before administration of the drug was 119 ± 14 spikes/sec. For a
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given cell, there was no relationship between the mean frequency of the spontaneous firing rate and the magnitude of the discharge obtained by natural stimulation. For 8 of the 10 cells studied, the administration of morphine induced a marked depression of the responses to natural stimulation (Fig. 3). The mean initial discharge frequency, which was of 119 ~ 14 spikes/sec, was decreased to 60 ~: 10 spikes/sec. This difference was highly significant (t9 = 4.90; P < 0,001). Thus, the responses to intense natural peripheral stimulation were reduced to 50 % of their initial value after morphine administration. As has been pointed out for the action of morphine on the spontaneous activity, there was no relationship between the magnitude of the discharge induced by natural stimulation and the depressive effect of morphine. For a given cell, the time course of the effect observed on the responses was almost similar to those described for the depressive effect induced on spontaneous activity: the duration of the depressive effect was at least 40 min, but was generally longer (Fig. 4). In order to assess the eventual relationship between the depressive effect of morphine and the size of the excitatory receptive field, each cell was affected with a coefficient proportional to the surface of the receptive field. It was found that the larger the receptive field, the more effective was the depressive effect of morphine on responses evoked by strong natural stimulation (Fig. 5). These effects were significant (ts -----2.57; r = 0.67; P < 0.05).
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..el Fig. 6. Effects o f morphine on responses to intense electrical stimulation (25 V; 1 msec) applied within the center o f the excitatory cutaneous receptive field. As shown in A and B the second c o m p o n e n t o f this response clearly appeared during repetitive stimulation (3/sec) (wind-up). A: examples o f individual responses; a: response 1, 2 and 3; b: response 40, 41 and 42; c: response 100, 101 and 102. B: dot-display analysis o f 180 successive stimulations (from top to bottom). C: poststimulus histograms (180 trials) showing the effects o f morphine and naloxone on the same activity•
Fig. 7. Effects of morphine and naloxone upon responses to electrical stimulations. A : the response of long duration induced by supramaximal stimulation (20 V; 1 msec) is strongly depressed by morphine. This effect is reversed by naloxone. B: the response of the same unit to threshold stimulation (0.8 V; 0.2 msec) is not affected.
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(3) Effects of morphine on excitatory responses evoked by electrical stimulation The latency of the responses obtained with supramaximal electrical stimulation applied in the center of the excitatory receptive field was 6-7 msec, but the magnitude of these responses showed a great variability between cells since their duration and their number of spikes ranged respectively from 30 to 3000 msec and from 5 to more than 250 spikes. Three schematic patterns of discharge have been found. (a) Certain cells presented short unimodal responses with a few potentials; the discharge of these cells was not affected by increasing the stimulation frequency. (b) A second group of cells presented bimodal responses which consist of a short initial component (10-30 msec) followed after a period of relative inhibition by a late longer component (40-400 msec) (Fig. 6); as shown in Fig. 6A and B, this second component is clearly revealed by using repetitive stimulation; this phenomenon, called 'wind-up' by Mendell al, is due to the activation of small fibers inputsat,a6, 49. (c) The third group of cells presented very long unimodal responses of several hundred milliseconds (Fig. 7A). In this case, it was not necessary to apply repetitive stimulations to obtain long duration responses. For l0 out of 16 cells, the responses to supramaximal stimulation were markedly depressed by morphine without latency modification. It is important to point out that the 6 unaffected cells belong to the first group of units which present responses of short duration with few spikes. On the contrary, long unimodal responses are strongly depressed (Fig. 7); in a similar way, the late component of the second group units induced by repetitive stimulation was depressed (Fig. 6B). The relationship between the degree of the depressive effect and the magnitude of the initial response is plotted in Fig. 8. For the 16 cells studied, the mean control response before morphine was 65 4- 20 spikes and was 43 4- 13 spikes after administration of the drug. Thus, the responses of lamina V cells to supramaximal electrical stimulation were significantly reduced to 67 % of their initial value (t 15 = 2.72; P < 0.02). As shown in Figs. 6C and 7A, morphine did not affect the first component of the
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responses. In a similar way, m o r p h i n e a d m i n i s t r a t i o n did not alter the small responses o f these cells to threshold stimulation (Fig. 7B).
(4) Effeet of antagonists (nalorphine or naloxone) on spontaneous and evoked activities In order to examine the specificity o f the m o r p h i n e effects, attempts to reverse its depressive action were made on a few cats by i.v. injections of n a l o r p h i n e (! mg/ kg) or naloxone (0.05-0.2 mg/kg).
273 In each case, the depressive effects of morphine were reversed. These modifications occurred rapidly (Fig. 9A) and were observed on spontaneous activity (Fig. 9B) as well as on responses to natural (Fig. 9C) or to electrical stimulation (Figs. 6 and 7). In some cases, after the administration of the antagonist, the activities were slightly higher than those observed at the beginning of the experiment. Nevertheless, we observed, in additional 4 experiments, that the antagonists given alone did not alter significantly lamina V cells activities. DISCUSSION
This study of the action of systemic injection of morphine (2 mg/kg) on lamina V type cells activities in the spinal cat indicates that this drug substantially reduces both spontaneous and evoked activities. These effects occur rapidly, reaching their maximum in less than 5 rain, and lasting at least 40 min. The spontaneous firing rate was reduced to 49 ~ of its mean inital value. Responses to intense natural stimulation were decreased by 50 ~ ; in this case, we found that the depressive effects of morphine were more pronounced on cells driven from larger peripheral cutaneous receptive fields. Responses to supramaximal electrical stimulation were reduced to 6 7 ~ of their mean initial value; in this case, we must emphasize the fact that morphine preferentially depresses responses of long duration which are certainly due to the activation of small A0 and C fibers 15,31,~6,49. Several studies have shown that lamina V type cells received convergent inputs from large and thin fibers18,31,36,49,50; by using supramaximal and threshold stimulation we found that morphine did not affect the first short lasting component of the responses which is due to the activation of large fibers. The entire depressive effects of morphine are specific since they are immediately reversed by administration of opiate antagonist (nalorphine or naloxone). Our results clearly show direct depressive effects of morphine at the spinal level, since, in this study, a possible supraspinal site of action of this drug was excluded by the C1 spinal transection. This data is in good agreement with previous reports on spinal reflexes and more recent experiments on dorsal horn interneurons. In the spinal animal, polysynaptic nociceptive reflexes are depressed by low doses of morphineS,2°,24,27,~l,5~ while stronger doses are needed to depress monosynaptic reflexes~5. Microphysiological studies have also emphasized a direct action of i.v. injection of morphinomimetics on dorsal interneurons: Iwata and Sakai 21 reported that fentanyl (20-40/~g/kg) suppressed the discharges of spinal interneurons evoked by stimulation of A0 cutaneous fibers in the spinal cat, while the responses of these cells to low intensity stimulation were unaffected by this drug. Similarly, Besson e t al. 7 mentioned that phenoperidine was more effective in depressing the responses of lamina V cells to intense electrical or natural stimuli than it was for responses to innocuous stimuli. Recently, Kitahata e t aL 2a described in the spinal cat a depressive effect of morphine upon lamina I and V units, but these authors mainly considered spontaneous activity of these cells. Moreover, the observed modifications were of very short duration since, even with 2 mg/kg of morphine, their time course curves clearly showed a total
