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Supraspinal tonic influence on spinal reflexes and involvement in the effect of chlorpromazine
Gen. Pharmac. Vol. 15, No. 2, pp. 155-158, 1984 Printed in Great Britain. All rights reserved
0306-3623/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd
SUPRASPINAL TONIC INFLUENCE ON SPINAL REFLEXES A N D INVOLVEMENT IN THE EFFECT OF CHLORPROMAZINE MASATAKA HINO, HIDEKI ONO a n d HIDEOMI FUKUDA* Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan (Received 6 July 1983)
Abstract--1. Chlorpromazine (CPZ) depressed mono-(MSR) and polysynaptic reflexes in anesthetized
rats. 2. The MSR remained unaltered after spinal transection (CI). After spinal transection, the inhibitory effect of CPZ on the MSR disappeared. After CPZ injection, spinal transection enhanced the MSR to pre-drug level. 3. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR. Phenoxybenzamine inhibited the MSR and, when pre-injected, markedly attenuated the effect of CPZ on the MSR. 4. These results suggest that in anesthetized rats, CPZ blocks the c~-adrenoceptor-mediated facilitatory component of the spontaneous tonic influence on the MSR.
INTRODUCTION C h l o r p r o m a z i n e (CPZ) inhibits spinal reflexes in intact animals (Krivoy, 1957; Carp and A n d e r s o n , 1981), but has no effect on the m o n o s y n a p t i c reflex ( M S R ) in spinal animals (Hudson, 1966; Preston, 1956; Barker a n d A n d e r s o n , 1970). Therefore, this drug has been considered to act on tonic influences from the brain rather t h a n on the spinal cord itself. Experimental evidence from the studies, in which "cold b l o c k " is used, indicates the presence of facilitatory tonic influences on l u m b e r m o t o n e u r o n e s from supraspinal structure. D u r i n g "cold b l o c k " in u p p e r spinal segments, eliminating tonic influences from the brain, the l u m b e r m o t o n e u r o n e hyperpolarizes (Barnes et al., 1962; Sinclair a n d Sastry, 1974; Sastry a n d Sinclair, 1977; Schadt a n d Barnes, 1980). However, C a r p a n d A n d e r s o n (1981) f o u n d no significant differences between M S R amplitudes o f intact a n d spinal animals. In this case, the supraspinal tonic influences (STIs) on the M S R a p p e a r to be negligible. W e a t t e m p t e d to quantitatively assess STIs on the spinal reflexes a n d the C P Z effects on the STIs, by m e a n s o f spinal transection a n d "cold b l o c k " d u r i n g reflex recording. The n e u r o t r a n s m i t t e r involved in the C P Z effects on the spinal reflexes is unclear, as this drug has a diversity of effects on the nervous systems. Several p a t h w a y s involved in the m o d u l a t i o n o f the M S R have been proposed. Electrical stimulation of the locus coeruleous ( F u n s a n d Barnes, 1981), a nucleus o f noradrenergic cells, or the s u b s t a n t i a nigra (York, 1972; Blinn et al., 1980), a nucleus of dopaminergic cells, facilitates the M S R a n d C P Z blocks these artificially evoked facilitations (York, 1973; Strahle n d o r f et al., 1980). However, since it is u n k n o w n *Author to whom all correspondence should be addressed. 155
whether these p a t h w a y s are tonically activated, it is unclear which n e u r o t r a n s m i t t e r is involved in the C P Z effect on M S R . The n e u r o t r a n s m i t t e r specificity of the effect of C P Z on spinal reflexes was also given attention. METHODS
Male Wistar rats (250-400g) were anesthetized with urethane (1 g/ks, i.p.) and a-chloralose (25ms/ks, i.p.). Laminectomy was performed in the lumbo-sacral region of the spinal cord. Ventral and dorsal roots of the segment L5 were isolated. A skin pouch was formed at the site of the dissection and the exposed tissues were covered with liquid paraffin thermoregulated at 37 + I°C. Rectal temperature was also maintained at 3 7 + I°C. The animals were artificially ventilated and the arterial blood pressure was monitored from the femoral artery. The dorsal root of the L5 segment was placed on a bipolar silver electrode for stimulation (0.2 Hz, 0.05 msec, supramaximal). For recording, the ventral root of the L5 segment was placed on a bipolar or monopolar silver electrode. When the monopolar electrode was used, a steel clip, as an indifferent electrode, was attached to the clamp that fixed the vertebra. The MSR and the PSR were amplified with a.c.-equipment and were recorded as an average of eight consecutive reflexes, using an averaging computer (Nihonkohden ATAC-350). In the experiments of spinal transection, the vagi were cut bilaterally before laminectomy. The spinal cord was cut at the C 1 segment with a spatula. "Cold block" was performed by the method similar to that described by Wall (1967). The cervical region of the spinal cord was exposed and a small cube of frozen physiological saline was placed on the cord. "Cold block" was performed more than twice before CPZ injection to examine the reversibility. Reserpine (Apoplon Injection, Daiichi) was given to the rats subcutaneously 24hr before the experiments. Chlorpromazine hydrochloride (Rhodia), phenoxybenzamine hydrochloride (Tokyo Kasei) and haloperidol (Serenace Injection, Dainippon) were dissolved in physiological saline and given into the femoral vein.
