Brain Research, 170 (1979) 209-213 © Elsevier/North-Holland Biomedical Press
209
Capsaicin-evoked release of substance P from primary sensory neurons
ELIZABETH THERIAULT*, MASANORI OTSUKA and THOMAS JESSELL** Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ka, Tokyo 113 (Japan)
(Accepted March 8th, 1979)
The presence of the peptide substance P in small unmyelinated primary sensory neurons 5 appears to be related to the transmission of nociceptive impulses at the level of the spinal cord. Release of substance P has been demonstrated from the rat spinal cord, in vitro, in response to electrical stimulation of the dorsal roots 1~. Dorsal horn neurons that respond to noxious peripheral stimuli are excited by the iontophoretic application of substance p3,ts and the peptide also produces a depolarization of ventral roots in the isolated rat spinal cord preparation 12,t6. Substance P-containing sensory neurons may then represent one target for the pharmacological modification of pain transmission within the spinal cord. The analgesic properties of the opiates, in particular, may be mediated by the inhibition of substance P release from primary sensory neuronsg, ~3. Although it has been known for many years that repeated administration of the homovanillylamide derivative, capsaicin, renders animals insensitive to painful chemogenic stimuli 8, the mechanism of action of this compound has remained obscure. Recently chronic capsaicin treatment has been shown to produce a dramatic and specific depletion of substance P within primary sensory neurons in the rat dorsal horn 10 suggesting a functional association between the effects of capsaicin and substance P. Moreover, the acute effects of capsaicin administration in vivo are characterized by intense pain which might be consistent with a capsaicin-evoked stimulation of substance P release from the central terminals of primary sensory neurons. To investigate this possibility we have examined the effects of capsaicin in the isolated rat spinal cord preparation 14 and on the release of substance P from spinal cord tissue. Spinal cords were removed from 0-4-day-old Wistar rats according to the method of Otsuka and Konishi is. Hemisected cords were superfused with artificial cerebrospinal fluid (CSF) at 27 °C. The composition of artificial CSF was as follows (mM): NaCI 138.6 ; KCI 3.35 ; CaC12 1.26 ; MgClz 1.16 ; NaHCO3 21.0 ; NaH2PO4 0.58 ; glucose 10 ; gassed with 95 ~o 02--5 ~o CO2. Potential changes generated in mote* Present address: Department of Neurosciences,McMaster University, Hamilton, Ontario, Canada ** Present address and address for correspondence: Department of Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston Mass. 02115, U.S.A.
210 a
i
ii
iii
~
i
ii
iii
I
m
Imin
L Imin
Fig. 1. The effect of substance P and capsaicin on the potential recorded from the ventral root of the isolated rat spinal cord. Drugs were applied for 40 sec as marked over records, a: depolarization of ventral roots induced by substance P (5 x 10 7 M ) - - ( i ) response in artificial CSF; (ii) response in artificial CSF containing 0.1 mM calcium and 2.0 mM magnesium; (iii) recovery of response after return to normal CSF. b: depolarization of ventral root induced by capsaicin (5 × 10 7 M ) - - (J), (ii) and (iii) as in (a). c: effect of baclofen (2.3 x 10-6 M) on ventral root depolarization induced by substance P, (i) depolarization of ventral root by substance P (2 x 10-7 M), (ii) response to substance P after addition of baclofen, (iii) recovery after removal of baclofen, d: effect of baclofen on capsaicininduced depolarization, (i) depolarization evoked by capsaicin (5 × I0 7 M) in artificial CSF, (ii) response after addition of baclofen, (iii) recovery after removal of baclofen. neurons were recorded with extracellular suction electrodes, as described previously 14, from ventral roots (L3-Ls) during bath application of drug solutions. Capsaicin solution was prepared according to the method o f Jancso et al.L Application o f substance P (5 x 10 -7 M ) t o the superfusion medium caused a marked depolarization o f the ventral root (Fig. ! a) in agreement with previous observations 18. Ventral root depolarization occurred within 3-5 sec o f addition of substance P to the recording chamber. Addition o f capsaicin (5 x 10 -7 M ) t o the superfusion medium produced a depolarization o f the ventral root which closely resembled the response to substance P (Fig. 1b). Similar results were obtained in 7 separate experiments. The depolarization of ventral roots induced by capsaicin and substance P was dose-dependent over a concentration range of 5 × 10-s M to 10 -6 M. To examine whether the depolarizing action of capsaicin is mediated by a direct effect on spinal m o t o n e u r o n s or by a transsynaptic action, both substance P and capsaicin were applied in artificial C S F in which the calcium ion concentration had been reduced to 0.1 m M and the magnesium ion concentration raised to 2.0 mM. In every experiment this change in the divalent cation concentration was sufficient to abolish ventral root depolarization evoked by dorsal root stimulation. The substance P-induced depolarization was unaffected (often a slight potentiation) by a reduction in calcium ion concentration (Fig. la). In contrast the depolarization of motoneurons induced by capsaicin was almost completely abolished in artificial C S F containing 0.1 m M calcium and 2.0 m M magnesium (Fig. lb). The depolarizing action ofcapsaicin was completely restored by superfusing the hemisected cord with artificial CSF containing 1.26 m M calcium and 1.16 m M magnesium (Fig. lb). It seems likely, therefore, that the capsaicin-induced depolarization o f spinal motoneurons is mediated transsynaptically and not by a direct action on motoneurons.