274 recovery 10 min after administration of the drug. In our study, the depressive effects ol" morphine on both spontaneous and evoked activities are of longer duration (at least 40 rain) and are more consistent with analgesia obtained in therapeutical practice. A direct effect of morphine on dorsal horn interneurons has been confirmed by using iontophoretic approaches; Dostrovsky and Pomeranz t4 have shown that morphine reduced excitation of dorsal horn interneurons induced by direct application of glutamate and aspartate. However, it is difficult to understand why, in their report, iontophoretically applied morphine was unable to depress the evoked activities induced by peripheral stimulation since a recent study 12 has shown that responses of dorsal horn interneurons to noxious radiant heat were strongly depressed by the same application. Although no conclusive evidence can be advanced to explain the depressive effects of morphine on spinal interneurons, all these macro- and microphysiological data show a direct action of morphine and related compounds at this level. Our results showing in the spinal preparation a strong depressive effect of morphine on various activities of lamina V cells could in part explain the analgesic properties of morphine, since we have mentioned in the Introduction that these cells were strongly activated by small afferent fibers from various origins and that some of them were at the origin of several ascending pathways in cat and monkey. In the same way, Grossman and Jurna 16 recently found that morphine, at a dose as low as 0.5 mg/kg, depressed the activity of axons of the ventrolateral tract. However, our findings contrast with those of Takagi et al. 4a and Satoh and Takagi 4t who compared, in intact, decerebrate and spinal animals, the effects of large doses of morphine (4-10 mg/kg i.v.) on polysynaptic reflexes and on responses evoked in the ventrolateral funiculus by splanchnic stimulation. These authors concluded that morphine's site of action is located in the brain stem, acting on spinal transmission by reinforcing descending inhibitory controls. Nevertheless, from our results and from those previously mentioned, it seems difficult to refute a direct action of morphine at the spinal level. Moreover, certain depressive effects described by Takagi e t al. 4a and Satoh and Takagi 4t are of short duration and have been obtained by considering macrophysiological responses, the interpretation of which is not easy. Furthermore, they obtained similar results on unanesthetized or anesthetized preparations and there is no doubt that, owing to the doses of barbiturate used, the activities of spinal interneurons evoked by noxious stimulation are. in their experiments, totally suppressed 6,a°. However, our results do not exclude morphine's supraspinal sites of action. Indeed, strong analgesia has been observed by morphine application at ventricular and periventricular levels 3,17,22,a3,46,4v. These experiments seem to agree with the initial hypothesis of Takagi et al. 4a suggesting an increase of descending inhibitory control after morphine administration. Furthermore, some recent behavioral and pharmacological data has underlined the relationship between morphine analgesia and analgesia induced by stimulation of the periaqueductal gray matter of the rat 1,29,3° and of raphe nuclei in the cat 3z,37 (see also refs. 39, 40 and 48). These effects could be due in part to descending inhibitory effects on the transmission of nociceptive messages at the spinal cord level 32. Thus, several findings are in favor of supraspinal sites of action of narcotic
275
analgesics. It seems likely that morphine depresses spinal transmission both by a direct depressive effect at the spinal level and by activating descending inhibitory pathways. Studies of the action of morphine on spinal structures involved in pain processes, such as lamina V, in decerebrate and intact animals, would be useful to determine the relative importance of these two mechanisms. ACKNOWLEDGEMENTS
This work was supported by l'Institut National de la Sant6 et de la Recherche m6dicale (INSERM) ATP Pharmacod6pendance, Contrat No. 71-152015.
REFERENCES 1 AKIL, H., MAYER, D. J., ET LIEBESKIND,J. C., Comparaison chez le rat entre l'analg6sie induite par stimulation de la substance grise p6ri-aqueducale et I'analg~sie morphinique, C.R. Acad. Sci. (Paris), 274 (1972) 3603-3605. 2 ALBE-F,ESSARD,O., LEVANTE, A., AND LAMOUR, Y., Origin of spino-thalamic tract in monkeys, Brain Research, 65 (1974) 503-509. 3 ALaus, K., SCHOTr, M., ANt) HERZ, A., Interaction between morphine and morphine antagonists after systematic and intraventricular application, Europ. J. Pharmacol., 12 (1970) 53-64. 4 BENELLi, G. C., BESSON, J. M., GUILBAUD,G., ET LOMBARD, M. C., R6ponses des interneurones de la corne dorsale thoracique ~ des stimulations cutan6es et ~tdes stimulations visc6rales nociceptives, J. Physiol. (Paris), 69 (1974) 221A. 5 BESSON,J. M., CONSEILLER,C., HAMANN, K. F., AND MMLLARD, M. C., Modification of dorsal horn cell activities in the spinal cord, after intra-arterial injection of bradykinin, J. Physiol. (Lond.), 221 (1972) 189-205. 6 BESSON,J. M., Rlvor, J. P., ABDELMOUMENE,M., ET ALEONARD,P., Etude de l'action des anesth6siques sur l'activit6 des cellules, de la corne dorsale de la moelle, J. Physiol. (Paris), 62 (1970) 350-351. 7 BESSON, J. M., WYON-MAILLARD,M. C., BENOIST,J. M., CONSEILLER, C., AND HAMANN, K. F., Effects of phenoperidine on lamina V cells in the cat dorsal horn, J. Pharmacol. exp. Ther., 187 (1973) 239-245. 8 BODO, R. C., AND BROOKS, C. McC., The effect of morphine on blood sugar and reflex activity in the chronic spinal cat, J. Pharmacol. exp. Ther., 61 (1937) 82-88. 9 BROWN, A. G., AND F'RANZ,O. N., Responses of spinocervical tract neurones to natural stimulation of identified cutaneous receptors, Exp. Brain Res., 7 (1969) 231-249. 10 BRYAN, R. N., COULTER, J. D., AND WILLIS, W. D., Cells of origin of the spinovercical tract in the monkey, Exp. Neurol., 42 (1974) 574-586. 11 BRYAN, R. N., TREVINO, D. L., COULTER,J. D., AND WILLIS, W. D., Location and somatotopic organization of the cells of origin of the spinocervical tract, Exp. Brain Res., 17 (1973) 177-181. 12 CALVILLO,O., HENRY, J. U, AND NEUMAN, R. S., Effects of morphine and naloxone on dorsal horn neurones in the cat, Canad. J. Physiol. Pharmacol., 52 (1974) 1207-1211. 13 DILLY, P. N., WALL, P. D., AND WEBSTER,K. E., Cells of origin of the spinothalamic tract in the cat and rat, Exp. NeuroL, 21 (1968) 550-562. 14 DOSTROVSKY,J., ANt) POMERANZ,B., Morphine blockade of amino acid putative transmitters on cat spinal cord sensory interneurones, Nature New Biol., 246 (1973) 222-224. 15 GREGOR, M., AND ZIMMERMANN, M., Characteristics of spinal neurones responding to cutaneous myelinated and unmyelinated fibres, J. Physiol. (Lond.), 221 (1972) 255-276. 16 GROSSMANN,W., AND JURNA, I., Depression by morphine of activity in the ventrolateral tract evoked from cutaneous A-fibres, Europ. J. Pharmacol., 29 (1974) 171-174. 17 HERZ, A., ALnUS, K., METY~, J., SCSUBERT,P., AND TESCHEMACHER, HJ., On the central sites for the antinociceptive action of morphine and fentanyl, Neuropharmacology, 9 (1970) 539-551. 18 HILLMAN, P., AND WALL, P. D., Inhibitory and excitatory factors influencing the receptive fields of lamina V spinal cord cells, Exp. Brain Res., 9 (1969) 284-306.
