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Effects of catecholamines on spinal motoneurones and spinal reflex discharges in the isolated spinal cord of the newborn rat
Developmental Brain Research, 19 (1985) 31-36
31
Elsevier BRD 50173
Effects of Catecholamines on Spinal Motoneurones and Spinal Reflex Discharges in the Isolated Spinal Cord of the Newborn Rat TAKIO KITAZAWA, KOJI SAITO* and AKIRA OHGA
Department of Pharmacology, Facultyof Veterinary Medicine, Hokkaido University, Sapporo 060 (Japan) (Accepted September 17th, 1984)
Key words: isolated spinal cord - - spinal motoneurons - - spinal reflex discharges - - catecholamines - - tyramine - - adamantanamine
The effects of catecholamines on spinal motoneurones and spinal reflex discharges were investigated in the isolated spinal cord of newborn rat. Noradrenaline (NA), adrenaline (Adr), dopamine (DA) and isoproterenol (Iso) caused depolarization of the motoneurones in a dose-dependent manner. The depolarizing action persisted in Ca2+-deficient Krebs solution. The order of potency was Adr >NA>DA~,Iso. The effects of NA and Adr on the monosynaptic reflex discharge varied; depression, potentiation or depression followed by potentiation. The polysynaptic reflex discharge was consistently depressed. DA depressed both the mono- and polysynaptic reflex discharges in all the preparations. Tyramine and adamantanamine induced a response similar to that to DA rather than to NA. Depolarization of the motoneurones and the effects on the spinal reflex discharges induced by all the catecholamines were decreased by phentolamine or phenoxybenzamine but not by propranolol or haloperidol. It is suggested that the endogenous catecholamines, mainly DA, depolarize the motoneurones and depress the mono- and polysynaptic reflex discharges through an a-adrenoceptor in the spinal cord of the newborn rat. INTRODUCTION
tic reflex, which was blocked by phenoxybenzamine2S.
The presence of n o r a d r e n e r g i c fibers has been d e m o n s t r a t e d in the spinal cord of rat 3-5,9,23,34, mouse 3,9 and cat 9,19 by using fluorescent histochemical and immunocytochemical techniques. These nerve fibres originate mainly from the locus coeruleUS4,5,23,34, subcoeruleus 34 and medial and lateral para-
There have been inconsistent results concerning the effecl;s of noradrenaline ( N A ) on the spinal motoneurone. N A applied iontophoretically caused hyperpolarization12. 25 and depression of the excitability of the m o t o n e u r o n e in the cat in situ 25,33, while N A a d d e d to a perfusion m e d i u m depolarized the m o t o n e u r o n e in the isolated spinal cord of the rat 1 and iontophoretically applied N A enhanced the excitability of the m o t o n e u r o n e of the rat in situ2, 35. The effects on the spinal reflexes of exogenous N A were also varied; depression of the k n e e - j e r k reflex22; potentiation of the flexor 11 and C-fibre reflexes 21, or no effects on the monosynaptic reflex discharge 8. Thus, the physiological roles of N A in the spinal cord are not clear at present. Recently, the presence of other descending fibres containing d o p a m i n e ( D A ) have been demon-
brachial nuclei in the pons 34. T h e y terminate near a - m o t o n e u r o n e s , interneurones in the ventral horn and interneurones in the superficial layer of the dorsal horn5,19,23,34. Close contact between n o r a d r e n e r gic fibers and a-motoneuronesSA9, 34 suggests functional synapses between them. F r o m these findings, it was p r o p o s e d that descending n o r a d r e n e r g i c fibers from the higher central nervous system play a role in the regulation of the spinal locomotion circuitryl6. In fact, electrical stimulation of the locus coeruleus in the cat caused facilitation of the l u m b a r monosynap-
* Present address: Division of Cell Biology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo 187, Japan. Correspondence: T. Kitazawa. Present address: Department of Veterinary Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
32
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strated 4,6A7 but there are few reports concerning the effects of DA on the motoneurone and spinal reflex activity. Much of the knowledge about the effects of catecholamines in the spinal cord was obtained for the cat in situ where catecholamines were applied iontophoretically or intravenously. In the present experiments, the effects of catecholamines and endogenous catecholamine releasers on the spinal motoneurone and the segmental reflex discharges were investigated in the isolated spinal cord of the newborn rat. In this preparation, drugs can be applied at known concentrations for a required period and the ionic environment of the neurone can be modified easily24.