211 In the isolated rat spinal cord preparation ventral root reflexes evoked by dorsal root stimulation are depressed by the drug baclofen 19. In the isolated spinal cord, substance P-induced depolarization of ventral roots is antagonized by bath application of baclofen 17,19 although the specificity of this compound seems to be critically dependent on the site and mode of administration (see discussion in refs. 2 and 17). The actions of substance P and capsaicin were compared in the presence of baclofen (2.3 × 10-e M); a concentration sufficient to abolish ventral root reflexes evoked by dorsal root stimulation 17,19. Depolarization of ventral roots in response to substance P was greatly depressed by addition of baclofen (Fig. lc) and capsaicin-induced depolarization was completely abolished (Fig. ld). Removal of baclofen resulted in complete recovery of substance P and capsaicin-induced depolarization (Fig. lc, d). The ability of capsaicin to release substance P from nerve terminals in the isolated rat spinal cord was also examined. Hemisected cords (T5 to caudal extremity) were placed in test tubes containing artificial CSF and then washed for 10 min in artificial CSF (normal or low calcium) and then transferred to tubes containing 1.0 ml of incubation solution for 30 min. During incubation the solution was gassed with 95 % 02-5 % CO2. Dithiothreitol (6 × 10-e M) was added to all samples to avoid oxidation of substance P. Incubation samples were immediately frozen at -80 °C, lyophilized, and the substance P content in each sample measured by radioimmunoassay with a sensitivity of 8 fmol/sample 2°. The unstimulated effiux of substance P from one hemisected spinal cord over a 30-min period was 56.5 -4- 29.6 fmol (Table 1). Incubation with capsaicin (10 -6 M) resulted in a 5-fold increase in substance P release to 287.3 _4_ 33.9 fmol/30 min. In the presence of capsaicin in artificial CSF containing 0.1 mM calcium and 2.0 mM magnesium, the release of substance P was 60.8 ± 24.2 fmol/30 min. Thus the capsaicin-evoked release of substance P was almost completely abolished by reducing the calcium concentration in the superfusing medium. These results clearly demonstrate that capsaicin produces a calcium-dependent release of substance P from rat spinal cord. The depolarization of spinal motoneurons by capsaicin may also be mediated by the release of substance P from nerve telminals. TABLE I Capsaicin-evoked release of substance P from newborn rat spinal cord
Each value is the mean 4- S.E.M. from 3 separate experiments. Incubating solution
fmol substance P released during 30 min period
Normal ACSF 56.5 4- 29.6 Normal ACSF +10 -6 M capsaicin 287.3i 33.9* ACSF containing: 0.1 mM Ca 2+2.0 mM M g 2+ + 1 0 -6 M
capsaicin
60.8 d: 24.2**
* P < 0.01 when compared with the value in normal ACSF. ** P < 0.01 when compared with the value in normal ACSF and capsaicin.