156
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I
al.
150 -
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II ,,~ - " - - A I ,, _ . . . . ~ , , ~ . ' _ _ ~ , ,
A
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l===~ft
~
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5O
PSR
IE
¢x 50 E
CPZ IA) 0
I
I
I
1
-30
0
30
60
Time
ST I 0
I 20
CPZ t 60
I 40
L 80
I I00
( min )
Fig. 2. Changes in spinal reflexes after spinal transection (ST) and non-effectiveness of subsequent CPZ injection. Closed and open symbols represent MSR and PSR, respectively. In the experiments represented by closed triangles, only spinal transection was performed. CPZ HC1 (1 mg/kg) was given intravenously 60 min after spinal transection. Each point represents reflex amplitude (the mean -t- SEM of 4-6 experiments) calculated as a percentage of the value just prior to spinal transection.
50-
(B) I -3o
I -20
Time
A
o
I -40
( rain )
I00
n," If/ n
0
CPZ 0
I
30 Time
I 60
( rain l
150CPZ
Fig. 1. Inhibitory effects of chlorpromazine (CPZ) on (A) mono-(MSR) and (B) polysynaptic reflexes (PSR). CPZ was given intravenously at the arrow. The doses of CPZ HC1 examined were 0.01 mg/kg (©), 0.1 mg/kg (A), 1 mg/kg (A) and 10 mg/kg ( I ) . Saline was injected for the control (0). Each point represents reflex amplitude (the mean 4- SEM of 4-6 experiments) calculated as a percentage of the value just prior to CPZ injection.
ST
MSR
o~ ~ Ioo o = ~. E 50
PSR 0
I 0
-30
I 30
Time
I 60
( min )
RESULTS
CPZ dose-dependently suppressed the M S R and the PSR in intact rats (Fig. 1). This suppression was rapidly saturated, long-lasting and was never attenuated even 6 0 m i n after injection. The effect of C P Z - H C I at l m g / k g was maximal, because CPZ-HC1 at a higher dose (10 mg/kg) had the same potency. The maximal effects of CPZ were approximately 55 and 75% suppression of the M S R and PSR, respectively. After spinal transection, which eliminates tonic influences from the brain, the level of the M S R changed little, while that of the PSR decreased to approximately 50% (Fig. 2). CPZ injection after spinal transection had no effect on either the M S R or the PSR. Spinal transection after CPZ injection increased the M S R to the pre-drug level but did not alter the PSR (Fig. 3). These results show that CPZ changes the supraspinal tonic influence (STI) on the M S R into inhibition and abolishes the facilitatory STI on the PSR. " C o l d block" before CPZ injection reduced the M S R but after CPZ injection this inhibition by "cold block" was markedly weakened (Fig. 4). To assess the neurotransmitter specificity of the effect of CPZ on the M S R , several drugs were preadministered. Pretreatment of reserpine, which depletes monoamines, converted the inhibition by
Fig. 3. Effects of spinal transection (ST) on spinal reflexes after CPZ injection. Closed and open circles represent MSR and PSR, respectively. Spinal transection was performed 30 rain after CPZ-HCI (1 mg/kg, i.v.). Each point represents reflex amplitude (the mean 4-SEM of 4.6 experiments) calculated as a percentage of the value just prior to CPZ injection.
,oo 4 Yr~;, /4/'~'-~
I
t Cold
0 -40
" L+
-4-+-
CPZ block
Cold
L -20
I 0 Time
I 20
block I 40
( min )
Fig. 4. Effect of "cold block" on the MSR before and after CPZ injection. The bars in the bottom of the figure represent "cold block". CPZ HC1 (1 mg/kg) was given intravenously. Each point represents MSR amplitude (the mean 4- SEM of 4 experiments) calculated as a percentage of the value just prior to CPZ injection.
Chlorpromazine and spinal reflexes
o< v
IOO
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0 -30
1
L
I
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30
60
Fig. 5. Effect of CPZ-HCI (1 mg/kg, i.v.) on MSR in reserpine treated rats. Reserpine (2 mg/kg, s.c.) was administered 24 hr before the experiments. Each point represents MSR amplitude (the mean 4- SEM of 4 experiments) calculated as a percentage of the valuejust prior to CPZ injection.