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MATERIALS AND METHODS
Both sexes of newborn Wistar rats (0-5-days-old) were used. The preparation was made by methods similar to those described previously24. 26. The spinal cord was isolated from a newborn rat anesthetized with ether, and then hemisected sagittally. The isolated preparation was placed in a chamber (0.3 ml) which was perfused with a nutrient medium at 3 ml/min. Krebs solution of the following composition (mM) was used as the nutrient medium: NaC1, 124; KCI, 5; MgSO 4, 1.3; CaC12, 2.4; KH2PO 4, 1.24; NaHCO3, 26 and glucose, 10. The solution was kept at 30 + 1 °C and bubbled with a 95% 0 2 and 5% CO 2 gas mixture. A Ca2+-deficient solution was prepared by increasing MgSO4 to 3.5 mM and decreasing CaC12 to 0.1 mM. Drugs were dissolved in the Krebs solution and applied by means of perfusion. The potential changes generated in the spinal motoneurones were recorded extracellularly from the L3 ventral root with a suction electrode. To evoke the segmental reflex discharges, the corresponding dorsal root was stimulated electrically (400/~s duration and supramaximai intensity).
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RESULTS
Effects of catecholamines on the spinal motoneurones Application of NA (10-7-5 × 10-5 M), adrenaline (Adr, 5×10-8-5x10 -5 M) and DA (10-6-2x 10-4 M) to the perfusing solution for 30 s caused depolarization of the motoneurones (Fig. 1Ba-Da). Many small spike discharges appeared near and at
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Fig. 2. Depolarization of the motoneurones induced by catecholamines. Symbols show responses induced by noradrenaline (O), adrenaline (©), dopamine ([3), isoproterenol (A), tyramine (11) and adamantanamine (&) in the normal Krebs solution (mean + S.E., n = 5-7). The amplitude of the depolarization (ordinate) is expressed as a percentage of that induced by 130 mM K ÷.
33 the peak of the depolarization when the response was large. These depolarizations were reproducible on 10 min interval application. The relationships between the amplitude of depolarization and the concentration of the drugs are shown in Fig. 2. Each catecholamine caused depolarization in a concentration dependent manner to induce the maximum response. The EDs0 values were 3x 10-6 M for NA, 10 -6 M for Adr and 5 x 10-5 M for DA. Isoproterenol (Iso) caused only slight depolarization. The order of potency for depolarization of the motoneurones was A d r > N A > D A - > I s o . The time courses of depolarization induced by the catecholamines were compared by employing concentrations at which about the same amplitude of depolarization occurred with each amine. The times (s) between the half maximum response were 55.7 + 1.7 (mean + S.E., n = 7) for NA (5×10 -6 M), 83.0 ___4.4 (n = 5) for Adr (10 -6 M) and 97.2 + 4.8 (n = 5) for D A (5 x 10-5 M). In the Ca2+-deficient solution (Ca 2+, 0.1 mM; Mg 2÷, 3.5 mM), segmental reflex discharges recorded from the L3 ventral root were almost completely abolished (Fig. l a b ) . In this condition, however, the amplitude of the depolarization of the motoneurones induced by NA, Adr and D A was nearly completely unaffected (Fig. 1Bb-Db). It was reported that ~-aminobutyric acid (GABA) acted directly on the isolated dorsal root of the frog and generated potential changes TM. This possibility was examined in the ventral root with catecholamines. The depolarization induced by the addition of 5 mM K ÷ was observed in the ordinary way, and then the ventral root was cut off close to the spinal cord. After this, the K+-induced depolarization in the cut off root remained at more than 70% of the control level. On the other hand, an appreciable potential change was not induced by NA, Adr or DA.
