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Selective and non-selective inhibition by enkephalins and noradrenaline of nociceptive response of lamina V type neurons in the spinal dorsal horn of the rabbit
384
Brain Research, 177 (1979) 384-387 © Elsevier/North-HollandBiomedicalPress
Selective and non-selective inhibition by enkephalins and noradrenaline of nociceptive response of lamina V type neurons in the spinal dorsal horn of the rabbit
MASAMICHI SATOH, SHIN-ICHI KAWAJIR[, YOJIRO UKA! and MASAKI YAMAMOTO Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606 (Japan)
(Accepted July 26th, 1979)
Methionine-enkephalin (M-Enk) and leucine-enkephalin (L-Enk) have been shown to be contained in moderate to high levels in interneurons of the spinal dorsal horn 5 and to produce a definite analgesic effect when intrathecally injected in large doses (200-400/~g)11. However, there are conflicting reports as to the effects of M- and L-Enk on single neuronal activities in the dorsal horn z,12. Previous studies in which minute doses of morphine were injected into the nucleus reticularis gigantocellularis of the medulla oblongata showed that noradrenaline (NA) probably is the particular transmitter involved in mediating descending inhibitions to the spinal cord 6. In the present experiments, M- and L-Enk and NA microelectrophoretically applied to the lamina V type neurons of the dorsal horn were examined and their actions on nociceptive and non-nociceptive responses compared in these neurons. Experiments were performed on ether-anesthetized, male albino rabbits (2.5-3.5 kg), the spinal cords of which were exposed by lumbar laminectomy and transected at the L2 level. All animals were immobilized with gallamine triethiodide and artificially respired. For the peripheral noxious stimulation, single injections of bradykinin (BK, Protein Research Foundation, Mino, Japan; 0.5-5.0 #g) were given into the femoral artery at intervals of 5 min 9. Tactile stimulation, in the same direction as the hair or skin at the center of the receptive field, once a see for 16 see with intervals of 5 min, was used as innocuous stimuli, The methods for extracellular recording and microelectrophoresis were described elsewhere s. Solutions for phoresis were: monosodium-L-glutamate (1 M), M- and L-Enk (kindly provided by Prof. H. Yajima, Kyoto University, Kyoto, Japan; 10 mM in 165 mM NaC1), (--)Sodium bitartarate (0.1 M, pH 4.3), naloxone HC1 (a kind gift from Endo Laboratories, Garden City, U.S.A. ; 50 mM in 165 mM NaC1) and NaC1 (3 M). The pHs of these solutions except for NA were about 5.0. Lamina V type cells were distinguished according to the electrophysiological properties described by WalP °. We selected only units having a signal-noise ratio of over 3, which did not change in spike shape for 30-90 min and which were excited by intra-arterial injection of BK and by glutamate phoretically applied (less than 200 nA). The difference between the numbers of spikes for 64 sec after and before each BK
385 injection was as BK-induced activity. The 95 ~ confidence limits were calculated from at least 3 values of BK-induced activities before electrophoresis of a test substance. If the values during and/or after such an application were either below or above the limits and then returned within the limits, the effect was regarded as being inhibitory or excitatory. A similar analysis of data was made for assessing the effects on the nonnociceptive response evoked by tactile stimulation. In this analysis, the difference between the numbers of spikes for 16 sec during and immediately before the stimulation was regarded as tactile-induced activity. Results were obtained from 51 lamina V type neurons of the spinal dorsal horn. Nineteen of the 51 neurons consistently responded with bursts of spikes to the tactile stimulation. As M- and L-Enk resulted in almost identical effects in this study, the data obtained with both substances were pooled. M- and L-Enk phoretically applied (mean 140 nA ~ 12 nA S.E. (n----31), 4-5 min) significantly inhibited the BK-induced activity in 25 of 31 lamina V type neurons tested, but had no influence on the other 6 neurons. The tactile-induced activity was not altered by either enkephalin in any of 8 lamina V type neurons tested, in 7 of which the BK response was inhibited as shown in the upper chart of Fig. 1. The inhibitory action on the nociceptive response appeared within 2 min, disappeared within 3 min after termination of phoresis and was blocked in 6 of 8 neurons when naloxone was simultaneously applied phoretically (100-200 nA), and in all of 4 neurons when the drug was intravenously administered (0.2-0.5 mg/kg), indicating that such an inhibitory effect of M- and L-Enk was mediated by specific opiate receptors. NA phoretically applied (mean 94 nA -4- 6 nA S.E. (n=20), 4-5 min) suppressed the BK-induced activity in 12 of 20 lamina V type neurons and the tactile-induced activity in 7 of 11. In all of the latter 7 neurons, the BK response was also inhibited as shown in the lower chart of Fig. 1. The suppressive effect of NA appeared within 2 min, disappeared 10-30 min after termination of phoresis and was not blocked by naloxone phoretically (200 nA) or intravenously (0.5 mg/kg) administered in any of 8 neurons examined. All the actions of phoretically applied M- and LEnk, and NA described above were obtained without changes in spike height and shape. The present experiments demonstrated that phoretically applied M- and L-Enk, naturally occurring pentapeptides, selectively inhibited the response of lamina V type neurons to noxious stimulation through specific opiate receptors. Such selective inhibition of nociceptive reponse was seen even in the same neuron and suggests the participation of presynaptic action of the enkephalins, as was also suggested by recent electrophysiological studies 7. Judging from the nature of the tips of electrodes used in the present experiments (the recording electrode protruded beyond the tip of the micropipette by 20-30 #m), the enkephalins ejected from the pipette may not have reached the substantia gelatinosa but preferentially may have been applied to the more proximal dendritic region of the lamina V type neuron. Duggan et a13 reported that M-Enk itself administered in the substantia gelatinosa did not influence the nociceptive response in 6 of 8 dorsal horn neurons tested, although an analogue of M-Enk (MEnk-amide) ejected in the same region reduced such a response in 19 of 29. From these findings it is conceivable that a site of action of naturally occurring M- and L-Enk in
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Fig. 1. Effects of electrophoreticallyapplied leucine-enkephalin (L-E; upper chart) and noradrenaline (NA ; lower chart) on the bradykinin (BK; noxious)- and tactile (innocuous)-evoked responses of the lamina V type neurons of the spinal dorsal horn. Intra-arterial injections of BK (1.0/~g) and tactile stimulation for 16 see were alternately given at thin and thick arrows, respectively, with fixed intervals. In each neuron, 3 pairs of noxious and innocuous stimuli were given as control trials before electrophoresis, but the responses to the first pair of stimuli were omitted in this figure. Stars indicate a significant decrease in the BK- or tactile-induced activity. The intermission period in the lower chart is 10 min. Note: L-E selectively depressed the BK-induced activity, while NA non-selectively suppressed both the BK- and tactile-induced activities. the spinal d o r s a l h o r n m a y n o t be p r i m a r i l y located in the s u b s t a n t i a gelatinosa b u t in the m o r e p r o x i m a l dendritic region o f the l a m i n a V type neuron. In the present experiments using the same type o f electrodes, phoretically a p p l i e d N A p r o d u c e d non-selective i n h i b i t i o n o f nociceptive a n d non-nociceptive responses in the same l a m i n a V type neurons, suggesting the involvement o f p o s t s y n a p t i c action o f N A which was s h o w n b y an intracellular r e c o r d i n g technique 3. Recently Belcher et al. 1 r e p o r t e d t h a t N A phoretically a p p l i e d n e a r the cell bodies i n h i b i t e d nociceptive response o f the d o r s a l h o r n interneurons, b u t did n o t describe the effect o f N A on the activity evoked by i n n o c u o u s stimuli in the same neurons. O n the other hand, o t h e r workers 4 showed t h a t N A ejected in the s u b s t a n t i a gelatinosa p r o d u c e d a selective r e d u c t i o n o f nociceptive response o f the d o r s a l h o r n neurons. These discrepancies m a y
387 result f r o m the differences in m e t h o d s for a p p l i c a t i o n o f N A a n d / o r c o m p l e x i t y o f the effects o f N A on the d o r s a l h o r n neurons. I n conclusion, o u r findings indicate t h a t M - a n d L - E n k , a n d N A phoretically a p p l i e d preferentially to the p r o x i m a l d e n d r i t i c r e g i o n o f the l a m i n a V type n e u r o n i n h i b i t nociceptive response o f such a n e u r o n , p r o b a b l y t h r o u g h p r e s y n a p t i c m e c h a n isms with M - a n d L - E n k , a n d p o s t s y n a p t i c m e c h a n i s m s with N A , respectively. W e t h a n k Prof. H. T a k a g i , K y o t o University, for v a l u a b l e c o m m e n t s a n d criticism in this study a n d M. O h a r a , K y o t o University, for assistance with the manuscript. This study was s u p p o r t e d in p a r t b y a Scientific F u n d (167279) f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e o f J a p a n .