CPZ into facilitation (Fig. 5). Since the inhibitory effect of CPZ on the MSR disappeared by reserpine pretreatment, monoaminergic systems were considered to be involved in the CPZ suppression of MSR. Phenoxybenzamine (PBZ), an irreversible longlasting ~-blocker, inhibited the MSR to much the same extent seen with CPZ, and pre-injection of PBZ markedly attenuated the inhibitory effect of CPZ on the MSR (Fig. 6). Haloperidol (HAL), a dopamine receptor antagonist, enhanced the MSR and preinjection of HAL did not alter the inhibitory effect of CPZ (Fig. 6). DISCUSSION
After spinal transection in rats, the level of the MSR remained unchanged (Fig. 2), thereby implying that there is little tonic influence on the MSR from the brain. These results are consistent with the report in which the amplitude of the MSR is the same between spinal and intact anesthetized cats (Carp & Anderson, 1981). However, the studies using "cold block" in decerebrated cats have shown that facilitatory supraspinal tonic influences (STIs) are consid150
PZB~
0 -30
CPZ
I
[
I
O
30
60
T i m e ( rain ) Fig. 6. Influence o f p h e n o x y b e n z a m i n e ( P B Z ) and halo-
peridol (HAL)
on the inhibitory
effect o f C P Z
on MSR.
PBZ-HC1 (5mg/kg, i.v.) or HAL (0.5 mg/kg, i.v.) was administered 30 min before CPZ HC1 (1 mg/kg, i.v.). In the experiments represented by closed triangles, only CPZ was injected, as the control. Each point represents MSR amplitude (the mean + SEM of 4-6 experiments) calculated as a percentage of the value just prior to CPZ injection.
157
erably potent on the MSR (Sinclair and Sastry, 1974; Sastry and Sinclair, 1977; Schadt and Barnes, 1980). We considered that this discrepancy might arise from the difference between anesthetized and decerebrated animals. In order to examine this point, "cold block" was performed in anesthetized rats. Contrary to our expectations, "cold block" reduced the MSR (Fig. 4). While this reduction in the MSR by "cold block" may not reflect total elimination of the STIs, it does suggest the existence of a potent facilitatory component in STIs on the MSR in anesthetized rats. The non-changed level of the MSR after spinal transection, which completely eliminates the influences from the brain, indicates that the STI on the MSR contains both facilitatory and inhibitory components to a similar extent, and that "cold block" does not block all tonic influences from the brain. The finding that the level of the PSR was markedly reduced after spinal transection (Fig. 2) indicates that the STI on the PSR is on the whole facilitatory. After spinal transection, CPZ had no effect on spinal reflexes (Fig. 2). These results confirm the view that CPZ acts selectively on tonic influences from supraspinal structures. After CPZ injection, spinal transection enhanced the MSR but did not alter the PSR (Fig. 3). These results show that after CPZ injection, the STI on the MSR is inhibitory as a whole and that the STI on the PSR is negligible. A simple explanation of the CPZ effect on the STIs is that CPZ blocks the facilitatory component of the supraspinal influences on the MSR and the PSR. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR (Fig. 5), thus monoaminergic systems are probably involved in the inhibitory effect of CPZ. Phenoxybenzamine (PBZ) pre-injection attenuated almost completely the inhibitory effect of CPZ on the MSR (Fig. 6), thereby suggesting a common mechanism in MSR inhibitions by PBZ and CPZ. This mechanism may be ~-adrenoceptor blockade, because both drugs are potent ~-blockers. Noradrenaline is probably involved in the inhibitory effect of CPZ. There is evidence that several noradrenergic nuclei in the brain stem send fibers to the spinal cord (Dahlstr6m and Fuxe, 1965; Nygren and Olson, 1977). Locus coeruleus, one of these nuclei, when electrically stimulated, exerts facilitatory effects on the MSR, and this facilitation is mediated by c~-adrenoceptor (Strahlendorf et al., 1980; Fung and Barnes, 1981). This pathway may be tonically activated in anesthetized rats and CPZ may block this facilitatory noradrenergic pathway. In such a case, the site of action of CPZ exists within the spinal cord. With regard to the L-DOPA-induced enhancement of the MSR in spinal cats, it is clear that CPZ acts within the spinal cord and blocks the enhancement (Barker and Anderson, 1970). Haloperidol increased the MSR (Fig. 6). This drug has no effect on the MSR in spinal rats (Ono & Fukuda, 1982) and seems to act selectively on tonic influences from the brain. A dopaminergic pathway to the spinal cord has been suggested (Commissiong et al., 1978, 1979; Gentleman et al., 1981); however, the effect of this pathway on the MSR is equivocal. Electrical stimulation of the substantia nigra facilitates the MSR (York, 1972, 1973; Blinn et al., 1980),
158
MASATAKAHINO et al.
