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Effect of substance P in cat dorsal horn neurones activated by noxious stimuli
164
Brain Research, 128 (1977) 164-169 © Elsevier/North-Holland Biomedical Press
Effect of substance P in cat dorsal horn neurones activated by noxious stimuli
MIRJANA RANDI(~ and VJEKOSLAV MILETI(~ Department of Veterinary Anatomy, Pharmacology and Physiology, Iowa State University of Science and Technology, Ames, Iowa 50011 (U.S.A.)
(Accepted February 10th, 1977)
Substance P, a peptide originally isolated from the horse brain and gut by yon Euler and Gaddum 4 and recently structurally characterized 2, was found at a higher concentration in the dorsal than in ventral roots1,5,13,16. This fact led Lembeck 13 to suggest that substance P may be the chemical transmitter released from primary afferent fibres. A new support for this concept has been provided by Otsuka and his collaborators who demonstrated in detailed electrophysiological and biochemical studies that: (1) the highest concentration of substance P is present in the dorsal horn of the cat spinal cord, the region where the primary afferent fibres terminate; (2) following ligation and/or sectioning of the dorsal roots the substance P level of the dorsal horn was markedly reduced; and (3) exogenously applied substance P has a depolarizing effect on both frog motoneurones and motoneurones of the newborn rat 10,11,15,1s,ag. In addition, evidence has been presented that microiontophoretic application of synthetic substance P has a strong, although slow, excitatory action in about half of the units tested in the dorsal horn of the cat spinal cord and cuneate nucleus 7,12. Interestingly, substance P also depolarizes the intramedullary portion of the primary afferents, although the functional significance of this observation is unclear 14. The cellular localization of substance P has recently been examined by immunohistochemical techniques by H6kfelt et al.a, 9. They have demonstrated the occurrence of a very dense network of substance P-positive fibres particularly in Lissauer's fasciculus and laminae l-lII of the cat lumbosacral spinal cord. In addition they have found that after local application of colchicine or compression of the dorsal root close to the spinal ganglion, substance P-positive fluorescence was observed in some of the small neuronal cell bodies. In agreement with the bioassay studies of Takahashi and Otsuka, they found a marked decrease in the number of substance P-positive fibres in Lissauer's tract and laminae I-III after a dorsal root rhizotomy x°,19. The physiological role of substance P-positive fibres in Lissauer's fasciculus and laminae I-III is at persent unknown. However, there is a recent suggestion that they may be related to pain preception 6. Henry 6 has found that microelectrophoretic application of substance P to single units, located in Rexed's laminae IV-VI of the cat
165 spinal cord, excited about half of the units tested. The units activated by noxious thermal stimulation usually responded to one other adequate stimulus as well. If substance P is involved in excitation of neurones concerned with pain spinal mechanisms, its failure to excite all nociceptive neurones is difficult to understand. Henry has offered two possible explanations: (1) low rate of substance P release from the micropipettes, and (2) the possibility that substance P is involved in neurotransmission at the first afferent synapse and therefore the neurones receiving inputs from second or higher order neurones would not be excited. The finding of H6kfelt et al.S, 9 that the substance P-positive fibres seem to terminate predominantly in lamina I, where the neurones principally or exclusively excited by an input in nociceptor afferent fibres have been reported to exist by Christensen and Perl 3, prompted us to study the central effects of substance P by applying it microelectrophoretically to dorsal horn neurones selectively activated by noxious stimuli at the level of Rexed's laminae I-III. The present paper will illustrate that substance P causes strong excitation of all tested units selectively activated by high threshold mechanical and/or thermal