276 19 HONGO, T., JANKOWSKA, E.. AND LUNDSEr(h A., Postsynaptic excitation and inhibition from primary afferents in neurones of the spino-cervical tract, J. Physiol. (Lond.), 199 (1968) 569-592. 20 IRWIN, S., HOUDE, R. W., BENNETT, D. R.. HENDERSHOT, L. C., AND SEEVERS, M. H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pkarmacol. exp. Ther., 101 (1951) 132-143. 21 IWATA, N., AND SAKAI, Y., Effects of fentanyl upon the spinal interneurons activated by A~5 afferent fibers of the cutaneous nerve of the cat, Jap. J. Pkarmacol., 21 (1971 ) 413-416. 22 JACQUET, Y. ]-'., AND LAJTHA. A., Morphine action at central nervous systern sites in rat: analgesia or hyperalgia depending on site and dose, Science, 182 (I 973) 490-492. 23 K~TAHATA, L. M., KOSAKA, Y., TAUB, A., BONIKOS, K., AND HOFFERT, M., Lamina specific suppression of dorsal-horn unit activity by morphine sulfate, Anesthesiology, 41 (1974) 39-48. 24 KOLL, W., HAASE, J., BLOCK, G., AND MUHLBERG, B., The predilective action of small doses of morphine on nociceptive spinal reflexes of low spinal cats, Int. J. Neuropkarmacol., 2 (1963) 57 65. 25 KmvoY, W., KROEGER, D., AND ZIMMERMANN,E., Actions of morphine on the segmental reflex ot decerebrate-spinal cat, Brit. J. Pharmacol., 47 (I 973) 457-464. 26 LEVANTE, A., LAMOUR, Y., ET ALBE-FESSARD, D., Localisation dans la couche V de Rexed de cellules d'origine d'un faisceau spino-thalamique crois6 chez le macaque, C.R, Acad. Sci. (Paris), 276 (1973) 1589-1592. 27 MCCLANE, T. K., AND MARTIN, W. R., Effects of morphine, nalorphine, cyclazocine and naloxone on the flexor reflex, Int. J. Neuropharmacol., 6 (1967) 89-98. 28 MCKENZIE, J. S., AND BEECHEY, N. R., The effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia, Electroenceph. clin. Neurophysiol., 14 (1962) 501-519. 29 MAYER, D. J., AND LIEBESKINO, J. C., Pain reduction of focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Research, 68 (1974) 73-94. 30 MAYER, D. J., WOLFLE, T. L., AKIL, H., CARDER, B,, AND LIEBESKIND, J. C., Analgesia from electrical stimulation in the brain stem of the rat, Science, 174 (1971) 1351-1354. 31 MENOELL, L. M., Physiological properties of unmyelinated fibre projection to the spinal cord, Exp. Neurol., 16 (1966) 316-332. 32 OUVERAS, J. L., BESSON, J. M., GUlLSAUD, G., ANt) LJEBESKIND, J. C., Behavioral and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat, Exp. Brain Res., 20 (1974) 32 44. 33 PERT, A., AND YAKSH, T., Sites of morphine induced analgesia in the prirnate brain: relation to pain pathways, Brain Research, 80 (1974) 135-140. 34 POMERANZ,B., WALL, P. D., AND WEnER, W. V., Cord cells responding to fine myelinated afferents from viscera, muscle and skin, J. Physiol. (Lond.), 199 (1968) 511-532. 35 PRICE, D. D., AND BROWE, A. C., Responses of spinal cord neurons to graded noxious and nonnoxious stimuli, Brain Research, 64 (1973) 425-529. 36 PRICE, D. D., AND WAGMAN, 1. H., Physiological roles of A and C fibre inputs to the spinal dorsal horn of Macaca nttdalta, Exp. Neurol., 29 (1970) 383-399. 37 REDJEMI, F., OLIVERAS, J. L., GUILBAUD, G., ET BESSON, J. M., Analgesic induite par la stimulation du noyau central inf6rieur du raph6 chez le chat, C.R. Acad. Sci. (Paris), 279 (1974) 1105-1107. 38 REXED, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 16 (1952) 415-495. 39 SAMANIN, R., GUMULKA, W., AND VALZELH, L., Reduced effect of morphine in midbrain raphe lesioned rats, Earop. J. Pharmacol., 10 (1970) 339-343. 40 SAMANIN, R., ANt) VALZELLL L., Increase of morphine-induced analgesia by stimulation of the nucleus raphe dorsalis, Europ. J. Pharmacol., 16 (1971) 298-302. 41 SATOH, M., AND TAKAGI, H., Enhancement by morphine of the central descending inhibitory influence on spinal sensory transmission, Europ. J. Pharmacol., 14 (1971) 60-65. 42 SELZER, M., AND SPENCVR, W. A., Convergence of visceral and cutaneous afferent pathways in the lumbar spinal cord, Brain Research, 14 (1969) 331-348. 43 TAKAGI, n., MATSUNARA, M., YANAI, m., AND OGIU, K., The effect of analgesics on the spinal reflex activity of the cat, Jap. J. Pharmacol., 4 (1955) 176-187. 44 TAUB, A., AND BISHOP, P. O., The spinocervical tract: dorsal column linkage, conduction velocity, primary afferent spectrum, Exp. Neurol., 13 (1965) 1-21. 45 TREVINO, D. L., COULTER, J. D., AND WILLIS, W. D., Location of cells of origin of spinothalamic tract in lumbar enlargement of the monkey, J. Neurophysiol., 36 (1973) 750-761.