1 Belcher, G., Ryail, R. W. and Schaffner, R., The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurons in the cat, Brain Research, 151 (1978) 307-321. 2 Duggan, A. W., Hall, J. G. and Headley, P. M., Enkephalins and dorsal horn neurons of the cat: effects on responses to noxious and innocuous skin stimuli, Brit. J. Pharmacol., 61 (1977) 399--408. 3 Engberg, I. and Marshall, K. C., Mechanism of noradrenaline hyperpolarization in spinal cord motoneurones of the cat, Actaphysiol. scand., 83 (1971) 142-144. 4 Headley, P. M., Duggan, A. W. and Griersmith, B. T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones, Brain Research, 145 (1978) 185-189. 5 H6kfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3081-3085. 6 Kuraishi, Y., Harada, Y., Satoh, M. and Takagi, H., Antagonism by phenoxybenzamine of the analgesic effect of morphine injected into the nucleus reticularis gigantocellularis of the rat, Neuropharmacology, 18 (1979) 107-110. 7 Macdonald, R. L. and Nelson, P. G., Specific opiate-induced depression of transmitter release from dorsal root ganglion cells in culture, Science, 199 (1978) 1449-1451. 8 Satoh, M., Akaike, A. and Takagi, H., Excitation by morphine and enkephalin of single neurons of nucleus reticularis paragigantocellularis in the rat: a probable mechanism of analgesic action of opioids, Brain Research, 169 (1979) 406-410. 9 Satoh, M., Nakamura, N. and Takagi, H., Effect of morphine on bradykinin-induced unitary discharges in the spinal cord of the rabbit, Europ. J. Pharmacol., 16 (1971) 245-247. 10 Wall, P. D., The laminar organization of the dorsal horn and effects of descending impulses, J. PhysioL (Lond.), 188 (1967) 403-423. 11 Yaksh, T. L., Huang, S. P., Rudy, T. A. and Frederickson, R. C. A., The direct and specific opiatelike effect of MetS-enkephalin and analogues on the spinal cord, Neuroscience, 2 (1977) 593-596. 12 Zieglghnsberger, W. and Tulloch, I. F., The effects of methionine- and leucine-enkephalin of spinal neurons of the cat, Brain Research, 167 (1979) 53-64.
Brain Research, 177 (1979) 384-387 © Elsevier/North-HollandBiomedicalPress
Selective and non-selective inhibition by enkephalins and noradrenaline of nociceptive response of lamina V type neurons in the spinal dorsal horn of the rabbit
MASAMICHI SATOH, SHIN-ICHI KAWAJIR[, YOJIRO UKA! and MASAKI YAMAMOTO Department of Pharmacology, Faculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606 (Japan)
(Accepted July 26th, 1979)
Methionine-enkephalin (M-Enk) and leucine-enkephalin (L-Enk) have been shown to be contained in moderate to high levels in interneurons of the spinal dorsal horn 5 and to produce a definite analgesic effect when intrathecally injected in large doses (200-400/~g)11. However, there are conflicting reports as to the effects of M- and L-Enk on single neuronal activities in the dorsal horn z,12. Previous studies in which minute doses of morphine were injected into the nucleus reticularis gigantocellularis of the medulla oblongata showed that noradrenaline (NA) probably is the particular transmitter involved in mediating descending inhibitions to the spinal cord 6. In the present experiments, M- and L-Enk and NA microelectrophoretically applied to the lamina V type neurons of the dorsal horn were examined and their actions on nociceptive and non-nociceptive responses compared in these neurons. Experiments were performed on ether-anesthetized, male albino rabbits (2.5-3.5 kg), the spinal cords of which were exposed by lumbar laminectomy and transected at the L2 level. All animals were immobilized with gallamine triethiodide and artificially respired. For the peripheral noxious stimulation, single injections of bradykinin (BK, Protein Research Foundation, Mino, Japan; 0.5-5.0 #g) were given into the femoral artery at intervals of 5 min 9. Tactile stimulation, in the same direction as the hair or skin at the center of the receptive field, once a see for 16 see with intervals of 5 min, was used as innocuous stimuli, The methods for extracellular recording and microelectrophoresis were described elsewhere s. Solutions for phoresis were: monosodium-L-glutamate (1 M), M- and L-Enk (kindly provided by Prof. H. Yajima, Kyoto University, Kyoto, Japan; 10 mM in 165 mM NaC1), (--)Sodium bitartarate (0.1 M, pH 4.3), naloxone HC1 (a kind gift from Endo Laboratories, Garden City, U.S.A. ; 50 mM in 165 mM NaC1) and NaC1 (3 M). The pHs of these solutions except for NA were about 5.0. Lamina V type cells were distinguished according to the electrophysiological properties described by WalP °. We selected only units having a signal-noise ratio of over 3, which did not change in spike shape for 30-90 min and which were excited by intra-arterial injection of BK and by glutamate phoretically applied (less than 200 nA). The difference between the numbers of spikes for 64 sec after and before each BK