whereas i.v. administration of dopamine agonists inhibits the MSR, in spinal cats (Carp and Anderson, 1982). The present results, in which haloperidol enhanced the M S R , suggest that a tonically activated inhibitory dopaminergic pathway is present in anesthetized rats. CPZ suppression of the M S R was not affected by pre-injection of haloperidol (Fig. 6), therefore, the dopaminergic pathway appears to be indifferent to the CPZ effect on the MSR. Methysergide, a serotonin antagonist, inhibited the M S R in spinal rats to the same extent as in intact rats and pre-injection of methysergide did not alter the inhibitory effect of CPZ on the M S R (unpublished data). We consider that serotonergic systems are probably not involved in the CPZ effect on the MSR. M S R inhibitions by CPZ and PBZ were accompanied by a decrease in blood pressure. But, it is unlikely that decrease in blood pressure causes a decrease in the MSR, because: (1) Haloperidol which enlarged the M S R also decreased blood pressure. (2) In preliminary experiments, hexamethonium, which cannot pass the blood-brain barrier and decreases peripheral blood pressure, did not alter the MSR, and after hexamethonium injection, the CPZ inhibitory effect was intact. In conclusion, the present findings suggest that in anesthetized rats, the STI on the M S R contains both facilitatory and inhibitory components, to a similar extent, that on the PSR is facilitatory as a whole, and CPZ blocks the e-adrenoceptor-mediated facilitatory component of the STI on the MSR. Our results provide data on supraspinal modulation of spinal reflexes. SUMMARY
Spinal transection and "cold block", which eliminate supraspinal tonic influence (STI), were performed in anesthetized rats, in order to quantitatively assess the STI on spinal reflexes and the effect of chlorpromazine (CPZ) on the STI. The monosynaptic reflex (MSR) remained unaltered after spinal transection but was reduced during "cold block". After spinal transection, the inhibitory effect of CPZ on the M S R disappeared. After CPZ injection, spinal transection enhanced the M S R and the reduction of the M S R by "cold block" was markedly attenuated. To clarify the neurotransmitter specificity of the inhibitory effect of CPZ on the MSR, several drugs were pretreated. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR. Phenoxybenzamine inhibited the M S R and, when preinjected, markedly attenuated the effect of CPZ on the MSR. These results suggest that in anesthetized rats, the STI on the M S R contains both facilitatory and inhibitory components, to a similar extent, and CPZ blocks the c~-adrenoceptor-mediated facilitatory component of the STI on the MSR. Acknowledgements--This work was supported in part by a
Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. We thank M. Ohara, Kyushu University, for critical reading of the manuscript. REFERENCES
Barker R. B. and Anderson E. G. (1970) The antagonism
of the effects of L-3,4-dihydroxyphenylalanine on spinal reflexes by adrenergic blocking agents. J. Pharmac. exp. Ther. 173, 224-231. Barnes C. D., Joynt R. J. and Schottelius B. A. (1962) Motoneuron resting potentials in spinal shock. Am. J. Physiol. 203, 1113-1116. Blinn G., Heinz G. and Jurna I. (1980) Effects of substantia nigra stimulation on suralis-evoked spinal reflex activity: comparison with the effects of morphine and stimulation in the periaqueductal gray matter. Neuropharmacology 19, 75-85. Carp J. S. and Anderson R. J. (1981) Modification of spinal cord transmission by an interaction of chlorpromazine and phenytoin. J. Pharmac. exp. Ther. 216, 270-274. Carp J. S. and Anderson R. J. (1982) Dopamine receptormediated depression of spinal monosynaptic transmission. Brain Res. 242, 247-254. Commissiong J. W., Galli C. L. and Neff N. H. (1978) Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord. J. Neurochem. 30, 1095-1099. Commissiong J. W., Gentleman S. and Neff N. H. (1979) Spinal cord dopaminergic neurons: evidence for an uncrossed nigrospinal pathway. Neuropharmacology 18, 565-568. Dahlstr6m A. and Fuxe K. (1965) Experimentally induced changes in the intraneuronal amine levels of bulbospinal neuron systems. Acta physiol, scand. 