277 46 Tsou, K., AND JANG, C. S., Studies on the site of analgesic action of morphine by intracerebral microinjections, Scientia Sinica, 13 (1964) 1099-1109. 47 VIOOURET,J., TESCHEMACHER,HJ., ALBUS,K., AND HERZ, A., Differentiation between spinal and supraspinal sites of action of morphine when inhibiting the hindleg flexor reflex in rabbits, Neuropharmacology, 12 (1973) 111-121. 48 VOGT,M., The effect of lowering the 5-hydroxytryptamine content of the rat spinal cord on analgesia produced by morphine, J. Physiol. (Lond.), 236 (1974) 483--498. 49 WAGMANN,I. H., AND PRICE, D. D., Responses of dorsal horn cells of Macaca mulatta to cutaneous and sural nerve A and C fibre stimuli, J. Neurophysiol., 32 (1969) 803-817. 50 WALL, P. n., The laminar organization of dorsal horn and effects of descending impulses, J. Physiol. (Lond), 188 0967) 403-423. 51 WIKLER,A., Studies on the action of morphine on the central nervous system of the cat, J. Pharmacol, exp. Ther., 80 (1944) 176-187. 52 WIKLER,k., AND FRANK, K., Hindlimb reflexes of chronic spinal dogs during cycles of addiction to morphine and methadone, J. Pharmacol. exp. Ther., 94 (1948) 382-400. 53 WILLIS,W. D., TREVINO,D. L., COULTER,J. D., AND MAUNZ,R. A., Response of primate spinothalamic tract neurons to natural stimulation of hindlimb, J. Neurophysiol., 37 (1974) 358-372.
261
DEPRESSIVE EFFECTS OF MORPHINE UPON LAMINA V CELLS ACTIVITIES IN THE DORSAL HORN OF THE SPINAL CAT
D. LE BARS, D. MENI~TREY, C. CONSEILLER AND J. M. BESSON Laboratoire de Physiologie des Centres Nerveux, Universit~ Pierre et Marie Curie, avenue GordonBennett, Paris 75016 (France)
(Accepted May 5th, 1975)
SUMMARY
The effects of morphine upon the transmission of nociceptive messages at the spinal level have been investigated in spinal cats by studying its effects on the activities of lamina V dorsal horn interneurons. Morphine (2 mg/kg i.v.) induced a direct depressive action at the spinal level, since it strongly reduced both spontaneous and evoked activities of lamina V cells. The spontaneous firing rate and the responses elicited by natural nociceptive stimulation were decreased by 50 %. The responses of these units evoked by supramaximal electrical stimulation were reduced to 67 % of their initial value; in this case, the depressive effect was much more prominent on the late component of the long duration responses. The observed depressive effects are specific since they are immediately reversed by administration of opiate antagonists (nalorphine or naloxone).
INTRODUCTION
Two hypotheses have been advanced to explain the depressive effects of morphine on various spinal activities: (1) a direct depressive action reported in the spinal animalS,20,24,~7,51,5~ and (2) an indirect effect by strengthening descending inhibitory controls of brain stem origin41,4a. Nevertheless, these studies were mainly concerned with motor reflexes and were not necessarily related to analgesia. The aim of the present study is to consider the first hypothesis by studying the effects of morphine on lamina V dorsal horn interneurons in the spinal cat. The role that lamina V dorsal horn cells play in the transmission of pain has been
262 emphasized by various authors: these cells are preferentially activated by small cutaneous afferent fibers and respond in a graded fashion to progressively more intense natural stimuli, including those at highly noxious levelslS,3~,49,50,.~)a; they receive visceral afferents4,a4, 42 and are strongly activated by intra-arterial injection of bradykinin 4,~. Concerning ascending projections of lamina V cells, several studies have shown that some lamina V cells are at the origin of the spino-cervico-thalamic tract 9-11,19,44 and of the spinothalamic tract 2,13,26,45,~3. From the available data, it can be concluded that lamina V cells are of special interest in the study of an eventual depressive effect of morphine on the transmission of nociceptive messages at the spinal level. Because of the particular behavioral reactivity of the cat to larger doses of morphine, we used a single dose of 2 mg/kg. With such a dose this drug has essentially analgesic properties. It is only at larger doses (4-6 mg/kg and above) that a 'bizarre motor behavior becomes dominant' (see ref. 28). METHODS
Twenty-seven adult cats of both sexes, each weighing 2-3.2 kg were used. After halothane anesthesia, they were immobilized by gallamine triethiodide (Flaxedil), artificially ventilated and placed in a Horsley-Clarke stereotaxic apparatus. A spinal cord section was performed at C1 and the carotid arteries were ligatured; anesthesia was then withdrawn and recordings were begun more than 4 h later. Arterial blood pressure, end-tidal CO2 and rectal temperature were continuously monitored; special care was taken to keep these physiological parameters under control: blood pressure always being above 80 mm Hg, central temperature being maintained at around 38 °C and end-tidal CO2 being adjusted to between 4 and 4.5 ~. Alter laminectomy, the dura mater was opened and the cord exposed from L4 to SI and covered with paraffin oil. The dura was retracted with 4 stitches to form a sling and used to lift the cord slightly in order to reduce the effects of respiratory movements. Under microscopic control small openings were made in the pia using fine forceps, to allow microelectrode penetration. Extracellular unitary recordings were made in the L6 and L7 segments using 3 M KCI and Pontamine blue filled glass microelectrodes, the resistance of which remained between 2 and 5 MfL The only cells considered in this analysis were those presenting no major alterations in spike amplitude or waveform during a period of study which lasted at least one hour before and one hour after morphine injection. Potentials were amplified, displayed on an oscilloscope and recorded on magnetic tape; firing frequency was continuously recorded through a spike integrator. Analysis of recorded activities were performed on an intertechnique apparatus (interspike histogram, post-stimuli histogram). Characterization of cells was made by using electrophysiological criteria described by Wall 5° and Hillman and WalP 8. These criteria allow a relatively precise differentiation between the units of laminae type IV, V and VI. Lamina IV type cells
263
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Fig. 1. Characterization of a lamina V type cell. A: location (see arrow) within the dorsal horn by extracellular injection of Pontamine blue (the subdivision in 10 layers has been added according to Rexed48). B: cutaneous excitatory receptive field (E.F.) of this unit. C: cell response to a pinch of long duration (time base 500 msec).