385 injection was as BK-induced activity. The 95 ~ confidence limits were calculated from at least 3 values of BK-induced activities before electrophoresis of a test substance. If the values during and/or after such an application were either below or above the limits and then returned within the limits, the effect was regarded as being inhibitory or excitatory. A similar analysis of data was made for assessing the effects on the nonnociceptive response evoked by tactile stimulation. In this analysis, the difference between the numbers of spikes for 16 sec during and immediately before the stimulation was regarded as tactile-induced activity. Results were obtained from 51 lamina V type neurons of the spinal dorsal horn. Nineteen of the 51 neurons consistently responded with bursts of spikes to the tactile stimulation. As M- and L-Enk resulted in almost identical effects in this study, the data obtained with both substances were pooled. M- and L-Enk phoretically applied (mean 140 nA ~ 12 nA S.E. (n----31), 4-5 min) significantly inhibited the BK-induced activity in 25 of 31 lamina V type neurons tested, but had no influence on the other 6 neurons. The tactile-induced activity was not altered by either enkephalin in any of 8 lamina V type neurons tested, in 7 of which the BK response was inhibited as shown in the upper chart of Fig. 1. The inhibitory action on the nociceptive response appeared within 2 min, disappeared within 3 min after termination of phoresis and was blocked in 6 of 8 neurons when naloxone was simultaneously applied phoretically (100-200 nA), and in all of 4 neurons when the drug was intravenously administered (0.2-0.5 mg/kg), indicating that such an inhibitory effect of M- and L-Enk was mediated by specific opiate receptors. NA phoretically applied (mean 94 nA -4- 6 nA S.E. (n=20), 4-5 min) suppressed the BK-induced activity in 12 of 20 lamina V type neurons and the tactile-induced activity in 7 of 11. In all of the latter 7 neurons, the BK response was also inhibited as shown in the lower chart of Fig. 1. The suppressive effect of NA appeared within 2 min, disappeared 10-30 min after termination of phoresis and was not blocked by naloxone phoretically (200 nA) or intravenously (0.5 mg/kg) administered in any of 8 neurons examined. All the actions of phoretically applied M- and LEnk, and NA described above were obtained without changes in spike height and shape. The present experiments demonstrated that phoretically applied M- and L-Enk, naturally occurring pentapeptides, selectively inhibited the response of lamina V type neurons to noxious stimulation through specific opiate receptors. Such selective inhibition of nociceptive reponse was seen even in the same neuron and suggests the participation of presynaptic action of the enkephalins, as was also suggested by recent electrophysiological studies 7. Judging from the nature of the tips of electrodes used in the present experiments (the recording electrode protruded beyond the tip of the micropipette by 20-30 #m), the enkephalins ejected from the pipette may not have reached the substantia gelatinosa but preferentially may have been applied to the more proximal dendritic region of the lamina V type neuron. Duggan et a13 reported that M-Enk itself administered in the substantia gelatinosa did not influence the nociceptive response in 6 of 8 dorsal horn neurons tested, although an analogue of M-Enk (MEnk-amide) ejected in the same region reduced such a response in 19 of 29. From these findings it is conceivable that a site of action of naturally occurring M- and L-Enk in
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Fig. 1. Effects of electrophoreticallyapplied leucine-enkephalin (L-E; upper chart) and noradrenaline (NA ; lower chart) on the bradykinin (BK; noxious)- and tactile (innocuous)-evoked responses of the lamina V type neurons of the spinal dorsal horn. Intra-arterial injections of BK (1.0/~g) and tactile stimulation for 16 see were alternately given at thin and thick arrows, respectively, with fixed intervals. In each neuron, 3 pairs of noxious and innocuous stimuli were given as control trials before electrophoresis, but the responses to the first pair of stimuli were omitted in this figure. Stars indicate a significant decrease in the BK- or tactile-induced activity. The intermission period