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Fig. 3. Effects of noradrenaline and dopamine on the monosynaptic reflex discharges (L3). Noradrenaline (5x10-6M) caused depression (A), depression followed by potentiation (B) and potentiation (C) of the monosynaptic reflex discharges ((3). The polysynaptic reflex discharges (0) were depressed (A-C) in all the preparations. Dopamine (5 x 10-5 M) also depressed monosynaptic reflex discharges (D,©). This effect was antagonized by phentolamine (2.7x 10-5 M) (D,II). Noradrenaline and dopamine were added to the bath during the periods indicated by the horizontal bars (3 min).
preparation. Polysynaptic reflex discharges were decreased in all the preparations examined (Fig. 3A-C). D A (10-6-5×10 -4 M) depressed both mono- and polysynaptic reflex discharges to almost the same extent and potentiation was never seen (Fig. 3D). Iso slightly depressed the mono- and polysynaptic reflex discharges at high concentrations (10-4-10 -3 M). Fig. 4 is a summary of the depressant effects of the catecholamines on the monosynaptic ioo
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Effects of catecholamines on the segmental reflex discharges NA (5×10-7-10 -4 M) and Adr (10-7-10 -4 M) had various effects on the amplitude of the monosynaptic reflex discharge; depression (NA, 18/27 preparations; Adr, 20/28), potentiation (NA, 2/27; Adr, 2/28) or depression followed by potentiation (NA, 7/27; Adr, 6/28) (Fig. 3 A - C ) . The response was not altered to one of the other types with time in the same
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Fig. 4. Depression of the monosynaptic reflex discharges by catecholamines. The symbolsshow the effects of noradrenaline (O), adrenaline (C)), dopamine (D), isoproterenol (A), tyramine (11) and adamantanamine (A) in the normal Krebs solution (mean _+S.E., n = 3-5). Only the inhibitory response concerning adrenaline and noradrenaline was summarized (see the text).
34 reflex discharge. The effects of NA and Adr were maximal at 10-5-10 -4 M, at which the discharge was reduced to about half of the control level. D A inhibited the discharge more extensively than Adr or NA and depressed it almost completely at 5 x 10-a M. Strychnine (10-6g/ml) and picrotoxin (5×10 -6 g/ml) did not reduce the depressant effect of the catecholamines.
Effects of receptor blocking agents Phentolamine (2.7x10-6-2.7x10-SM) or phenoxybenzamine (5 x 10-6 M), which did not produce any appreciable potential changes in the motoneurones, decreased the depolarization induced by NA (5x10 -6 M), Adr (5x10 -6 M), D A (5x10-5 M) and Iso (10 .3 M). Depolarization induced by substance P (2x10 -7 M) or serotonin (5x10 -6 M) was not affected by phentolamine (2.7x10-6 M) (Fig. 5). Propranolol (5 x 10-6 M) did not affect all the depolarizations. The inhibitory effects of catecholamines on the monosynaptic reflex discharges were also decreased by phentolamine (2.7x 10-5 M) but not by propranolol (5 x 10-6 M). Fig. 3D shows the effect of phentolamine on the depressant effect of DA. Both receptor blocking agents did not influence the inhibition induced by serotonin (5x10 -6 M) or the reflex discharge itself at the concentrations employed. The potentiation of the monosynaptic reflex discharges induced by NA and Adr in some preparations was also blocked by phentolamine.
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Fig. 5. Effects of phentolamine (2.7x 10-6 M) on the depolarization of the motoneurones induced by catecholamines. The traces show the depolarization of the motoneurones recorded from the L3 ventral root induced by A, noradrenaline (5X10 -6 M); B, adrenaline (5x10 -6 M); C, dopamine (5 x 10-5 M); D, serotonin (5 x 10-6 M). Depolarizations in the normal Krebs solution are shown in a and in the Krebs solution containing phentotamine (2.7x10-6 M) in b. Drugs were added to the bath during the periods indicated by the horizontal bars (30 s).