stimuli or by afferent volleys in A~ and C fibres, located in laminae I-IlL In contrast, the units adequately activated by light pressure applied to the skin of the tail were either weakly depressed or not affected by substance P. The preliminary results of our findings were communicated to the Society of Neuroscience 17. The experiments were performed on 20 adult cats initially anaesthetized by halothane. The brain was anaemically destroyed by bilateral occlusion of the common carotid and vertebral arteries. The spinal cord was transected at the first cervical level. Thereafter, the animal was artificially respired and immobilized by gallamine triethiodide. Body temperature, blood pressure and end-tidal CO2 concentration were monitored and maintained at optimal levels throughout the experiment. Laminectomy was made at the sacral and caudal spinal levels. A coccygeal dorsal root (or rootlet) was freed and placed on a bipolar stimulating electrode distally and a bipolar recording electrode centrally, leaving both peripheral and central connections intact. This electrode arrangement served for electrical stimulation of the dorsal root and recording of compound action potentials of different kinds of myelinated and unmyelinated afferent fibres. A multiple micropipette was inserted at the root entry zone while an electrical stimulus was repeatedly applied to the corresponding dorsal root at a rate of 1/sec. The electrical stimulus was made sufficiently strong so that even the smallest unmyelinated fibres in the root were activated. The electrical stimulus thus acted as a search stimulus for units activated by afferent volleys conducted in A~ and C fibre groups. High intensity mechanical stimuli were delivered to the skin of the tail (e.g. pressure from sharply pointed objects; grasping a fold of skin with calibrated forceps. When the latter type of mechanical stimulus was used on human skin, it provoked a highly painful sensation). Occasionally noxious heat (radiant source) was applied to the skin of the tail. The activity of the dorsal horn neurones in Rexed's laminae I-Ill, selectively activated either by noxious mechanical and/or thermal stimulation or by a volley in A6 or C fibres, was recorded extracellularly through the central barrel of a multi-
166 TABLE I Excitatory effects of substance P in cat dorsal horn neurones selectively activated by noxious stimuli Total number o[ units
lnput
22
High threshold 1 3 × 10 SA 100 (2 20-fold mechanical and/ increase) or thermal stimulation Low threshold 2-5 s 10-8 A 14 (1.5-fold mechanical increase)
7
Substance P
Number of units excited ( % o[" total)
Number of units Number of units depressed not affected ( % of total) ( % of total)
57 (slight)
29
barrelled (2- or 3-barrelled) glass micropipette filled with a saturated solution of Fast green (FCF, Matheson, Coleman and Bell) in 3 M sodium chloride. The site o f recording was marked by iontophoresis o f the dye. Conventional microelectrophoretic technique was used to study the effect o f substance P (0.5 mg was dissolved in 0.1 ml o f 20 m M acetic acid, giving a final concentration o f about 3.7 m M and a p H near 5.5) on the spontaneous and evoked firing o f the dorsal horn neurones selectively activated by noxious stimuli located in Rexed's laminae I-I11. A control solution was prepared containing 20 m M sodium acetate acidified to a similar p H with acetic acid. The 3barrelled micropipettes were allowed to stand at 4 °C for 3 6 4 8 h before experiments. In some experiments omega-dot triple micropipettes were filled with substance P prior to the chemical unit analysis. The microelectrophoretic unit used contained current balancing circuit. The results o f present experiments are shown in Table I. They are based on the data obtained f r o m 22 units activated either by noxious mechanical and/or thermal
20
a z
isP3On sP3i! i"aAc'O
o I.U O3
10
03 U,I
ii
Of)
I
I
2
i
MINUTES
I
4
I
I
6
Fig. 1. Excitation of a silent lamina I cell (see insert) produced by microelectrophoretic application of substance P (sP, 30 nA). Sodium acetate (NaAc, 40 NA) is without effect. Unit activity was recorded on moving film and the discharge frequency counted.