have a narrow peripheral field and are only driven by low intensity stimuli (touch, hair movements, airjet); their responses adapt rapidly to such natural stimulation. Lamina V type cells have a much larger receptive field (Fig. 1), respond to various stimuli (touch, pressure, pinch), increase their discharge in relation to the stimulus intensity and have a threshold gradient between the center and the periphery of the receptive field: low intensity stimuli are effective in the center, while more intense stimuli are generally necessary at the periphery. A prolonged natural stimulation of
264 the receptive field of these cells induces a tonic response (Fig. l). Lamina VI type cells present characteristics of lamina V type cells, since they are polysynaptically activated from the latter cells by cutaneous afferents, but they are also driven by muscular and articular afferents and generally present a more regular spontaneous firing rate '~0. In addition to the electrophysiological criteria, we used anatomical techniques in order to localize the recorded cells : at the end of most of the experiments, extracellular injections of Pontamine blue were performed (Fig. 1). Although the great majority of lamina V type cells, characterized by physiological criteria, are located in lamina V, as described by Rexed as, we noticed that some of them were in the ventral part of lamina IV and the dorsal part of lamina VI. The effects of morphine were studied on various types of activities: spontaneous activity, responses to natural cutaneous stimulus of strong intensity (intense pinching of the skin with a clamp placed in the peripheral excitatory receptive field), responses to supramaximal (10-25 V intensity; 0.3 or I msec duration) and to threshold electrical stimulation (1-3 V intensity; 0.3 or I msec duration) applied in the center of the peripheral receptive field. Student t-tests were used for statistical analysis. Morphine sulfate was slowly injected (3 min) in single doses of 2 mg/kg and attempts to reverse its action were made in some experiments by i.v. injections of nalorphine (1 mg/kg) (Clin-Comar Laboratories, Paris, France) or naloxone (0.05-0.2 mg/kg) (Endo Laboratories, Garden City, U.S.A.). RESULTS
(1) Effects of morphine on spontaneous activity The spontaneous activity of lamina V cells shows great variability between cells since the mean frequency ranged in these experiments from 0 to more than 40 spikes/ sec.
For the 19 cells studied, the mean value of the initial spontaneous firing rate was 19.6 ~ 3.2 spikes/sec. Among these 19 cells, 16 had a spontaneous activity high enough to evaluate an eventual depressive effect. For 15 of these 16 cells, injection of morphine sulfate induced a marked decrease of the spontaneous activity. It is of interest to note that the unaffected cell presented the lowest initial firing rate (3.1 spikes/ see).
For the 16 cells which initially showed spontaneous activity, the mean value of the firing rate was 22.3 + 2.9 spikes/sec before, and 11.4 -k 2.0 spikes/sec after morphine administration. This difference was highly significant (t15 ..... 6.92; P <./ 0.001); thus, the spontaneous firing rate of lamina V cells was reduced to 49 ~ of its initial value after morphine administration. No relationship between the level of initial spontaneous activity and the degree of the observed depressive effect had been found. The depression occurred rapidly, reaching its maximum less than 5 min after beginning the injection (Fig. 2A). Recovery was never observed before 50 min. However, as shown in Fig. 2B, the time course of the depressive effect showed large variations from one cell to another. In two cases, strong depressive effects were still present 2
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(2) Effects of morphine on activities evoked by natural stimulation In the cells studied, the excitatory receptive fields were located on the distal part o f the hindlimb. The size o f these fields was never restricted to less than two toes, Natural nociceptive stimulation, strong pinches applied within the center o f the re-
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ceptive field, induced an intense increase of the firing rate. From a methodological point of view, it is of interest to emphasize the fact that the responses are easily reproduced by successive pinches (Fig. 3). In this study, we used stimulations of 5, 10 and 20 sec duration. For the 10 cells studied with this type of stimulation, the mean frequency of the responses before administration of the drug was 119 ± 14 spikes/sec. For a
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given cell, there was no relationship between the mean frequency of the spontaneous firing rate and the magnitude of the discharge obtained by natural stimulation. For 8 of the 10 cells studied, the administration of morphine induced a marked depression of the responses to natural stimulation (Fig. 3). The mean initial discharge frequency, which was of 119 ~ 14 spikes/sec, was decreased to 60 ~: 10 spikes/sec. This difference was highly significant (t9 = 4.90; P < 0,001). Thus, the responses to intense natural peripheral stimulation were reduced to 50 % of their initial value after morphine administration. As has been pointed out for the action of morphine on the spontaneous activity, there was no relationship between the magnitude of the discharge induced by natural stimulation and the depressive effect of morphine. For a given cell, the time course of the effect observed on the responses was almost similar to those described for the depressive effect induced on spontaneous activity: the duration of the depressive effect was at least 40 min, but was generally longer (Fig. 4). In order to assess the eventual relationship between the depressive effect of morphine and the size of the excitatory receptive field, each cell was affected with a coefficient proportional to the surface of the receptive field. It was found that the larger the receptive field, the more effective was the depressive effect of morphine on responses evoked by strong natural stimulation (Fig. 5). These effects were significant (ts -----2.57; r = 0.67; P < 0.05).
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Fig. 7. Effects of morphine and naloxone upon responses to electrical stimulations. A : the response of long duration induced by supramaximal stimulation (20 V; 1 msec) is strongly depressed by morphine. This effect is reversed by naloxone. B: the response of the same unit to threshold stimulation (0.8 V; 0.2 msec) is not affected.