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Fig. 6. Effects of tyramine and adamantanamine on the spinal motoneurones. The traces show potential changes in the motoneurones recorded from the L3 ventral root; A, depolarization induced by tyramine (5x10 -5 M) and B, by adamantanamine (5×10-3 M). Responses in the normal Krebs solution arc shown in a, in the Ca2+-deficient Krebs solution (Ca2", 0.1 mM; Mg2÷, 3.5 mM) in b, and in the Krebs solution containing phentolamine (2.7x 10-6 M) in c. The effect of adamantanamine in the Ca2+-deficientsolution was restored in the normal solution before adding phentolamine (not shown). Tyramine and adamantanamine were added to the bath during the periods indicated by the horizontal bars (30 s).
Haloperidol (10-7-10 -5 M) did not decrease the depolarization of the motoneurones or the inhibition of the reflex discharges evoked by DA. At higher concentrations than 10-5 M, haloperidol itself depressed the reflex discharges.
Effects of endogenous catecholamine releasers Tyramine, a releaser of endogenous NA 29,3°, depolarized the motoneurones in a concentration dependent manner (5×10-6-5×10 -4 M) (Fig. 2, II). Adamantanamine (2x10-4-10 -2 M), an endogenous D A releaser 31,32, also depolarized the motoneurones (Fig. 2, A). Fig. 6 shows the effects of tyramine (5x10-5 M) and adamantanamine (5x10 -3 M). In the Ca2+-deficient solution, tyramine-induced depolarization was scarcely affected but depolarization induced by adamantanamine was abolished almost completely, suggesting that adamantanamine depolarized the motoneurones trans-synapticalty. The effect of adamantanamine or tyramine was decreased by phentolamine (2.7x 10-6 M). Tyramine (2x 10-6-5x 10-4 M) and adamantanamine (2x10-4-10 -2 M) decreased the amplitude of the mono- and polysynaptic reflex discharges. The inhibition of the monosynaptic reflex discharge was almost complete (Fig. 4). The depressant effects of tyramine and adamantanamine were antagonized by phentolamine (2.7x 10-5 M) but not by propranolol (5 x 10-6 M) or haloperidol (10 -s M). Potentiation of
35 the monosynaptic reflex discharge was observed with neither of the drugs as in the case of NA or Adr. These effects of tyramine and adamantanamine on the monosynaptic reflex were thus similar to those of DA. DISCUSSION
Effects of catecholamines on motoneurones The present experiments showed that catecholamines directly depolarized the spinal motoneurones. As the axons of the motoneurones (ventral root) showed negligible response to catecholaminesl the sites of action of catecholamines may be on the cell body or dendrites of the motoneurone. Previous histological studies showed that fluorescent varicosities of NA surrounded the perikaryon and dendrites of the a-motoneurones 5A9,34. Radioligand binding studies demonstrated that there were many a-adrenoceptors and few fl-adrenoceptors in the cat 7 and rat TMspinal cord. The present results indicated that NA, Adr and even Iso exerted their effects through a-adrenoceptor activation. Therefore, a-adrenoceptors may be dominant in the rat spinal cord. In the cat ~5 and rat 2 spinal cord, responses induced by D A were reported to be mediated by a D A receptor which was separate from a- or fl-adrenoceptors. In radioligand binding studies, however, the presence 10 and absence 7 of D A receptors have been reported in the spinal cord of rat and cat, respectively. The present experiments showed that the effects of DA were decreased by phentolamine but not by haloperidol, suggesting that the functional D A receptor was not involved in the effects of D A in the spinal cord of the newborn rat.
Effects on segmental reflex discharges It has been reported that NA potentiates11, 21 or depresses 22 the spinal reflexes. NA and Adr not only inhibited but also potentiated the monosynaptic reflex discharge in the present work. Because of this potentiating effect, NA and Adr may not depress the monosynaptic reflex discharge completely. Although the potentiation induced by NA and Adr, as well as the depression, was shown to result from a-adrenoceptor activation, we could not obtain any further information accounting for the mechanism of the potentiating effects.