247 (Suppl.), 1 36. Fung S. J. and Barnes C. D. (1981) Evidence of facilitatory coerulospinal action in lumber motoneurons of cats. Brain Res. 216, 299-311. Gentleman S., Parenti M., Commissiong J. W. and Neff N. H. (1981) Dopamine-activated adenylate cyclase of spinal cord: supersensitivity following transection of the cord. Brain Res. 210, 271-275. Hudson R. D. (1966) Effects of chlorpromazine on spinal cord reflex mechanisms. Neuropharmacology 5, 43-58. Krivoy W. A. (1957) Actions of chlorpromazine and of reserpine on spinal reflex activity in the cat. Proc. Soc. exp. Biol. 96, 18-20. Nygren L.-G. and Olson L. (1977) A new major projection from locus coeruleus: the main source of noradrenergic nerve terminals in the ventral and dorsal columns of the spinal cord. Brain Res. 132, 85-93. Ono H. and Fukuda H. (1982) Ventral root depolarization and spinal reflex augmentation by a TRH analog in rat spinal cord. Neuropharmacology 21, 739-744. Preston J. B. (1956) Effects of chlorpromazine on the central nervous system of the cat: a possible neural basis for action. J. Pharmac. 118, 100~115. Sastry B. S. R. and Sinclair J. G. (1977) Tonic inhibitory influence of a supraspinal monoaminergic system on presynaptic inhibition of an extensor monosynaptic reflex. Brain Res. 124, 109 120. Schadt J. C. and Barnes C. D. (1980) Motoneuron membrane changes associated with spinal shock and the Schiff-Sherrington phenomenon. Brain Res. 201,373-383. Sinclair J. G. and Sastry B. S. R. (1974) Supraspinal monoaminergic involvement in the blockade of recurrent inhibition of the monosynaptic reflex. Neuropharmacology 13, 741-747. Strahlendorf J. C., Strahlendorf H. K., Kingsley R. E., Gintautas J. and Barnes C. D. (1980) Facilitation of the lumber monosynaptic reflexes by locus coeruleus stimulation. Neuropharmacology 19, 225-230. Wall P. D. (1967) The laminar organization of dorsal horn and effects of descending impulses. J. Physiol. 188, 403-423. York D. H. (1972) Potentiation of lumbo-sacral monosynaptic reflexes by the substantia nigra. Exp. Neurol. 36, 437-448. York D. H. (1973) Antagonism of descending effects of the substantia nigra on lumbo-sacral monosynaptic reflexes. Neuropharmacology 12, 629-636.
0306-3623/84 $3.00 + 0.00 Copyright © 1984 Pergamon Press Ltd
SUPRASPINAL TONIC INFLUENCE ON SPINAL REFLEXES A N D INVOLVEMENT IN THE EFFECT OF CHLORPROMAZINE MASATAKA HINO, HIDEKI ONO a n d HIDEOMI FUKUDA* Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan (Received 6 July 1983)
Abstract--1. Chlorpromazine (CPZ) depressed mono-(MSR) and polysynaptic reflexes in anesthetized
rats. 2. The MSR remained unaltered after spinal transection (CI). After spinal transection, the inhibitory effect of CPZ on the MSR disappeared. After CPZ injection, spinal transection enhanced the MSR to pre-drug level. 3. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR. Phenoxybenzamine inhibited the MSR and, when pre-injected, markedly attenuated the effect of CPZ on the MSR. 4. These results suggest that in anesthetized rats, CPZ blocks the c~-adrenoceptor-mediated facilitatory component of the spontaneous tonic influence on the MSR.
INTRODUCTION C h l o r p r o m a z i n e (CPZ) inhibits spinal reflexes in intact animals (Krivoy, 1957; Carp and A n d e r s o n , 1981), but has no effect on the m o n o s y n a p t i c reflex ( M S R ) in spinal animals (Hudson, 1966; Preston, 1956; Barker a n d A n d e r s o n , 1970). Therefore, this drug has been considered to act on tonic influences from the brain rather t h a n on the spinal cord itself. Experimental evidence from the studies, in which "cold b l o c k " is used, indicates the presence of facilitatory tonic influences on l u m b e r m o t o n e u r o n e s from supraspinal structure. D u r i n g "cold b l o c k " in u p p e r spinal segments, eliminating tonic influences from the brain, the l u m b e r m o t o n e u r o n e hyperpolarizes (Barnes et al., 1962; Sinclair a n d Sastry, 1974; Sastry a n d Sinclair, 1977; Schadt a n d Barnes, 1980). However, C a r p a n d A n d e r s o n (1981) f o u n d no significant differences between M S R amplitudes o f intact a n d spinal animals. In this case, the supraspinal tonic influences (STIs) on the M S R a p p e a r to be negligible. W e a t t e m p t e d to quantitatively assess STIs on the spinal reflexes a n d the