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(3) Effects of morphine on excitatory responses evoked by electrical stimulation The latency of the responses obtained with supramaximal electrical stimulation applied in the center of the excitatory receptive field was 6-7 msec, but the magnitude of these responses showed a great variability between cells since their duration and their number of spikes ranged respectively from 30 to 3000 msec and from 5 to more than 250 spikes. Three schematic patterns of discharge have been found. (a) Certain cells presented short unimodal responses with a few potentials; the discharge of these cells was not affected by increasing the stimulation frequency. (b) A second group of cells presented bimodal responses which consist of a short initial component (10-30 msec) followed after a period of relative inhibition by a late longer component (40-400 msec) (Fig. 6); as shown in Fig. 6A and B, this second component is clearly revealed by using repetitive stimulation; this phenomenon, called 'wind-up' by Mendell al, is due to the activation of small fibers inputsat,a6, 49. (c) The third group of cells presented very long unimodal responses of several hundred milliseconds (Fig. 7A). In this case, it was not necessary to apply repetitive stimulations to obtain long duration responses. For l0 out of 16 cells, the responses to supramaximal stimulation were markedly depressed by morphine without latency modification. It is important to point out that the 6 unaffected cells belong to the first group of units which present responses of short duration with few spikes. On the contrary, long unimodal responses are strongly depressed (Fig. 7); in a similar way, the late component of the second group units induced by repetitive stimulation was depressed (Fig. 6B). The relationship between the degree of the depressive effect and the magnitude of the initial response is plotted in Fig. 8. For the 16 cells studied, the mean control response before morphine was 65 4- 20 spikes and was 43 4- 13 spikes after administration of the drug. Thus, the responses of lamina V cells to supramaximal electrical stimulation were significantly reduced to 67 % of their initial value (t 15 = 2.72; P < 0.02). As shown in Figs. 6C and 7A, morphine did not affect the first component of the
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responses. In a similar way, m o r p h i n e a d m i n i s t r a t i o n did not alter the small responses o f these cells to threshold stimulation (Fig. 7B).
(4) Effeet of antagonists (nalorphine or naloxone) on spontaneous and evoked activities In order to examine the specificity o f the m o r p h i n e effects, attempts to reverse its depressive action were made on a few cats by i.v. injections of n a l o r p h i n e (! mg/ kg) or naloxone (0.05-0.2 mg/kg).
273 In each case, the depressive effects of morphine were reversed. These modifications occurred rapidly (Fig. 9A) and were observed on spontaneous activity (Fig. 9B) as well as on responses to natural (Fig. 9C) or to electrical stimulation (Figs. 6 and 7). In some cases, after the administration of the antagonist, the activities were slightly higher than those observed at the beginning of the experiment. Nevertheless, we observed, in additional 4 experiments, that the antagonists given alone did not alter significantly lamina V cells activities. DISCUSSION
This study of the action of systemic injection of morphine (2 mg/kg) on lamina V type cells activities in the spinal cat indicates that this drug substantially reduces both spontaneous and evoked activities. These effects occur rapidly, reaching their maximum in less than 5 rain, and lasting at least 40 min. The spontaneous firing rate was reduced to 49 ~ of its mean inital value. Responses to intense natural stimulation were decreased by 50 ~ ; in this case, we found that the depressive effects of morphine were more pronounced on cells driven from larger peripheral cutaneous receptive fields. Responses to supramaximal electrical stimulation were reduced to 6 7 ~ of their mean initial value; in this case, we must emphasize the fact that morphine preferentially depresses responses of long duration which are certainly due to the activation of small A0 and C fibers 15,31,~6,49. Several studies have shown that lamina V type cells received convergent inputs from large and thin fibers18,31,36,49,50; by using supramaximal and threshold stimulation we found that morphine did not affect the first short lasting component of the responses which is due to the activation of large fibers. The entire depressive effects of morphine are specific since they are immediately reversed by administration of opiate antagonist (nalorphine or naloxone). Our results clearly show direct depressive effects of morphine at the spinal level, since, in this study, a possible supraspinal site of action of this drug was excluded by the C1 spinal transection. This data is in good agreement with previous reports on spinal reflexes and more recent experiments on dorsal horn interneurons. In the spinal animal, polysynaptic nociceptive reflexes are depressed by low doses of morphineS,2°,24,27,~l,5~ while stronger doses are needed to depress monosynaptic reflexes~5. Microphysiological studies have also emphasized a direct action of i.v. injection of morphinomimetics on dorsal interneurons: Iwata and Sakai 21 reported that fentanyl (20-40/~g/kg) suppressed the discharges of spinal interneurons evoked by stimulation of A0 cutaneous fibers in the spinal cat, while the responses of these cells to low intensity stimulation were unaffected by this drug. Similarly, Besson e t al. 7 mentioned that phenoperidine was more effective in depressing the responses of lamina V cells to intense electrical or natural stimuli than it was for responses to innocuous stimuli. Recently, Kitahata e t aL 2a described in the spinal cat a depressive effect of morphine upon lamina I and V units, but these authors mainly considered spontaneous activity of these cells. Moreover, the observed modifications were of very short duration since, even with 2 mg/kg of morphine, their time course curves clearly showed a total
274 recovery 10 min after administration of the drug. In our study, the depressive effects ol" morphine on both spontaneous and evoked activities are of longer duration (at least 40 rain) and are more consistent with analgesia obtained in therapeutical practice. A direct effect of morphine on dorsal horn interneurons has been confirmed by using iontophoretic approaches; Dostrovsky and Pomeranz t4 have shown that morphine reduced excitation of dorsal horn interneurons induced by direct application of glutamate and aspartate. However, it is difficult to understand why, in their report, iontophoretically applied morphine was unable to depress the evoked activities induced by peripheral stimulation since a recent study 12 has shown that responses of dorsal horn interneurons to noxious radiant heat were strongly depressed by the same application. Although no conclusive evidence can be advanced to explain the depressive effects of morphine on spinal interneurons, all these macro- and microphysiological data show a direct action of morphine and related compounds at this level. Our results showing in the spinal preparation a strong depressive effect of morphine on various activities of lamina V cells could in part explain the analgesic properties of morphine, since we have mentioned in the Introduction that these cells were strongly activated by small afferent fibers from various origins and that some of them were at the origin of several ascending pathways in cat and monkey. In the same way, Grossman and Jurna 16 recently found that morphine, at a dose as low as 0.5 mg/kg, depressed the activity of axons of the ventrolateral tract. However, our findings contrast with those of Takagi et al. 4a and Satoh and Takagi 4t who compared, in intact, decerebrate and spinal animals, the effects of large doses of morphine (4-10 mg/kg i.v.) on polysynaptic reflexes and on responses evoked in the ventrolateral funiculus by splanchnic stimulation. These authors concluded that morphine's site of action is located in the brain stem, acting on spinal transmission by reinforcing descending inhibitory controls. Nevertheless, from our results and from those previously mentioned, it seems difficult to refute a direct action of morphine at the spinal level. Moreover, certain depressive effects described by Takagi e t al. 4a and Satoh and Takagi 4t are of short duration and have been obtained by considering macrophysiological responses, the interpretation of which is not easy. Furthermore, they obtained similar results on unanesthetized or anesthetized preparations and there is no doubt that, owing to the doses of barbiturate used, the activities of spinal interneurons evoked by noxious stimulation are. in their experiments, totally suppressed 6,a°. However, our results do not exclude morphine's supraspinal sites of action. Indeed, strong analgesia has been observed by morphine application at ventricular and periventricular levels 3,17,22,a3,46,4v. These experiments seem to agree with the initial hypothesis of Takagi et al. 4a suggesting an increase of descending inhibitory control after morphine administration. Furthermore, some recent behavioral and pharmacological data has underlined the relationship between morphine analgesia and analgesia induced by stimulation of the periaqueductal gray matter of the rat 1,29,3° and of raphe nuclei in the cat 3z,37 (see also refs. 39, 40 and 48). These effects could be due in part to descending inhibitory effects on the transmission of nociceptive messages at the spinal cord level 32. Thus, several findings are in favor of supraspinal sites of action of narcotic
275
analgesics. It seems likely that morphine depresses spinal transmission both by a direct depressive effect at the spinal level and by activating descending inhibitory pathways. Studies of the action of morphine on spinal structures involved in pain processes, such as lamina V, in decerebrate and intact animals, would be useful to determine the relative importance of these two mechanisms. ACKNOWLEDGEMENTS
This work was supported by l'Institut National de la Sant6 et de la Recherche m6dicale (INSERM) ATP Pharmacod6pendance, Contrat No. 71-152015.