167 stimulation or by a volley in A~ or C fibres. In addition, 7 units activated by low threshold mechanical stimulation were tested with microelectrophoretically applied substance P. Substance P was applied as a cation with currents ranging from 10 to 50 nA for periods up to 2 min. It is clear from the results presented in Table I that all units activated either by noxious mechanical and/or thermal stimulation or by a volley in A~ or C fibres located in Rexed's laminae I-III were strongly excited by substance P. In contrast to this finding 6 out of 7 cells activated by low threshold mechanical stimulation (touching by resting a finger lightly on the skin; brushing with a camel hair brush) located in laminae I-III were weakly depressed or not affected by microelectrophoretically applied substance P. Excitation was observed as initiation of firing in a previously quiescent cell (Fig. 1) or as an increase in the rate of spontaneous firing (Fig. 2). The ongoing firing rate was usually raised 2-20-fold with substance P. The unit illustrated in Fig. 1 did not show any background activity, however, it was activated by noxious mechanical and thermal stimulation of the skin of the tail. A continuous application of substance P (30 nA) for 1 min caused excitation within 7 sec. On turning off the current expelling substance P, the discharge persisted for more than 1 min. The second application of substance P evoked similar or somewhat larger response. "Positive" current of 40 nA applied through a sodium acetate barrel consistently failed to elicit comparable excitatory response. Since the sodium acetate solution was prepared at a similar pH as substance P, this test also practically eliminated a rather unlikely effect of pH. In addition, we have found that substance P produced a facilitation of the response of this unit to noxious stimulation. On histological examination of the recording site, we found that the unit was located in the marginal cell layer (Rexed's lamina I) as illustrated in the insert of Fig. 1. Another example of substance P-produced slow excitation in a spontaneously firing unit activated by noxious mechanical stimulation is shown in Fig. 2. Here, a continuous application of substance P (20 nA) for 72 sec caused excitation within 16
0
2
4
6
8
MINUTES
Fig. 2. Excitation of a spontaneously active cell, located in the border zone between laminae 1I and III (see insert), produced by microelectrophoreticapplication of substance P (10 and 20 nA). Sodium acetate is without effect.
168 A 30
15
a z 0
0
I
LU
if) m
30]
0
t
0
1
t
,
2
,
MINUTES
4
,
6
Fig. 3. Effect of substance P (5 hA) on activity of two units located in lamina II. A: occasionally responding to low threshold mechanical stimulation, was weakly depressed by substance P (tipper trace). B: responding exclusively to high threshold mechanical stimulation, was excited by substance P (lower trace).
sec and a prolonged after-discharge lasting almost 2 min. The maximum increase in the firing rate in this cell amounted to 363 ~ of the control level. Second application of a smallel dose of substance P (10 nA) still produced almost a 3-fold increase in the firing level. On histological examination of the recording site the unit was found to be located in the border zone between laminae I1 and I11. A particularly interesting result is shown in Fig. 3 where activity of two units located in lamina |I, unit A (occasionally responding to low threshold mechanical stimulation) and unit B (responding exclusively to high threshold mechanical stimulation), was simultaneously studied with substance P. As can be seen from Fig. 3, while unit B was excited by substance P (5 hA), unit A responded with weak depression. In conclusion we have demonstrated in this paper that: (1) iontophoretic application of synthetic substance P causes strong excitation of all tested units selectively activated by high threshold mechanical and/or thermal stimuli or by afferent volleys in A 6 and C fibres, located in laminae I-II l; (2) substance P facilitated the responses of nociceptive neurones to adequate stimulation; (3) the majority of units adequately activated by light pressure applied to the skin of the tail were either weakly depressed or not affected by substance P. However, more extensive analysis of chemical, anatomical and stimulus specificity of substance P excitatory action is in progress and will hopefully confirm the preliminary results published in this paper. The strong excitant effect of substance P upon all tested spinal neurones selectively activated by noxious stimuli, located at the level of Rexed's laminae l-IlI, seems to suggest that this peptide may have a physiological role of an excitatory transmitter or modulator at the synapses between the primary afferent fibres and second order neurones in spinal pain pathways. Similar conclusion was reached
169 recently by H e n r y 6 w h o f o u n d t h a t m i c r o e l e c t r o p h o r e t i c ap p l i cat i o n o f synthetic substance P to single units, in a different p o p u l a t i o n o f spinal neurones located in l a m i n a e I V - V I o f the cat spinal cord, causes a slow excitation o f a p p r o x i m a t e l y h a l f o f the nociceptive units tested. I f substance P is physiologically i n v o l v e d in excitation o f n e u r o n e s c o n c e r n e d with spinal pain mechanisms, its failure to excite all nociceptive n e u r o n e s is difficult to explain. This w o r k was s u p p o r t e d by P H S R e s e a r c h G r a n t NS12972-01, I o w a State U n i v e r s i t y R e s e a r c h F o u n d a t i o n a n d Salsbury F o u n d a t i o n .
