Brain Research, 97 (1975) 177-180 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
177
Antagonism between Lioresal and substance P in rat spinal cord
KOJi SA1TO*, SHIRO KONISHI AND MASANOR10TSUKA Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental Uniw,rsity, Yushima, Bunkyo-ku, Tokyo (Japan)
(Accepted June 24th, 1975)
There is considerable evidence implicating the undecapeptide substance P (see ref. 1) in the primary afferent transmission in the spinal cord. Substance P is present in the dorsal root of bovine and feline spinal nerves in a concentration 9-27 times higher than that in the ventral root6, 9,10. Substance P is highly concentrated in the dorsal part of dorsal horn of cat spinal cord, and its level in this region is greatly reduced after the section of dorsal roots10. Synthetic substance P exerts a strong depolarizing action on the spinal motoneurons of the frog and the rat3,4, 6. Lioresal (Ciba-Geigy, fl-(4-chlorophenyl)-~-aminobutyric acid) has recently been reported to suppress the spinal monosynaptic and polysynaptic reflexes without affecting the electrical properties of spinal motoneurons in the cat 7,8. If substance P or a related peptide is a transmitter of primary afferent neurons, a possible mechanism of the action of Lioresal is its antagonism toward the peptide transmitter inducing the depolarization of spinal neurons. This possibility was examined in the present study. The spinal cord of 0-7-day-old Wistar rats was isolated, hemisected sagittally and placed in a bath perfused with Krebs solution (27 ~: 1 °C). The solution was equilibrated with a mixture of 95 ~, 02 and 5 ~o COz. Potentials were recorded from the ventral or dorsal root (L3-L5) with a suction electrode of the type described in a previous paperL The recording electrode was connected through a preamplifier to an oscilloscope or a pen-recorder. The bath was grounded through a reference calomel electrode. When a dorsal root was stimulated by a single shock, typical mono- and polysynaptic reflexes were recorded from the corresponding ventral root (Fig. I A~). The application of synthetic substance P or L-glutamic acid produced a depolarization of motoneurons, this being recorded as a positive potential at the electrode recording from the cut end of the ventral root (Fig. IB and C). After the bath was flushed with normal Krebs solution, the potential returned to the original level. From the comparison of the dose-response curves of substance P and e-glutamate at the level of 0.5 * On leave from the Department of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
178
a
A
~~dlUlm
b
a
;'0 rnsec: ] 2 my
D
b
l%~!ns~
i
B
.•
2rnin " ]05/ I mV
E
....--]1 mV
C
Fig. 1. Effects of Lioresal on the reflex activities and the responses to substance P and amino acids. A-C: recordings from L3 ventral root. A: mono- and polysynaptic reflexes; B: responses to substance P (2 ~. 10-7 M); C: responses to L-glutamate (10-3 M). D and E: recordings from L5 dorsal root. D: dorsal root potentials induced by a single volley in L4 dorsal root; E: responses to GABA (3 x 10 ~',M). Horizontal bars under records mark the periods of drug applications. In each pair of records, a was obtained in normal Krebs solution, and b in the solution containing Lioresal (5 . 10-8 M). Scales for B apply also to C. Positivity at the recording electrode upwards. mV potential change, as exemplified in Fig. 2, it was estimated that the depolarizing action of substance P was 1000-9000 times more potent than that of L-glutamate. The application of Lioresal in a concentration of 5 x 10-6 M d i d not produce any appreciable change of DC potential recorded from the ventral root, but resulted in a marked decrease of the size of monosynaptic reflex in a few minutes (Fig. 1Ah). Polysynaptic reflex was also depressed by the drug. Under such conditions, the depolarizing action of substance P was almost completely abolished (Figs. l Bb and 2). The depolarizing action of L-glutamate was also antagonized by Lioresal, but to a much smaller extent (Figs. ICh and 2). The effect of Lioresal was reversible; thus, after the removal of the drug from the perfusion medium, the mono- and polysynaptic reflexes as well as the responses to substance P and L-glutamate recovered completely within 30 min. When the preparation was soaked in a modified Krebs solution containing low Ca (0.7 mM) and high Mg (7 mM), the mono- and polysynaptic reflexes were completely suppressed, whereas the depolarizing responses to substance P and L-glutamate as recorded from the ventral root were only slightly reduced. Under such conditions, Lioresal again effectively antagonized the action of substance P to depolarize the motoneurons. These results indicate that both substance P and Lioresal act directly on the motoneurons. Fig. 1D and E shows the effect of Lioresal on the responses recorded from the dorsal root. When the potentials were recorded from a dorsal root and the adjacent
179 mV
05
0 10 -7
' 10 - 6
I )O -5
.