C P Z effects on the STIs, by m e a n s o f spinal transection a n d "cold b l o c k " d u r i n g reflex recording. The n e u r o t r a n s m i t t e r involved in the C P Z effects on the spinal reflexes is unclear, as this drug has a diversity of effects on the nervous systems. Several p a t h w a y s involved in the m o d u l a t i o n o f the M S R have been proposed. Electrical stimulation of the locus coeruleous ( F u n s a n d Barnes, 1981), a nucleus o f noradrenergic cells, or the s u b s t a n t i a nigra (York, 1972; Blinn et al., 1980), a nucleus of dopaminergic cells, facilitates the M S R a n d C P Z blocks these artificially evoked facilitations (York, 1973; Strahle n d o r f et al., 1980). However, since it is u n k n o w n *Author to whom all correspondence should be addressed. 155
whether these p a t h w a y s are tonically activated, it is unclear which n e u r o t r a n s m i t t e r is involved in the C P Z effect on M S R . The n e u r o t r a n s m i t t e r specificity of the effect of C P Z on spinal reflexes was also given attention. METHODS
Male Wistar rats (250-400g) were anesthetized with urethane (1 g/ks, i.p.) and a-chloralose (25ms/ks, i.p.). Laminectomy was performed in the lumbo-sacral region of the spinal cord. Ventral and dorsal roots of the segment L5 were isolated. A skin pouch was formed at the site of the dissection and the exposed tissues were covered with liquid paraffin thermoregulated at 37 + I°C. Rectal temperature was also maintained at 3 7 + I°C. The animals were artificially ventilated and the arterial blood pressure was monitored from the femoral artery. The dorsal root of the L5 segment was placed on a bipolar silver electrode for stimulation (0.2 Hz, 0.05 msec, supramaximal). For recording, the ventral root of the L5 segment was placed on a bipolar or monopolar silver electrode. When the monopolar electrode was used, a steel clip, as an indifferent electrode, was attached to the clamp that fixed the vertebra. The MSR and the PSR were amplified with a.c.-equipment and were recorded as an average of eight consecutive reflexes, using an averaging computer (Nihonkohden ATAC-350). In the experiments of spinal transection, the vagi were cut bilaterally before laminectomy. The spinal cord was cut at the C 1 segment with a spatula. "Cold block" was performed by the method similar to that described by Wall (1967). The cervical region of the spinal cord was exposed and a small cube of frozen physiological saline was placed on the cord. "Cold block" was performed more than twice before CPZ injection to examine the reversibility. Reserpine (Apoplon Injection, Daiichi) was given to the rats subcutaneously 24hr before the experiments. Chlorpromazine hydrochloride (Rhodia), phenoxybenzamine hydrochloride (Tokyo Kasei) and haloperidol (Serenace Injection, Dainippon) were dissolved in physiological saline and given into the femoral vein.
156
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I
al.
150 -
I00
II ,,~ - " - - A I ,, _ . . . . ~ , , ~ . ' _ _ ~ , ,
A
~oo (,f)
l===~ft
~
"
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IE
¢x 50 E
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I
I
I
1
-30
0
30
60
Time
ST I 0
I 20
CPZ t 60
I 40
L 80
I I00
( min )
Fig. 2. Changes in spinal reflexes after spinal transection (ST) and non-effectiveness of subsequent CPZ injection. Closed and open symbols represent MSR and PSR, respectively. In the experiments represented by closed triangles, only spinal transection was performed. CPZ HC1 (1 mg/kg) was given intravenously 60 min after spinal transection. Each point represents reflex amplitude (the mean -t- SEM of 4-6 experiments) calculated as a percentage of the value just prior to spinal transection.
50-
(B) I -3o
I -20
Time
A
o
I -40
( rain )
I00
n," If/ n
0
CPZ 0
I
30 Time
I 60
( rain l
150CPZ
Fig. 1. Inhibitory effects of chlorpromazine (CPZ) on (A) mono-(MSR) and (B) polysynaptic reflexes (PSR). CPZ was given intravenously at the arrow. The doses of CPZ HC1 examined were 0.01 mg/kg (©), 0.1 mg/kg (A), 1 mg/kg (A) and 10 mg/kg ( I ) . Saline was injected for the control (0). Each point represents reflex amplitude (the mean 4- SEM of 4-6 experiments) calculated as a percentage of the value just prior to CPZ injection.