REFERENCES 1 AKIL, H., MAYER, D. J., ET LIEBESKIND,J. C., Comparaison chez le rat entre l'analg6sie induite par stimulation de la substance grise p6ri-aqueducale et I'analg~sie morphinique, C.R. Acad. Sci. (Paris), 274 (1972) 3603-3605. 2 ALBE-F,ESSARD,O., LEVANTE, A., AND LAMOUR, Y., Origin of spino-thalamic tract in monkeys, Brain Research, 65 (1974) 503-509. 3 ALaus, K., SCHOTr, M., ANt) HERZ, A., Interaction between morphine and morphine antagonists after systematic and intraventricular application, Europ. J. Pharmacol., 12 (1970) 53-64. 4 BENELLi, G. C., BESSON, J. M., GUILBAUD,G., ET LOMBARD, M. C., R6ponses des interneurones de la corne dorsale thoracique ~ des stimulations cutan6es et ~tdes stimulations visc6rales nociceptives, J. Physiol. (Paris), 69 (1974) 221A. 5 BESSON,J. M., CONSEILLER,C., HAMANN, K. F., AND MMLLARD, M. C., Modification of dorsal horn cell activities in the spinal cord, after intra-arterial injection of bradykinin, J. Physiol. (Lond.), 221 (1972) 189-205. 6 BESSON,J. M., Rlvor, J. P., ABDELMOUMENE,M., ET ALEONARD,P., Etude de l'action des anesth6siques sur l'activit6 des cellules, de la corne dorsale de la moelle, J. Physiol. (Paris), 62 (1970) 350-351. 7 BESSON, J. M., WYON-MAILLARD,M. C., BENOIST,J. M., CONSEILLER, C., AND HAMANN, K. F., Effects of phenoperidine on lamina V cells in the cat dorsal horn, J. Pharmacol. exp. Ther., 187 (1973) 239-245. 8 BODO, R. C., AND BROOKS, C. McC., The effect of morphine on blood sugar and reflex activity in the chronic spinal cat, J. Pharmacol. exp. Ther., 61 (1937) 82-88. 9 BROWN, A. G., AND F'RANZ,O. N., Responses of spinocervical tract neurones to natural stimulation of identified cutaneous receptors, Exp. Brain Res., 7 (1969) 231-249. 10 BRYAN, R. N., COULTER, J. D., AND WILLIS, W. D., Cells of origin of the spinovercical tract in the monkey, Exp. Neurol., 42 (1974) 574-586. 11 BRYAN, R. N., TREVINO, D. L., COULTER,J. D., AND WILLIS, W. D., Location and somatotopic organization of the cells of origin of the spinocervical tract, Exp. Brain Res., 17 (1973) 177-181. 12 CALVILLO,O., HENRY, J. U, AND NEUMAN, R. S., Effects of morphine and naloxone on dorsal horn neurones in the cat, Canad. J. Physiol. Pharmacol., 52 (1974) 1207-1211. 13 DILLY, P. N., WALL, P. D., AND WEBSTER,K. E., Cells of origin of the spinothalamic tract in the cat and rat, Exp. NeuroL, 21 (1968) 550-562. 14 DOSTROVSKY,J., ANt) POMERANZ,B., Morphine blockade of amino acid putative transmitters on cat spinal cord sensory interneurones, Nature New Biol., 246 (1973) 222-224. 15 GREGOR, M., AND ZIMMERMANN, M., Characteristics of spinal neurones responding to cutaneous myelinated and unmyelinated fibres, J. Physiol. (Lond.), 221 (1972) 255-276. 16 GROSSMANN,W., AND JURNA, I., Depression by morphine of activity in the ventrolateral tract evoked from cutaneous A-fibres, Europ. J. Pharmacol., 29 (1974) 171-174. 17 HERZ, A., ALnUS, K., METY~, J., SCSUBERT,P., AND TESCHEMACHER, HJ., On the central sites for the antinociceptive action of morphine and fentanyl, Neuropharmacology, 9 (1970) 539-551. 18 HILLMAN, P., AND WALL, P. D., Inhibitory and excitatory factors influencing the receptive fields of lamina V spinal cord cells, Exp. Brain Res., 9 (1969) 284-306.