At some synapses in the peripheral and the central nervous system, a-adrenoceptors on the presynaptic nerve terminals (a2-adrenoceptors) decrease the release of the neurotransmitter and depress the synaptic transmission 20. Clonidine, a potent a2-adrenoceptor agonist20, 27, only slightly depressed the mono-and polysynaptic reflex discharges even at fairly high concentrations (4% and 35% inhibition at 10-4 and 10 -3 M, respectively), suggesting that NA, Adr and D A did not act through such a presynaptic a2-adrenoceptor. Depression of the reflex discharges may not result from depolarization of the motoneurone, since D A still evoked depression of the reflex discharges without further potential changes of the motoneurone when the motoneurone was continuously depolarized to some extent by such as glutamate. As the excitability of the spinal interneurones was decreased by iontophoretically applied NA 13,19, inhibition of the polysynaptic reflex discharge in the present experiment may have resulted from the depression of these interneurones.
Effects of catecholamine releasers In the present experiments, adamantanamine-induced depolarization of the motoneurones disappeared in the absence of Ca 2÷, indicating the indirect action on the motoneurone. Adamantanamine had effects similar to those of DA, that is, D A and adamantanamine depressed the segmental reflex discharges almost completely through a-adrenoceptors and did not potentiate the discharges as NA or Adr did. Therefore, it is possible that adamantanamine releases the endogenous D A in the spinal cord of the newborn rat. The depolarizing effect of tyramine on the motoneurones remained in the Ca2+-deficient solution. As tyramine was reported to release the endogenous NA without requiring the extracellular Ca 2÷ ions 30, it may be that tyramine acted indirectly on the motoneurone. The effect of tyramine on the monosynaptic reflex discharge was similar to the effect of D A rather than NA or Adr in that tyramine only depressed the mono- and polysynaptic reflex discharges almost completely. This observation is consistent with the report that tyramine releases not only NA but also DA32.
36 REFERENCES 1 Ault, B. and Evans, R. H., The action of catecholamines on the isolated hemisected spinal cord of the immature rat, J. Physiol. (Lond.), 278 (1978) 41p-42p. 2 Barasi, S. and Roberts, M. H. T., Responses of motoneurones to electrophoretically applied dopamine, Brit. J. Pharmacol., 60 (1977) 29-34. 3 Carlsson, A., Falck, B., Fuxe, K. and Hillarp, N., Cellular localization of monoamines in the spinal cord, Acta physiol. scand., 60 (1964) 112-119. 4 Commissiong, J, W., Galli, C. L. and Neff, N. H., Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord, J. Neurochem., 30 (1978) 1095-1099. 5 Commissiong, J. W., Hellstr6m, S. O. and Neff, N. H., A new projection from locus coeruleus to the spinal ventral columns: histochemical and biochemical evidence, Brain Res., 148 (1978) 207-213. 6 Commissiong, J. W. and Neff, N. H., Current status of dopamine in the mammalian spinal cord, Biochem. Pharmacol., 28 (1979) 1569-1573. 7 Coote, J. H., Fleetwood-Walker, S. M. and Mitchell, P. R., Catecholamine receptors in thoracic spinal cord, Brit. J. Pharmacol., 67 (1979) 136p-137p. 8 Curtis, D. R., Eccles, J. C. and Eccles, R. M., Pharmacological studies on spinal reflexes, J. Physiol. (Lond.), 136 (1957) 420-434. 