ST
MSR
o~ ~ Ioo o = ~. E 50
PSR 0
I 0
-30
I 30
Time
I 60
( min )
RESULTS
CPZ dose-dependently suppressed the M S R and the PSR in intact rats (Fig. 1). This suppression was rapidly saturated, long-lasting and was never attenuated even 6 0 m i n after injection. The effect of C P Z - H C I at l m g / k g was maximal, because CPZ-HC1 at a higher dose (10 mg/kg) had the same potency. The maximal effects of CPZ were approximately 55 and 75% suppression of the M S R and PSR, respectively. After spinal transection, which eliminates tonic influences from the brain, the level of the M S R changed little, while that of the PSR decreased to approximately 50% (Fig. 2). CPZ injection after spinal transection had no effect on either the M S R or the PSR. Spinal transection after CPZ injection increased the M S R to the pre-drug level but did not alter the PSR (Fig. 3). These results show that CPZ changes the supraspinal tonic influence (STI) on the M S R into inhibition and abolishes the facilitatory STI on the PSR. " C o l d block" before CPZ injection reduced the M S R but after CPZ injection this inhibition by "cold block" was markedly weakened (Fig. 4). To assess the neurotransmitter specificity of the effect of CPZ on the M S R , several drugs were preadministered. Pretreatment of reserpine, which depletes monoamines, converted the inhibition by
Fig. 3. Effects of spinal transection (ST) on spinal reflexes after CPZ injection. Closed and open circles represent MSR and PSR, respectively. Spinal transection was performed 30 rain after CPZ-HCI (1 mg/kg, i.v.). Each point represents reflex amplitude (the mean 4-SEM of 4.6 experiments) calculated as a percentage of the value just prior to CPZ injection.
,oo 4 Yr~;, /4/'~'-~
I
t Cold
0 -40
" L+
-4-+-
CPZ block
Cold
L -20
I 0 Time
I 20
block I 40
( min )
Fig. 4. Effect of "cold block" on the MSR before and after CPZ injection. The bars in the bottom of the figure represent "cold block". CPZ HC1 (1 mg/kg) was given intravenously. Each point represents MSR amplitude (the mean 4- SEM of 4 experiments) calculated as a percentage of the value just prior to CPZ injection.
Chlorpromazine and spinal reflexes
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60
Fig. 5. Effect of CPZ-HCI (1 mg/kg, i.v.) on MSR in reserpine treated rats. Reserpine (2 mg/kg, s.c.) was administered 24 hr before the experiments. Each point represents MSR amplitude (the mean 4- SEM of 4 experiments) calculated as a percentage of the valuejust prior to CPZ injection.
CPZ into facilitation (Fig. 5). Since the inhibitory effect of CPZ on the MSR disappeared by reserpine pretreatment, monoaminergic systems were considered to be involved in the CPZ suppression of MSR. Phenoxybenzamine (PBZ), an irreversible longlasting ~-blocker, inhibited the MSR to much the same extent seen with CPZ, and pre-injection of PBZ markedly attenuated the inhibitory effect of CPZ on the MSR (Fig. 6). Haloperidol (HAL), a dopamine receptor antagonist, enhanced the MSR and preinjection of HAL did not alter the inhibitory effect of CPZ (Fig. 6). DISCUSSION
After spinal transection in rats, the level of the MSR remained unchanged (Fig. 2), thereby implying that there is little tonic influence on the MSR from the brain. These results are consistent with the report in which the amplitude of the MSR is the same between spinal and intact anesthetized cats (Carp & Anderson, 1981). However, the studies using "cold block" in decerebrated cats have shown that facilitatory supraspinal tonic influences (STIs) are consid150
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CPZ
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60
T i m e ( rain ) Fig. 6. Influence o f p h e n o x y b e n z a m i n e ( P B Z ) and halo-
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effect o f C P Z
on MSR.
PBZ-HC1 (5mg/kg, i.v.) or HAL (0.5 mg/kg, i.v.) was administered 30 min before CPZ HC1 (1 mg/kg, i.v.). In the experiments represented by closed triangles, only CPZ was injected, as the control. Each point represents MSR amplitude (the mean + SEM of 4-6 experiments) calculated as a percentage of the value just prior to CPZ injection.