.
.
.
L~ 10 .4
. . . . .
• _ 10 ~ M
Fig. 2. Effects of Lioresal on the dose-response curves of substance P and L-glutamate. Ordinate:
potential change produced in L3 ventral root by substance P and L-glutamate. Abscissa: concentration of the drugs on logarithmic scale. © and 0 , responses to substance P. ~., and A, responses to L-glutamate. Open symbols indicate the responses in normal Krebs solution, and filled symbols indicate those in the solution containing Lioresal (5 /. 10 6 M). dorsal root was stimulated, a dorsal root potential was observed. The application of G A B A produced a depolarizing response o f p r i m a r y afferent fibers. Lioresal blocked the dorsal root potential, b u t did n o t affect the G A B A action. These results agree with the observations o f Davidoff and Sears in the frog spinal cord 2 a n d suggest that Lioresal blocks the excitatory t r a n s m i s s i o n at the p r i m a r y afferent synapses in the spinal cord. The present paper reports the first specific a n t a g o n i s t toward the excitatory action of substance P on the central neurons. T h a t an antagonist of substance P blocked the m o n o - and polysynaptic reflexes as well as the dorsal root potential again supports the hypothesis that substance P or a closely related peptide is a sensory t r a n s m i t t e r of spinal dorsal root fibers 3,4,6,9A°. We wish to t h a n k Drs. K. O b a t a a n d T. T a k a h a s h i for their helpful advice.
I CHANG, M. M., LEEMAN, S. E., AND NIALL, H. D., Amino-acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 2 DAVIDOFF, R. A., AND SEARS, E. S., The effects of Lioresal on synaptic activity in the isolated spinal cord, Neurology (Minneap.), 24 (1974) 957-963. 3 KONISHI, S., AND OTSUKA, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397~10. 4 KONISHI,S., AND OTSUKA, M., Excitatory action of hypothalamic substance P on spinal motoneurones of newborn rats, Nature (Lond.), 252 (1974) 734-735. 50TSUKA, M., AND KONISHI, S., Electrophysiology of mammalian spinal cord in vitro, Nature (Lond.), 252 (1974) 733-734. 60TSUKA, M., KONISH , S., AND TAKAHASHI, T., A further study of the motoneuron-depolarizing peptide extracted from dorsal roots of bovine spinal nerves, Proc. Jap. Acad., 48 (1972) 747-752. 7 PEDERSEN, E., ARL1EN-SOBORG, P., GRYNDERUP, V., AND HENRIKSEN, O., GABA derivative in spasticity (fl-(4-chlorophenyl)-~-aminobutyric acid, Ciba, 34.647-Ba), Acta neurol, stand., 46 (1970) 257-266. 8 PIERAU, F. K., AND ZIMMERMANN, P., Action of a GABA-derivative on postsynaptic potentials
180 and membrane properties of cats' spinal motoneurones, Brain Research, 54 (1973) 376 380. 9 TAKAt~ASHI, T., KONISHI, S., POWEr.L, D., LEEMAN, S. E., AND OTSt~KA, M., Identificatio~ of the motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P Brail Research. 73 (1974) 59 69. l0 TAKAHASm, T., AYD 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- I I.