212 A t present it is n o t clear whether the release o f substance P occurs solely f r o m the terminals o f small d i a m e t e r fibres in the d o r s a l h o r n o f the spinal cord. Release m a y also occur f r o m substance P-containing n e u r o n s which a p p e a r to synapse o n t o m o t o n e u rons in the ventral h o r n o f the spinal c o r d 4,5. The present findings, t a k e n with those o f Jessell et al. 10 suggest t h a t b o t h the acute a n d chronic effects o f capsaicin on nociception m a y be explained by an interaction with the central terminals o f p r i m a r y sensory n e u r o n s containing substance pT,s. The a d m i n i s t r a t i o n o f capsaicin has been shown to d i s r u p t t h e r m o r e g u l a t i o n in the h y p o t h a l a m u s 6, a region rich in substance p l l a n d also excites nociceptive n e u r o n s in the m e d i a l t h a l a m u s in a selective a n d opiate-sensitive m a n n e r 1. Capsaicin m a y therefore p r o v i d e a useful t o o l for the analysis o f substance P function at spinal a n d s u p r a s p i n a l sites involved in nociception a n d t h e r m o r e g u l a t i o n . E.T. was s u p p o r t e d by a g r a n t f r o m the M i n i s t r y o f H e a l t h a n d W e l f a r e o f Japan. T.J. was in receipt o f a R o y a l Society J a p a n P r o g r a m m e Fellowship. P a r t o f this w o r k was s u p p o r t e d by research grants f r o m the M i n i s t r y o f E d u c a t i o n o f J a p a n . 1 Ando, R., Onodera, K., Shima, K., Kisara, K., Takahashi, M. and Osawa, K., Effects of capsaicin on spontaneous unit discharges in medial thalamic single neurons of cats, Folia pharmacoL jap., 73 (1977) 955-959. 2 Fox, S., Krnjevid, K., Morris, M. E., Puil, E. and Werman, R., Action of baclofen on mammalian synaptic transmission, Neuroscience, 3 (1978) 495-515. 3 Henry, J. L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 4 H6kfelt, T., Johansson, J. O., Kellerth, J. O., Ljungdahl, A., Nilsson, G., Nyghrds, A. and Pernow, B., Immunohistochemical distribution of substance P. In U. S. von Euler and B. Pernow, (Eds.), Substance P Raven Press, 1977, pp. 117-145. 5 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 6 Jancso, N., Desensitization with capsaicin and related acylamides as a tool for studying the function of pain receptors. In Pharmacology of Pain, Proc. 3rd. Int. PharmacoL Meet., Pergamon Press, 1966, pp. 33-55. 7 Jancs6, G., Kiraly, E. and Jancs6-Gfibor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones, Nature (Lond.), 270 (1977) 741-743. 8 Jancs6, G. and Knyih~r, E., Functional linkage between nociception and fluoride-resistant acid phosphatase activity in the Rolando substance, Neurobiology, 5 (1975) 42-43. 9 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release in rat trigeminal nucleus, Nature (Lond.), 268 (1977) 549-551. 10 Jessell, T. M., Iversen, L. L. and Cuello, A. C., Capsaicin-induced depletion of substance P from primary sensory neurones, Brain Research, 152 (1978) 183-188. 11 Kanazawa, I. and Jessell, T. M., Post-mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Research, 117 (1976) 362-367. 12 Konishi, S. and Otsuka, M., Excitatory action of hypothalamic substance P on spinal motoneurones of newborn rats, Nature (Lond.), 252 (1974) 734-735. 13 Mudge, A. W., Leeman, S. E. and Fischbach, G. D., Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 526-530. 14 Otsuka, M. and Konishi, S., Electrophysiology of mammalian spinal cord in vitro, Nature (Lond.), 252 (1974) 733-734. 15 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat, Nature (Lond.), 264 (1976) 83-84. 16 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, CoM Spr. Harb. Symp. quant. BioL, 40 (1976) 135-143.
213 17 Otsuka, M. and Yanagisawa, M., The action of substance P on motoneurons of the isolated rat spinal cord, Proc. 7th Int. Congr. Pharmacol., Vol. 2, Pergamon Press, in press. 18 Randi~, M. and Mileti6, V., Effect of substance P in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 19 Saito, K., Konishi, S. and Otsuka, M., Antagonism between Lioresal and substance P in rat spinal cord, Brain Research, 97 (1975) 17%180. 20 Yanaihara, N., Yanaihara, C., Hirohashi, M., Sato, H., Iizuka, Y., Hashimoto, T. and Sakagami, M., Substance P analogs: synthesis and biological and immunological properties. In U. S. yon Euler and B. Pernow, (Eds.), Substance P, Raven Press, 1977, pp. 2%33.