1 Amin, A. H., Crawford, T. B. B. and Gaddum, J. H., The distribution of substance P and 5hydroxytryptamine in the central nervous system of the dog, J. Physiol. (Lond.), 126 (1954) 596-618. 2 Chang, M. M., Leeman, S. E. and Niall, H. D., Amino acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 3 Christensen, B.N.andPerl,E. R.,Spinalneuronesspecificallyexcitedby noxious or thermal stimuli: marginal zone of the dorsal horn, J. NeurophysioL, 33 (1970) 293-307. 4 Euler, U. S. von and Gaddum, J. H., An unidentified depressor substance in certain tissue extracts, J. PhysioL (Lond.), 72 (1931) 74-87. 5 Hellauer, H., Zur Charakterisierung der Erregungssubstanz sensibler Nerven, Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 234-241. 6 Henry, J. L., Effects of substance P on functionally identified units in cat spinal cord, Brain Research, 114 (1976) 439-451. 7 Henry, J. L., Krnjevi6, K. and Morris, M. E., Substance P and spinal neurones, Canad. J. Physiol. Pharmacol., 53 (1975) 423-432. 8 Hbkfelt, T., Elde, R., Johansson, O., Luft, R., Nilsson, G. andAr;mura, A., Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131-136. 9 Hbkfelt, 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. 10 Konishi, S. and Otsuka, M., Actions of certain polypeptides on frog spinal neurons, Jap. J. PharmacoL, 21 (1971) 685-687. 11 Konishi, S. and Otsuka, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397-410. 12 Krnjevi6, K. and Morris, M. E., An excitatory action of substance P on cuneate neurones, Canad. J. Physiol. Pharmacol., 52 (1974) 736-744. 13 Lembeck, F., Zur Frage der zentralen Obertragung afferenter Impulse. III. Mitteilung. Das Vorkommen und die Bedeutung, der Substanz P in den dorsalen Wurzeln des RiJckenmarks, NaunynSchmiedeberg's Arch. exp. Path. Pharmak., 219 (1953) 197-213. 14 Nicoll, R. A., Promising peptides. In J. A. Ferrendelli, B. S. McEwen and S. H. Snyder (Eds.), Neurotransmitters, Hormones and Receptors: Novel Approaches, Neurosci. Symp. 1Iol. 1, Society for Neuroscience, Bethesda, Md., 1976, pp. 99-122. 15 Otsuka, M., Konishi, S. and Takahashi, T., The presence of a motoneuron-depolarizing peptide in bovine dorsal roots of sp+nal nerves, Proc. Jap. Acad., 48 (1972) 342-346. 16 Pernow, B., Studies on substance P. Purification, occurrence and biological actions, ActaphysioL scand., 29, Suppl. 105 (1953) 1-90. 17 Randi6, M. and Mileti6, V., Chemical sensitivity of cat dorsal horn neurones activated by noxious stimuli, Neurosci. Abstr., 1I (1976) 1369. 18 Takahashi, T., Konishi, S., Powell, D., Leeman, S. E. and Otsuka, M., Identification of the motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P, Brain Research, 73 (1974) 59-69. 19 Takahashi, T. and Otsuka, M., Regional distribution of substance P in the spinal cord and nerve roots of the cat and the effect of dorsal root section, Brain Research, 87 (1975) 1-11.
Brain Research, 128 (1977) 164-169 © Elsevier/North-Holland Biomedical Press
Effect of substance P in cat dorsal horn neurones activated by noxious stimuli
MIRJANA RANDI(~ and VJEKOSLAV MILETI(~ Department of Veterinary Anatomy, Pharmacology and Physiology, Iowa State University of Science and Technology, Ames, Iowa 50011 (U.S.A.)