157
erably potent on the MSR (Sinclair and Sastry, 1974; Sastry and Sinclair, 1977; Schadt and Barnes, 1980). We considered that this discrepancy might arise from the difference between anesthetized and decerebrated animals. In order to examine this point, "cold block" was performed in anesthetized rats. Contrary to our expectations, "cold block" reduced the MSR (Fig. 4). While this reduction in the MSR by "cold block" may not reflect total elimination of the STIs, it does suggest the existence of a potent facilitatory component in STIs on the MSR in anesthetized rats. The non-changed level of the MSR after spinal transection, which completely eliminates the influences from the brain, indicates that the STI on the MSR contains both facilitatory and inhibitory components to a similar extent, and that "cold block" does not block all tonic influences from the brain. The finding that the level of the PSR was markedly reduced after spinal transection (Fig. 2) indicates that the STI on the PSR is on the whole facilitatory. After spinal transection, CPZ had no effect on spinal reflexes (Fig. 2). These results confirm the view that CPZ acts selectively on tonic influences from supraspinal structures. After CPZ injection, spinal transection enhanced the MSR but did not alter the PSR (Fig. 3). These results show that after CPZ injection, the STI on the MSR is inhibitory as a whole and that the STI on the PSR is negligible. A simple explanation of the CPZ effect on the STIs is that CPZ blocks the facilitatory component of the supraspinal influences on the MSR and the PSR. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR (Fig. 5), thus monoaminergic systems are probably involved in the inhibitory effect of CPZ. Phenoxybenzamine (PBZ) pre-injection attenuated almost completely the inhibitory effect of CPZ on the MSR (Fig. 6), thereby suggesting a common mechanism in MSR inhibitions by PBZ and CPZ. This mechanism may be ~-adrenoceptor blockade, because both drugs are potent ~-blockers. Noradrenaline is probably involved in the inhibitory effect of CPZ. There is evidence that several noradrenergic nuclei in the brain stem send fibers to the spinal cord (Dahlstr6m and Fuxe, 1965; Nygren and Olson, 1977). Locus coeruleus, one of these nuclei, when electrically stimulated, exerts facilitatory effects on the MSR, and this facilitation is mediated by c~-adrenoceptor (Strahlendorf et al., 1980; Fung and Barnes, 1981). This pathway may be tonically activated in anesthetized rats and CPZ may block this facilitatory noradrenergic pathway. In such a case, the site of action of CPZ exists within the spinal cord. With regard to the L-DOPA-induced enhancement of the MSR in spinal cats, it is clear that CPZ acts within the spinal cord and blocks the enhancement (Barker and Anderson, 1970). Haloperidol increased the MSR (Fig. 6). This drug has no effect on the MSR in spinal rats (Ono & Fukuda, 1982) and seems to act selectively on tonic influences from the brain. A dopaminergic pathway to the spinal cord has been suggested (Commissiong et al., 1978, 1979; Gentleman et al., 1981); however, the effect of this pathway on the MSR is equivocal. Electrical stimulation of the substantia nigra facilitates the MSR (York, 1972, 1973; Blinn et al., 1980),
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whereas i.v. administration of dopamine agonists inhibits the MSR, in spinal cats (Carp and Anderson, 1982). The present results, in which haloperidol enhanced the M S R , suggest that a tonically activated inhibitory dopaminergic pathway is present in anesthetized rats. CPZ suppression of the M S R was not affected by pre-injection of haloperidol (Fig. 6), therefore, the dopaminergic pathway appears to be indifferent to the CPZ effect on the MSR. Methysergide, a serotonin antagonist, inhibited the M S R in spinal rats to the same extent as in intact rats and pre-injection of methysergide did not alter the inhibitory effect of CPZ on the M S R (unpublished data). We consider that serotonergic systems are probably not involved in the CPZ effect on the MSR. M S R inhibitions by CPZ and PBZ were accompanied by a decrease in blood pressure. But, it is unlikely that decrease in blood pressure causes a decrease in the MSR, because: (1) Haloperidol which enlarged the M S R also decreased blood pressure. (2) In preliminary experiments, hexamethonium, which cannot pass the blood-brain barrier and decreases peripheral blood pressure, did not alter the MSR, and after hexamethonium injection, the CPZ inhibitory effect was intact. In conclusion, the present findings suggest that in anesthetized rats, the STI on the M S R contains both facilitatory and inhibitory components, to a similar extent, that on the PSR is facilitatory as a whole, and CPZ blocks the e-adrenoceptor-mediated facilitatory component of the STI on the MSR. Our results provide data on supraspinal modulation of spinal reflexes. SUMMARY
Spinal transection and "cold block", which eliminate supraspinal tonic influence (STI), were performed in anesthetized rats, in order to quantitatively assess the STI on spinal reflexes and the effect of chlorpromazine (CPZ) on the STI. The monosynaptic reflex (MSR) remained unaltered after spinal transection but was reduced during "cold block". After spinal transection, the inhibitory effect of CPZ on the M S R disappeared. After CPZ injection, spinal transection enhanced the M S R and the reduction of the M S R by "cold block" was markedly attenuated. To clarify the neurotransmitter specificity of the inhibitory effect of CPZ on the MSR, several drugs were pretreated. Reserpine pretreatment abolished the inhibitory effect of CPZ on the MSR. Phenoxybenzamine inhibited the M S R and, when preinjected, markedly attenuated the effect of CPZ on the MSR. These results suggest that in anesthetized rats, the STI on the M S R contains both facilitatory and inhibitory components, to a similar extent, and CPZ blocks the c~-adrenoceptor-mediated facilitatory component of the STI on the MSR. Acknowledgements--This work was supported in part by a
Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. We thank M. Ohara, Kyushu University, for critical reading of the manuscript. REFERENCES
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