209
Capsaicin-evoked release of substance P from primary sensory neurons
ELIZABETH THERIAULT*, MASANORI OTSUKA and THOMAS JESSELL** Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental University, Yushima, Bunkyo-ka, Tokyo 113 (Japan)
(Accepted March 8th, 1979)
The presence of the peptide substance P in small unmyelinated primary sensory neurons 5 appears to be related to the transmission of nociceptive impulses at the level of the spinal cord. Release of substance P has been demonstrated from the rat spinal cord, in vitro, in response to electrical stimulation of the dorsal roots 1~. Dorsal horn neurons that respond to noxious peripheral stimuli are excited by the iontophoretic application of substance p3,ts and the peptide also produces a depolarization of ventral roots in the isolated rat spinal cord preparation 12,t6. Substance P-containing sensory neurons may then represent one target for the pharmacological modification of pain transmission within the spinal cord. The analgesic properties of the opiates, in particular, may be mediated by the inhibition of substance P release from primary sensory neuronsg, ~3. Although it has been known for many years that repeated administration of the homovanillylamide derivative, capsaicin, renders animals insensitive to painful chemogenic stimuli 8, the mechanism of action of this compound has remained obscure. Recently chronic capsaicin treatment has been shown to produce a dramatic and specific depletion of substance P within primary sensory neurons in the rat dorsal horn 10 suggesting a functional association between the effects of capsaicin and substance P. Moreover, the acute effects of capsaicin administration in vivo are characterized by intense pain which might be consistent with a capsaicin-evoked stimulation of substance P release from the central terminals of primary sensory neurons. To investigate this possibility we have examined the effects of capsaicin in the isolated rat spinal cord preparation 14 and on the release of substance P from spinal cord tissue. Spinal cords were removed from 0-4-day-old Wistar rats according to the method of Otsuka and Konishi is. Hemisected cords were superfused with artificial cerebrospinal fluid (CSF) at 27 °C. The composition of artificial CSF was as follows (mM): NaCI 138.6 ; KCI 3.35 ; CaC12 1.26 ; MgClz 1.16 ; NaHCO3 21.0 ; NaH2PO4 0.58 ; glucose 10 ; gassed with 95 ~o 02--5 ~o CO2. Potential changes generated in mote* Present address: Department of Neurosciences,McMaster University, Hamilton, Ontario, Canada ** Present address and address for correspondence: Department of Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston Mass. 02115, U.S.A.
210 a
i
ii
iii
~
i
ii
iii
I
m
Imin
L Imin
Fig. 1. The effect of substance P and capsaicin on the potential recorded from the ventral root of the isolated rat spinal cord. Drugs were applied for 40 sec as marked over records, a: depolarization of ventral roots induced by substance P (5 x 10 7 M ) - - ( i ) response in artificial CSF; (ii) response in artificial CSF containing 0.1 mM calcium and 2.0 mM magnesium; (iii) recovery of response after return to normal CSF. b: depolarization of ventral root induced by capsaicin (5 × 10 7 M ) - - (J), (ii) and (iii) as in (a). c: effect of baclofen (2.3 x 10-6 M) on ventral root depolarization induced by substance P, (i) depolarization of ventral root by substance P (2 x 10-7 M), (ii) response to substance P after addition of baclofen, (iii) recovery after removal of baclofen, d: effect of baclofen on capsaicininduced depolarization, (i) depolarization evoked by capsaicin (5 × I0 7 M) in artificial CSF, (ii) response after addition of baclofen, (iii) recovery after removal of baclofen. neurons were recorded with extracellular suction electrodes, as described previously 14, from ventral roots (L3-Ls) during bath application of drug solutions. Capsaicin solution was prepared according to the method o f Jancso et al.L Application o f substance P (5 x 10 -7 M ) t o the superfusion medium caused a marked depolarization o f the ventral root (Fig. ! a) in agreement with previous observations 18. Ventral root depolarization occurred within 3-5 sec o f addition of substance P to the recording chamber. Addition o f capsaicin (5 x 10 -7 M ) t o the superfusion medium produced a depolarization o f the ventral root which closely resembled the response to substance P (Fig. 1b). Similar results were obtained in 7 separate experiments. The depolarization of ventral roots induced by capsaicin and substance P was dose-dependent over a concentration range of 5 × 10-s M to 10 -6 M. To examine whether the depolarizing action of capsaicin is mediated by a direct effect on spinal m o t o n e u r o n s or by a transsynaptic action, both substance P and capsaicin were applied in artificial C S F in which the calcium ion concentration had been reduced to 0.1 m M and the magnesium ion concentration raised to 2.0 mM. In every experiment this change in the divalent cation concentration was sufficient to abolish ventral root depolarization evoked by dorsal root stimulation. The substance P-induced depolarization was unaffected (often a slight potentiation) by a reduction in calcium ion concentration (Fig. la). In contrast the depolarization of motoneurons induced by capsaicin was almost completely abolished in artificial C S F containing 0.1 m M calcium and 2.0 m M magnesium (Fig. lb). The depolarizing action ofcapsaicin was completely restored by superfusing the hemisected cord with artificial CSF containing 1.26 m M calcium and 1.16 m M magnesium (Fig. lb). It seems likely, therefore, that the capsaicin-induced depolarization o f spinal motoneurons is mediated transsynaptically and not by a direct action on motoneurons.