(Accepted February 10th, 1977)
Substance P, a peptide originally isolated from the horse brain and gut by yon Euler and Gaddum 4 and recently structurally characterized 2, was found at a higher concentration in the dorsal than in ventral roots1,5,13,16. This fact led Lembeck 13 to suggest that substance P may be the chemical transmitter released from primary afferent fibres. A new support for this concept has been provided by Otsuka and his collaborators who demonstrated in detailed electrophysiological and biochemical studies that: (1) the highest concentration of substance P is present in the dorsal horn of the cat spinal cord, the region where the primary afferent fibres terminate; (2) following ligation and/or sectioning of the dorsal roots the substance P level of the dorsal horn was markedly reduced; and (3) exogenously applied substance P has a depolarizing effect on both frog motoneurones and motoneurones of the newborn rat 10,11,15,1s,ag. In addition, evidence has been presented that microiontophoretic application of synthetic substance P has a strong, although slow, excitatory action in about half of the units tested in the dorsal horn of the cat spinal cord and cuneate nucleus 7,12. Interestingly, substance P also depolarizes the intramedullary portion of the primary afferents, although the functional significance of this observation is unclear 14. The cellular localization of substance P has recently been examined by immunohistochemical techniques by H6kfelt et al.a, 9. They have demonstrated the occurrence of a very dense network of substance P-positive fibres particularly in Lissauer's fasciculus and laminae l-lII of the cat lumbosacral spinal cord. In addition they have found that after local application of colchicine or compression of the dorsal root close to the spinal ganglion, substance P-positive fluorescence was observed in some of the small neuronal cell bodies. In agreement with the bioassay studies of Takahashi and Otsuka, they found a marked decrease in the number of substance P-positive fibres in Lissauer's tract and laminae I-III after a dorsal root rhizotomy x°,19. The physiological role of substance P-positive fibres in Lissauer's fasciculus and laminae I-III is at persent unknown. However, there is a recent suggestion that they may be related to pain preception 6. Henry 6 has found that microelectrophoretic application of substance P to single units, located in Rexed's laminae IV-VI of the cat
165 spinal cord, excited about half of the units tested. The units activated by noxious thermal stimulation usually responded to one other adequate stimulus as well. If substance P is involved in excitation of neurones concerned with pain spinal mechanisms, its failure to excite all nociceptive neurones is difficult to understand. Henry has offered two possible explanations: (1) low rate of substance P release from the micropipettes, and (2) the possibility that substance P is involved in neurotransmission at the first afferent synapse and therefore the neurones receiving inputs from second or higher order neurones would not be excited. The finding of H6kfelt et al.S, 9 that the substance P-positive fibres seem to terminate predominantly in lamina I, where the neurones principally or exclusively excited by an input in nociceptor afferent fibres have been reported to exist by Christensen and Perl 3, prompted us to study the central effects of substance P by applying it microelectrophoretically to dorsal horn neurones selectively activated by noxious stimuli at the level of Rexed's laminae I-III. The present paper will illustrate that substance P causes strong excitation of all tested units selectively activated by high threshold mechanical and/or thermal stimuli or by afferent volleys in A~ and C fibres, located in laminae I-IlL In contrast, the units adequately activated by light pressure applied to the skin of the tail were either weakly depressed or not affected by substance