in the lower chart is 10 min. Note: L-E selectively depressed the BK-induced activity, while NA non-selectively suppressed both the BK- and tactile-induced activities. the spinal d o r s a l h o r n m a y n o t be p r i m a r i l y located in the s u b s t a n t i a gelatinosa b u t in the m o r e p r o x i m a l dendritic region o f the l a m i n a V type neuron. In the present experiments using the same type o f electrodes, phoretically a p p l i e d N A p r o d u c e d non-selective i n h i b i t i o n o f nociceptive a n d non-nociceptive responses in the same l a m i n a V type neurons, suggesting the involvement o f p o s t s y n a p t i c action o f N A which was s h o w n b y an intracellular r e c o r d i n g technique 3. Recently Belcher et al. 1 r e p o r t e d t h a t N A phoretically a p p l i e d n e a r the cell bodies i n h i b i t e d nociceptive response o f the d o r s a l h o r n interneurons, b u t did n o t describe the effect o f N A on the activity evoked by i n n o c u o u s stimuli in the same neurons. O n the other hand, o t h e r workers 4 showed t h a t N A ejected in the s u b s t a n t i a gelatinosa p r o d u c e d a selective r e d u c t i o n o f nociceptive response o f the d o r s a l h o r n neurons. These discrepancies m a y
387 result f r o m the differences in m e t h o d s for a p p l i c a t i o n o f N A a n d / o r c o m p l e x i t y o f the effects o f N A on the d o r s a l h o r n neurons. I n conclusion, o u r findings indicate t h a t M - a n d L - E n k , a n d N A phoretically a p p l i e d preferentially to the p r o x i m a l d e n d r i t i c r e g i o n o f the l a m i n a V type n e u r o n i n h i b i t nociceptive response o f such a n e u r o n , p r o b a b l y t h r o u g h p r e s y n a p t i c m e c h a n isms with M - a n d L - E n k , a n d p o s t s y n a p t i c m e c h a n i s m s with N A , respectively. W e t h a n k Prof. H. T a k a g i , K y o t o University, for v a l u a b l e c o m m e n t s a n d criticism in this study a n d M. O h a r a , K y o t o University, for assistance with the manuscript. This study was s u p p o r t e d in p a r t b y a Scientific F u n d (167279) f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d C u l t u r e o f J a p a n .
1 Belcher, G., Ryail, R. W. and Schaffner, R., The differential effects of 5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive and non-nociceptive dorsal horn interneurons in the cat, Brain Research, 151 (1978) 307-321. 2 Duggan, A. W., Hall, J. G. and Headley, P. M., Enkephalins and dorsal horn neurons of the cat: effects on responses to noxious and innocuous skin stimuli, Brit. J. Pharmacol., 61 (1977) 399--408. 3 Engberg, I. and Marshall, K. C., Mechanism of noradrenaline hyperpolarization in spinal cord motoneurones of the cat, Actaphysiol. scand., 83 (1971) 142-144. 4 Headley, P. M., Duggan, A. W. and Griersmith, B. T., Selective reduction by noradrenaline and 5-hydroxytryptamine of nociceptive responses of cat dorsal horn neurones, Brain Research, 145 (1978) 185-189. 5 H6kfelt, T., Ljungdahl, A., Terenius, L., Elde, R. and Nilsson, G., Immunohistochemical analysis of peptide pathways possibly related to pain and analgesia: enkephalin and substance P, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 3081-3085. 6 Kuraishi, Y., Harada, Y., Satoh, M. and Takagi, H., Antagonism by phenoxybenzamine of the analgesic effect of morphine injected into the nucleus reticularis gigantocellularis of the rat, Neuropharmacology, 18 (1979) 107-110. 7 Macdonald, R. L. and Nelson, P. G., Specific opiate-induced depression of transmitter release from dorsal root ganglion cells in culture, Science, 199 (1978) 1449-1451. 8 Satoh, M., Akaike, A. and Takagi, H., Excitation by morphine and enkephalin of single neurons of nucleus reticularis paragigantocellularis in the rat: a probable mechanism of analgesic action of opioids, Brain Research, 169 (1979) 406-410. 9 Satoh, M., Nakamura, N. and Takagi, H., Effect of morphine on bradykinin-induced unitary discharges in the spinal cord of the rabbit, Europ. J. Pharmacol., 16 (1971) 245-247. 10 Wall, P. D., The laminar organization of the dorsal horn and effects of descending impulses, J. PhysioL (Lond.), 188 (1967) 403-423. 11 Yaksh, T. L., Huang, S. P., Rudy, T. A. and Frederickson, R. C. A., The direct and specific opiatelike effect of MetS-enkephalin and analogues on the spinal cord, Neuroscience, 2 (1977) 593-596. 12 Zieglghnsberger, W. and Tulloch, I. F., The effects of methionine- and leucine-enkephalin of spinal neurons of the cat, Brain Research, 167 (1979) 53-64.