276 19 HONGO, T., JANKOWSKA, E.. AND LUNDSEr(h A., Postsynaptic excitation and inhibition from primary afferents in neurones of the spino-cervical tract, J. Physiol. (Lond.), 199 (1968) 569-592. 20 IRWIN, S., HOUDE, R. W., BENNETT, D. R.. HENDERSHOT, L. C., AND SEEVERS, M. H., The effects of morphine, methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation, J. Pkarmacol. exp. Ther., 101 (1951) 132-143. 21 IWATA, N., AND SAKAI, Y., Effects of fentanyl upon the spinal interneurons activated by A~5 afferent fibers of the cutaneous nerve of the cat, Jap. J. Pkarmacol., 21 (1971 ) 413-416. 22 JACQUET, Y. ]-'., AND LAJTHA. A., Morphine action at central nervous systern sites in rat: analgesia or hyperalgia depending on site and dose, Science, 182 (I 973) 490-492. 23 K~TAHATA, L. M., KOSAKA, Y., TAUB, A., BONIKOS, K., AND HOFFERT, M., Lamina specific suppression of dorsal-horn unit activity by morphine sulfate, Anesthesiology, 41 (1974) 39-48. 24 KOLL, W., HAASE, J., BLOCK, G., AND MUHLBERG, B., The predilective action of small doses of morphine on nociceptive spinal reflexes of low spinal cats, Int. J. Neuropkarmacol., 2 (1963) 57 65. 25 KmvoY, W., KROEGER, D., AND ZIMMERMANN,E., Actions of morphine on the segmental reflex ot decerebrate-spinal cat, Brit. J. Pharmacol., 47 (I 973) 457-464. 26 LEVANTE, A., LAMOUR, Y., ET ALBE-FESSARD, D., Localisation dans la couche V de Rexed de cellules d'origine d'un faisceau spino-thalamique crois6 chez le macaque, C.R, Acad. Sci. (Paris), 276 (1973) 1589-1592. 27 MCCLANE, T. K., AND MARTIN, W. R., Effects of morphine, nalorphine, cyclazocine and naloxone on the flexor reflex, Int. J. Neuropharmacol., 6 (1967) 89-98. 28 MCKENZIE, J. S., AND BEECHEY, N. R., The effects of morphine and pethidine on somatic evoked responses in the midbrain of the cat, and their relevance to analgesia, Electroenceph. clin. Neurophysiol., 14 (1962) 501-519. 29 MAYER, D. J., AND LIEBESKINO, J. C., Pain reduction of focal electrical stimulation of the brain: an anatomical and behavioral analysis, Brain Research, 68 (1974) 73-94. 30 MAYER, D. J., WOLFLE, T. L., AKIL, H., CARDER, B,, AND LIEBESKIND, J. C., Analgesia from electrical stimulation in the brain stem of the rat, Science, 174 (1971) 1351-1354. 31 MENOELL, L. M., Physiological properties of unmyelinated fibre projection to the spinal cord, Exp. Neurol., 16 (1966) 316-332. 32 OUVERAS, J. L., BESSON, J. M., GUlLSAUD, G., ANt) LJEBESKIND, J. C., Behavioral and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat, Exp. Brain Res., 20 (1974) 32 44. 33 PERT, A., AND YAKSH, T., Sites of morphine induced analgesia in the prirnate brain: relation to pain pathways, Brain Research, 80 (1974) 135-140. 34 POMERANZ,B., WALL, P. D., AND WEnER, W. V., Cord cells responding to fine myelinated afferents from viscera, muscle and skin, J. Physiol. (Lond.), 199 (1968) 511-532. 35 PRICE, D. D., AND BROWE, A. C., Responses of spinal cord neurons to graded noxious and nonnoxious stimuli, Brain Research, 64 (1973) 425-529. 36 PRICE, D. D., AND WAGMAN, 1. H., Physiological roles of A and C fibre inputs to the spinal dorsal horn of Macaca nttdalta, Exp. Neurol., 29 (1970) 383-399. 37 REDJEMI, F., OLIVERAS, J. L., GUILBAUD, G., ET BESSON, J. M., Analgesic induite par la stimulation du noyau central inf6rieur du raph6 chez le chat, C.R. Acad. Sci. (Paris), 279 (1974) 1105-1107. 38 REXED, B., The cytoarchitectonic organization of the spinal cord in the cat, J. comp. Neurol., 16 (1952) 415-495. 39 SAMANIN, R., GUMULKA, W., AND VALZELH, L., Reduced effect of morphine in midbrain raphe lesioned rats, Earop. J. Pharmacol., 10 (1970) 339-343. 40 SAMANIN, R., ANt) VALZELLL L., Increase of morphine-induced analgesia by stimulation of the nucleus raphe dorsalis, Europ. J. Pharmacol., 16 (1971) 298-302. 41 SATOH, M., AND TAKAGI, H., Enhancement by morphine of the central descending inhibitory influence on spinal sensory transmission, Europ. J. Pharmacol., 14 (1971) 60-65. 42 SELZER, M., AND SPENCVR, W. A., Convergence of visceral and cutaneous afferent pathways in the lumbar spinal cord, Brain Research, 14 (1969) 331-348. 43 TAKAGI, n., MATSUNARA, M., YANAI, m., AND OGIU, K., The effect of analgesics on the spinal reflex activity of the cat, Jap. J. Pharmacol., 4 (1955) 176-187. 44 TAUB, A., AND BISHOP, P. O., The spinocervical tract: dorsal column linkage, conduction velocity, primary afferent spectrum, Exp. Neurol., 13 (1965) 1-21. 45 TREVINO, D. L., COULTER, J. D., AND WILLIS, W. D., Location of cells of origin of spinothalamic tract in lumbar enlargement of the monkey, J. Neurophysiol., 36 (1973) 750-761.
277 46 Tsou, K., AND JANG, C. S., Studies on the site of analgesic action of morphine by intracerebral microinjections, Scientia Sinica, 13 (1964) 1099-1109. 47 VIOOURET,J., TESCHEMACHER,HJ., ALBUS,K., AND HERZ, A., Differentiation between spinal and supraspinal sites of action of morphine when inhibiting the hindleg flexor reflex in rabbits, Neuropharmacology, 12 (1973) 111-121. 48 VOGT,M., The effect of lowering the 5-hydroxytryptamine content of the rat spinal cord on analgesia produced by morphine, J. Physiol. (Lond.), 236 (1974) 483--498. 49 WAGMANN,I. H., AND PRICE, D. D., Responses of dorsal horn cells of Macaca mulatta to cutaneous and sural nerve A and C fibre stimuli, J. Neurophysiol., 32 (1969) 803-817. 50 WALL, P. n., The laminar organization of dorsal horn and effects of descending impulses, J. Physiol. (Lond), 188 0967) 403-423. 51 WIKLER,A., Studies on the action of morphine on the central nervous system of the cat, J. Pharmacol, exp. Ther., 80 (1944) 176-187. 52 WIKLER,k., AND FRANK, K., Hindlimb reflexes of chronic spinal dogs during cycles of addiction to morphine and methadone, J. Pharmacol. exp. Ther., 94 (1948) 382-400. 53 WILLIS,W. D., TREVINO,D. L., COULTER,J. D., AND MAUNZ,R. A., Response of primate spinothalamic tract neurons to natural stimulation of hindlimb, J. Neurophysiol., 37 (1974) 358-372.