P. The preliminary results of our findings were communicated to the Society of Neuroscience 17. The experiments were performed on 20 adult cats initially anaesthetized by halothane. The brain was anaemically destroyed by bilateral occlusion of the common carotid and vertebral arteries. The spinal cord was transected at the first cervical level. Thereafter, the animal was artificially respired and immobilized by gallamine triethiodide. Body temperature, blood pressure and end-tidal CO2 concentration were monitored and maintained at optimal levels throughout the experiment. Laminectomy was made at the sacral and caudal spinal levels. A coccygeal dorsal root (or rootlet) was freed and placed on a bipolar stimulating electrode distally and a bipolar recording electrode centrally, leaving both peripheral and central connections intact. This electrode arrangement served for electrical stimulation of the dorsal root and recording of compound action potentials of different kinds of myelinated and unmyelinated afferent fibres. A multiple micropipette was inserted at the root entry zone while an electrical stimulus was repeatedly applied to the corresponding dorsal root at a rate of 1/sec. The electrical stimulus was made sufficiently strong so that even the smallest unmyelinated fibres in the root were activated. The electrical stimulus thus acted as a search stimulus for units activated by afferent volleys conducted in A~ and C fibre groups. High intensity mechanical stimuli were delivered to the skin of the tail (e.g. pressure from sharply pointed objects; grasping a fold of skin with calibrated forceps. When the latter type of mechanical stimulus was used on human skin, it provoked a highly painful sensation). Occasionally noxious heat (radiant source) was applied to the skin of the tail. The activity of the dorsal horn neurones in Rexed's laminae I-Ill, selectively activated either by noxious mechanical and/or thermal stimulation or by a volley in A6 or C fibres, was recorded extracellularly through the central barrel of a multi-
166 TABLE I Excitatory effects of substance P in cat dorsal horn neurones selectively activated by noxious stimuli Total number o[ units
lnput
22
High threshold 1 3 × 10 SA 100 (2 20-fold mechanical and/ increase) or thermal stimulation Low threshold 2-5 s 10-8 A 14 (1.5-fold mechanical increase)
7
Substance P
Number of units excited ( % o[" total)
Number of units Number of units depressed not affected ( % of total) ( % of total)
57 (slight)
29
barrelled (2- or 3-barrelled) glass micropipette filled with a saturated solution of Fast green (FCF, Matheson, Coleman and Bell) in 3 M sodium chloride. The site o f recording was marked by iontophoresis o f the dye. Conventional microelectrophoretic technique was used to study the effect o f substance P (0.5 mg was dissolved in 0.1 ml o f 20 m M acetic acid, giving a final concentration o f about 3.7 m M and a p H near 5.5) on the spontaneous and evoked firing o f the dorsal horn neurones selectively activated by noxious stimuli located in Rexed's laminae I-I11. A control solution was prepared containing 20 m M sodium acetate acidified to a similar p H with acetic acid. The 3barrelled micropipettes were allowed to stand at 4 °C for 3 6 4 8 h before experiments. In some experiments omega-dot triple micropipettes were filled with substance P prior to the chemical unit analysis. The microelectrophoretic unit used contained current balancing circuit. The results o f present experiments are shown in Table I. They are based on the data obtained f r o m 22 units activated either by noxious mechanical and/or thermal
20
a z
isP3On sP3i! i"aAc'O
o I.U O3
10
03 U,I
ii
Of)
I
I
2
i
MINUTES
I
4
I
I
6
Fig. 1. Excitation of a silent lamina I cell (see insert) produced by microelectrophoretic application of substance P (sP, 30 nA). Sodium acetate (NaAc, 40 NA) is without effect. Unit activity was recorded on moving film and the discharge frequency counted.