211 In the isolated rat spinal cord preparation ventral root reflexes evoked by dorsal root stimulation are depressed by the drug baclofen 19. In the isolated spinal cord, substance P-induced depolarization of ventral roots is antagonized by bath application of baclofen 17,19 although the specificity of this compound seems to be critically dependent on the site and mode of administration (see discussion in refs. 2 and 17). The actions of substance P and capsaicin were compared in the presence of baclofen (2.3 × 10-e M); a concentration sufficient to abolish ventral root reflexes evoked by dorsal root stimulation 17,19. Depolarization of ventral roots in response to substance P was greatly depressed by addition of baclofen (Fig. lc) and capsaicin-induced depolarization was completely abolished (Fig. ld). Removal of baclofen resulted in complete recovery of substance P and capsaicin-induced depolarization (Fig. lc, d). The ability of capsaicin to release substance P from nerve terminals in the isolated rat spinal cord was also examined. Hemisected cords (T5 to caudal extremity) were placed in test tubes containing artificial CSF and then washed for 10 min in artificial CSF (normal or low calcium) and then transferred to tubes containing 1.0 ml of incubation solution for 30 min. During incubation the solution was gassed with 95 % 02-5 % CO2. Dithiothreitol (6 × 10-e M) was added to all samples to avoid oxidation of substance P. Incubation samples were immediately frozen at -80 °C, lyophilized, and the substance P content in each sample measured by radioimmunoassay with a sensitivity of 8 fmol/sample 2°. The unstimulated effiux of substance P from one hemisected spinal cord over a 30-min period was 56.5 -4- 29.6 fmol (Table 1). Incubation with capsaicin (10 -6 M) resulted in a 5-fold increase in substance P release to 287.3 _4_ 33.9 fmol/30 min. In the presence of capsaicin in artificial CSF containing 0.1 mM calcium and 2.0 mM magnesium, the release of substance P was 60.8 ± 24.2 fmol/30 min. Thus the capsaicin-evoked release of substance P was almost completely abolished by reducing the calcium concentration in the superfusing medium. These results clearly demonstrate that capsaicin produces a calcium-dependent release of substance P from rat spinal cord. The depolarization of spinal motoneurons by capsaicin may also be mediated by the release of substance P from nerve telminals. TABLE I Capsaicin-evoked release of substance P from newborn rat spinal cord
Each value is the mean 4- S.E.M. from 3 separate experiments. Incubating solution
fmol substance P released during 30 min period
Normal ACSF 56.5 4- 29.6 Normal ACSF +10 -6 M capsaicin 287.3i 33.9* ACSF containing: 0.1 mM Ca 2+2.0 mM M g 2+ + 1 0 -6 M
capsaicin
60.8 d: 24.2**
* P < 0.01 when compared with the value in normal ACSF. ** P < 0.01 when compared with the value in normal ACSF and capsaicin.