177
Antagonism between Lioresal and substance P in rat spinal cord
KOJi SA1TO*, SHIRO KONISHI AND MASANOR10TSUKA Department of Pharmacology, Faculty of Medicine, Tokyo Medical and Dental Uniw,rsity, Yushima, Bunkyo-ku, Tokyo (Japan)
(Accepted June 24th, 1975)
There is considerable evidence implicating the undecapeptide substance P (see ref. 1) in the primary afferent transmission in the spinal cord. Substance P is present in the dorsal root of bovine and feline spinal nerves in a concentration 9-27 times higher than that in the ventral root6, 9,10. Substance P is highly concentrated in the dorsal part of dorsal horn of cat spinal cord, and its level in this region is greatly reduced after the section of dorsal roots10. Synthetic substance P exerts a strong depolarizing action on the spinal motoneurons of the frog and the rat3,4, 6. Lioresal (Ciba-Geigy, fl-(4-chlorophenyl)-~-aminobutyric acid) has recently been reported to suppress the spinal monosynaptic and polysynaptic reflexes without affecting the electrical properties of spinal motoneurons in the cat 7,8. If substance P or a related peptide is a transmitter of primary afferent neurons, a possible mechanism of the action of Lioresal is its antagonism toward the peptide transmitter inducing the depolarization of spinal neurons. This possibility was examined in the present study. The spinal cord of 0-7-day-old Wistar rats was isolated, hemisected sagittally and placed in a bath perfused with Krebs solution (27 ~: 1 °C). The solution was equilibrated with a mixture of 95 ~, 02 and 5 ~o COz. Potentials were recorded from the ventral or dorsal root (L3-L5) with a suction electrode of the type described in a previous paperL The recording electrode was connected through a preamplifier to an oscilloscope or a pen-recorder. The bath was grounded through a reference calomel electrode. When a dorsal root was stimulated by a single shock, typical mono- and polysynaptic reflexes were recorded from the corresponding ventral root (Fig. I A~). The application of synthetic substance P or L-glutamic acid produced a depolarization of motoneurons, this being recorded as a positive potential at the electrode recording from the cut end of the ventral root (Fig. IB and C). After the bath was flushed with normal Krebs solution, the potential returned to the original level. From the comparison of the dose-response curves of substance P and e-glutamate at the level of 0.5 * On leave from the Department of Pharmacology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan.
178
a
A
~~dlUlm
b
a
;'0 rnsec: ] 2 my
D
b
l%~!ns~
i
B
.•
2rnin " ]05/ I mV
E
....--]1 mV
C
Fig. 1. Effects of Lioresal on the reflex activities and the responses to substance P and amino acids. A-C: recordings from L3 ventral root. A: mono- and polysynaptic reflexes; B: responses to substance P (2 ~. 10-7 M); C: responses to L-glutamate (10-3 M). D and E: recordings from L5 dorsal root. D: dorsal root potentials induced by a single volley in L4 dorsal root; E: responses to GABA (3 x 10 ~',M). Horizontal bars under records mark the periods of drug applications. In each pair of records, a was obtained in normal Krebs solution, and b in the solution containing Lioresal (5 . 10-8 M). Scales for B apply also to C. Positivity at the recording electrode upwards. mV potential change, as exemplified in Fig. 2, it was estimated that the depolarizing action of substance P was 1000-9000 times more potent than that of L-glutamate. The application of Lioresal in a concentration of 5 x 10-6 M d i d not produce any appreciable change of DC potential recorded from the ventral root, but resulted in a marked decrease of the size of monosynaptic reflex in a few minutes (Fig. 1Ah). Polysynaptic reflex was also depressed by the drug. Under such conditions, the depolarizing action of substance P was almost completely abolished (Figs. l Bb and 2). The depolarizing action of L-glutamate was also antagonized by Lioresal, but to a much smaller extent (Figs. ICh and 2). The effect of Lioresal was reversible; thus, after the removal of the drug from the perfusion medium, the mono- and polysynaptic reflexes as well as the responses to substance P and L-glutamate recovered completely within 30 min. When the preparation was soaked in a modified Krebs solution containing low Ca (0.7 mM) and high Mg (7 mM), the mono- and polysynaptic reflexes were completely suppressed, whereas the depolarizing responses to substance P and L-glutamate as recorded from the ventral root were only slightly reduced. Under such conditions, Lioresal again effectively antagonized the action of substance P to depolarize the motoneurons. These results indicate that both substance P and Lioresal act directly on the motoneurons. Fig. 1D and E shows the effect of Lioresal on the responses recorded from the dorsal root. When the potentials were recorded from a dorsal root and the adjacent
179 mV
05
0 10 -7
' 10 - 6
I )O -5
.