167 stimulation or by a volley in A~ or C fibres. In addition, 7 units activated by low threshold mechanical stimulation were tested with microelectrophoretically applied substance P. Substance P was applied as a cation with currents ranging from 10 to 50 nA for periods up to 2 min. It is clear from the results presented in Table I that all units activated either by noxious mechanical and/or thermal stimulation or by a volley in A~ or C fibres located in Rexed's laminae I-III were strongly excited by substance P. In contrast to this finding 6 out of 7 cells activated by low threshold mechanical stimulation (touching by resting a finger lightly on the skin; brushing with a camel hair brush) located in laminae I-III were weakly depressed or not affected by microelectrophoretically applied substance P. Excitation was observed as initiation of firing in a previously quiescent cell (Fig. 1) or as an increase in the rate of spontaneous firing (Fig. 2). The ongoing firing rate was usually raised 2-20-fold with substance P. The unit illustrated in Fig. 1 did not show any background activity, however, it was activated by noxious mechanical and thermal stimulation of the skin of the tail. A continuous application of substance P (30 nA) for 1 min caused excitation within 7 sec. On turning off the current expelling substance P, the discharge persisted for more than 1 min. The second application of substance P evoked similar or somewhat larger response. "Positive" current of 40 nA applied through a sodium acetate barrel consistently failed to elicit comparable excitatory response. Since the sodium acetate solution was prepared at a similar pH as substance P, this test also practically eliminated a rather unlikely effect of pH. In addition, we have found that substance P produced a facilitation of the response of this unit to noxious stimulation. On histological examination of the recording site, we found that the unit was located in the marginal cell layer (Rexed's lamina I) as illustrated in the insert of Fig. 1. Another example of substance P-produced slow excitation in a spontaneously firing unit activated by noxious mechanical stimulation is shown in Fig. 2. Here, a continuous application of substance P (20 nA) for 72 sec caused excitation within 16
0
2
4
6
8
MINUTES
Fig. 2. Excitation of a spontaneously active cell, located in the border zone between laminae 1I and III (see insert), produced by microelectrophoreticapplication of substance P (10 and 20 nA). Sodium acetate is without effect.
168 A 30
15
a z 0
0
I
LU
if) m
30]
0
t
0
1
t
,
2
,
MINUTES
4
,
6
Fig. 3. Effect of substance P (5 hA) on activity of two units located in lamina II. A: occasionally responding to low threshold mechanical stimulation, was weakly depressed by substance P (tipper trace). B: responding exclusively to high threshold mechanical stimulation, was excited by substance P (lower trace).
sec and a prolonged after-discharge lasting almost 2 min. The maximum increase in the firing rate in this cell amounted to 363 ~ of the control level. Second application of a smallel dose of substance P (10 nA) still produced almost a 3-fold increase in the firing level. On histological examination of the recording site the unit was found to be located in the border zone between laminae I1 and I11. A particularly interesting result is shown in Fig. 3 where activity of two units located in lamina |I, unit A (occasionally responding to low threshold mechanical stimulation) and unit B (responding exclusively to high threshold mechanical stimulation), was simultaneously studied with substance P. As can be seen from Fig. 3, while unit B was excited by substance P (5 hA), unit A responded with weak depression. In conclusion we have demonstrated in this paper that: (1) iontophoretic application of synthetic substance P causes strong excitation of all tested units selectively activated by high threshold mechanical and/or thermal stimuli or by afferent volleys in A 6 and C fibres, located in laminae I-II l; (2) substance P facilitated the responses of nociceptive neurones to adequate stimulation; (3) the majority of units adequately activated by light pressure applied to the skin of the tail were either weakly depressed or not affected by substance P. However, more extensive analysis of chemical, anatomical and stimulus specificity of substance P excitatory action is in progress and will hopefully confirm the preliminary results published in this paper. The strong excitant effect of substance P upon all tested spinal neurones selectively activated by noxious stimuli, located at the level of Rexed's laminae l-IlI, seems to suggest that this peptide may have a physiological role of an excitatory transmitter or modulator at the synapses between the primary afferent fibres and second order neurones in spinal pain pathways. Similar conclusion was reached
169 recently by H e n r y 6 w h o f o u n d t h a t m i c r o e l e c t r o p h o r e t i c ap p l i cat i o n o f synthetic substance P to single units, in a different p o p u l a t i o n o f spinal neurones located in l a m i n a e I V - V I o f the cat spinal cord, causes a slow excitation o f a p p r o x i m a t e l y h a l f o f the nociceptive units tested. I f substance P is physiologically i n v o l v e d in excitation o f n e u r o n e s c o n c e r n e d with spinal pain mechanisms, its failure to excite all nociceptive n e u r o n e s is difficult to explain. This w o r k was s u p p o r t e d by P H S R e s e a r c h G r a n t NS12972-01, I o w a State U n i v e r s i t y R e s e a r c h F o u n d a t i o n a n d Salsbury F o u n d a t i o n .
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