212 A t present it is n o t clear whether the release o f substance P occurs solely f r o m the terminals o f small d i a m e t e r fibres in the d o r s a l h o r n o f the spinal cord. Release m a y also occur f r o m substance P-containing n e u r o n s which a p p e a r to synapse o n t o m o t o n e u rons in the ventral h o r n o f the spinal c o r d 4,5. The present findings, t a k e n with those o f Jessell et al. 10 suggest t h a t b o t h the acute a n d chronic effects o f capsaicin on nociception m a y be explained by an interaction with the central terminals o f p r i m a r y sensory n e u r o n s containing substance pT,s. The a d m i n i s t r a t i o n o f capsaicin has been shown to d i s r u p t t h e r m o r e g u l a t i o n in the h y p o t h a l a m u s 6, a region rich in substance p l l a n d also excites nociceptive n e u r o n s in the m e d i a l t h a l a m u s in a selective a n d opiate-sensitive m a n n e r 1. Capsaicin m a y therefore p r o v i d e a useful t o o l for the analysis o f substance P function at spinal a n d s u p r a s p i n a l sites involved in nociception a n d t h e r m o r e g u l a t i o n . E.T. was s u p p o r t e d by a g r a n t f r o m the M i n i s t r y o f H e a l t h a n d W e l f a r e o f Japan. T.J. was in receipt o f a R o y a l Society J a p a n P r o g r a m m e Fellowship. P a r t o f this w o r k was s u p p o r t e d by research grants f r o m the M i n i s t r y o f E d u c a t i o n o f J a p a n . 1 Ando, R., Onodera, K., Shima, K., Kisara, K., Takahashi, M. and Osawa, K., Effects of capsaicin on spontaneous unit discharges in medial thalamic single neurons of cats, Folia pharmacoL jap., 73 (1977) 955-959. 2 Fox, S., Krnjevid, K., Morris, M. E., Puil, E. and Werman, R., Action of baclofen on mammalian synaptic transmission, Neuroscience, 3 (1978) 495-515. 3 Henry, J. L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 4 H6kfelt, T., Johansson, J. O., Kellerth, J. O., Ljungdahl, A., Nilsson, G., Nyghrds, A. and Pernow, B., Immunohistochemical distribution of substance P. In U. S. von Euler and B. Pernow, (Eds.), Substance P Raven Press, 1977, pp. 117-145. 5 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 235-252. 6 Jancso, N., Desensitization with capsaicin and related acylamides as a tool for studying the function of pain receptors. In Pharmacology of Pain, Proc. 3rd. Int. PharmacoL Meet., Pergamon Press, 1966, pp. 33-55. 7 Jancs6, G., Kiraly, E. and Jancs6-Gfibor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurones, Nature (Lond.), 270 (1977) 741-743. 8 Jancs6, G. and Knyih~r, E., Functional linkage between nociception and fluoride-resistant acid phosphatase activity in the Rolando substance, Neurobiology, 5 (1975) 42-43. 9 Jessell, T. M. and Iversen, L. L., Opiate analgesics inhibit substance P release in rat trigeminal nucleus, Nature (Lond.), 268 (1977) 549-551. 10 Jessell, T. M., Iversen, L. L. and Cuello, A. C., Capsaicin-induced depletion of substance P from primary sensory neurones, Brain Research, 152 (1978) 183-188. 11 Kanazawa, I. and Jessell, T. M., Post-mortem changes and regional distribution of substance P in the rat and mouse nervous system, Brain Research, 117 (1976) 362-367. 12 Konishi, S. and Otsuka, M., Excitatory action of hypothalamic substance P on spinal motoneurones of newborn rats, Nature (Lond.), 252 (1974) 734-735. 13 Mudge, A. W., Leeman, S. E. and Fischbach, G. D., Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 526-530. 14 Otsuka, M. and Konishi, S., Electrophysiology of mammalian spinal cord in vitro, Nature (Lond.), 252 (1974) 733-734. 15 Otsuka, M. and Konishi, S., Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat, Nature (Lond.), 264 (1976) 83-84. 16 Otsuka, M. and Konishi, S., Substance P and excitatory transmitter of primary sensory neurons, CoM Spr. Harb. Symp. quant. BioL, 40 (1976) 135-143.
213 17 Otsuka, M. and Yanagisawa, M., The action of substance P on motoneurons of the isolated rat spinal cord, Proc. 7th Int. Congr. Pharmacol., Vol. 2, Pergamon Press, in press. 18 Randi~, M. and Mileti6, V., Effect of substance P in cat dorsal horn neurones activated by noxious stimuli, Brain Research, 128 (1977) 164-169. 19 Saito, K., Konishi, S. and Otsuka, M., Antagonism between Lioresal and substance P in rat spinal cord, Brain Research, 97 (1975) 17%180. 20 Yanaihara, N., Yanaihara, C., Hirohashi, M., Sato, H., Iizuka, Y., Hashimoto, T. and Sakagami, M., Substance P analogs: synthesis and biological and immunological properties. In U. S. yon Euler and B. Pernow, (Eds.), Substance P, Raven Press, 1977, pp. 2%33.