.
.
.
L~ 10 .4
. . . . .
• _ 10 ~ M
Fig. 2. Effects of Lioresal on the dose-response curves of substance P and L-glutamate. Ordinate:
potential change produced in L3 ventral root by substance P and L-glutamate. Abscissa: concentration of the drugs on logarithmic scale. © and 0 , responses to substance P. ~., and A, responses to L-glutamate. Open symbols indicate the responses in normal Krebs solution, and filled symbols indicate those in the solution containing Lioresal (5 /. 10 6 M). dorsal root was stimulated, a dorsal root potential was observed. The application of G A B A produced a depolarizing response o f p r i m a r y afferent fibers. Lioresal blocked the dorsal root potential, b u t did n o t affect the G A B A action. These results agree with the observations o f Davidoff and Sears in the frog spinal cord 2 a n d suggest that Lioresal blocks the excitatory t r a n s m i s s i o n at the p r i m a r y afferent synapses in the spinal cord. The present paper reports the first specific a n t a g o n i s t toward the excitatory action of substance P on the central neurons. T h a t an antagonist of substance P blocked the m o n o - and polysynaptic reflexes as well as the dorsal root potential again supports the hypothesis that substance P or a closely related peptide is a sensory t r a n s m i t t e r of spinal dorsal root fibers 3,4,6,9A°. We wish to t h a n k Drs. K. O b a t a a n d T. T a k a h a s h i for their helpful advice.
I CHANG, M. M., LEEMAN, S. E., AND NIALL, H. D., Amino-acid sequence of substance P, Nature New Biol., 232 (1971) 86-87. 2 DAVIDOFF, R. A., AND SEARS, E. S., The effects of Lioresal on synaptic activity in the isolated spinal cord, Neurology (Minneap.), 24 (1974) 957-963. 3 KONISHI, S., AND OTSUKA, M., The effects of substance P and other peptides on spinal neurons of the frog, Brain Research, 65 (1974) 397~10. 4 KONISHI,S., AND OTSUKA, M., Excitatory action of hypothalamic substance P on spinal motoneurones of newborn rats, Nature (Lond.), 252 (1974) 734-735. 50TSUKA, M., AND KONISHI, S., Electrophysiology of mammalian spinal cord in vitro, Nature (Lond.), 252 (1974) 733-734. 60TSUKA, M., KONISH , S., AND TAKAHASHI, T., A further study of the motoneuron-depolarizing peptide extracted from dorsal roots of bovine spinal nerves, Proc. Jap. Acad., 48 (1972) 747-752. 7 PEDERSEN, E., ARL1EN-SOBORG, P., GRYNDERUP, V., AND HENRIKSEN, O., GABA derivative in spasticity (fl-(4-chlorophenyl)-~-aminobutyric acid, Ciba, 34.647-Ba), Acta neurol, stand., 46 (1970) 257-266. 8 PIERAU, F. K., AND ZIMMERMANN, P., Action of a GABA-derivative on postsynaptic potentials
180 and membrane properties of cats' spinal motoneurones, Brain Research, 54 (1973) 376 380. 9 TAKAt~ASHI, T., KONISHI, S., POWEr.L, D., LEEMAN, S. E., AND OTSt~KA, M., Identificatio~ of the motoneuron-depolarizing peptide in bovine dorsal root as hypothalamic substance P Brail Research. 73 (1974) 59 69. l0 TAKAHASm, T., AYD 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- I I.