9 Dahlstr6m, A. and Fuxe, K., Evidence for the existence of monoamine containing neurones in the central nervous system. II. Experimentally induced changes in the intraneurohal amine levels of bulbospinal neurone system, Aeta physiol. scand., 64 Suppl., 247 (1965) 1-36. 10 Demenge, P., Feuerstein, C., Mouchet, P. and Guerin, B., Stereospecific binding of 3H-haloperidol in rat dorsal spinal cord, Europ. J. Pharmacol., 66 (1980) 117-120. 11 Dhawan, B. N. and Sharma, J. N., Facilitation of the flexor reflex in the cat by intrathecal injection of catecholamines, Brit. J. Pharmacol. Chemother., 40 (1970) 237-248. 12 Engberg, I. and Marshall, K. C., Mechanism of noradrenaline hyperpolarization in spinal cord motoneurones of the cat, Actaphysiol. scand., 83 (1971) 142-144. 13 Engberg, I. and Ryall, R. W., The inhibitory action of noradrenaline and other monoamines on spinal neurones, J. Physiol. (Lond.), 185 (1966) 298-322. 14 Evans, R. H., Evidence supporting the indirect depolarization of primary afferent terminals in the frog by excitatory amino acids, J. Physiol. (Lond.), 298 (1980) 25-35. 15 Gallagher, J. P., lnokuchi, H. and Gallagher, P. S., Dopamine depolarisation of mammalian primary afferent neutones, Nature (Lond.), 283 (1980) 770-772. t6 Grillner, S., Locomotion in vertebrates: central mechanisms and reflex interaction, Physiol. Rev., 55 (1975) 247-304. 17 H6kfelt, T., Phillipson, O. and Goldstein, M., Evidence for a dopaminergic pathway in the rat descending from the A l l cell group to the spinal cord, Acta physiol, scand., 107 (1979) 393-395. 18 Jones, D. J., Kendall, D. E. and Enna, S. J., Adrenergic re-
ceptors in rat spinal cord, Neuropharmacology, 21 (1982) 191-195. 19 Jordan, L. M., McCrea, D. A., Steeves, J. D. and Menzies, J. E., Noradrenergic synapses and effects of noradrenaline on interneurons in the ventral horn of the cat spinal cord, Canad. J. Physiol. Pharmacol., 55 (1977) 399-412. 20 Langer, S. Z., Presynaptic receptors and their role in the regulation of transmitter release, Brit. J. Pharmacol., 60 (1977) 481-497. 21 Matsumiya, T., Berry, J. N. and Bell, J. A., The effect of intraspinal microinjection of dopamine on the C-fiber reflex in the acute decerebrate spinal cat, Life Sci., 24 (1979) 1153-1158. 22 McLennan, H., The effect of some catecholamines upon a monosynaptic reflex pathway in the spinal cord, J. Physiol. (Lond.), 158 (1961) 411-425. 23 Nyrgen, L. and Olson, L., 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 (1977) 85-93. 24 Otsuka, M. and Konishi, S., Electrophysiology of mammalian spinal cord in vitro, Nature (Lond.), 252 (1974) 733-734. 25 Phillis, J. W., Teb~cis, A. K. and York, D. H., Depression of spinal motoneurones by noradrenaline, 5-bydroxytryptamine and histamine, Europ. J. Pharmaeol., 4 (1968) 471-475. 26 Saito, K., Development of spinal reflexes in the rat fetus studied in vitro, J. Physiol. (Lond.), 294 (1979) 581-594. 27 Starke, K. and Altmann, K. P., Inhibition of adrenergic neurotransmission by clonidine: an action of prejunctional a-receptors, Neuropharmaeology, 12 (1973) 339-347. 28 Strablendorf, J. C., Strahlendorf, H. K., Kingsley, R. E., Gintautas, J. and Barnes, C. D., Facilitation of the lumbar monosynaptic reflexes by locus coeruleus stimulation, Neuropharmacology, 19 (1980) 225-230. 29 Thoa, N. B., Wooten, G. F., Axelrod, J. and Kopin, I. J., On the mechanism of release of norepinephrine from sympathetic nerves induced by depolarizing agents and sympathomimetic drugs, Molec. Pharmacol., 11 (1975) 10-18. 30 Thoenen, H., Huerlimann, A. and Haefely, W., Cation dependence of the noradrenaline-releasing action of tyramine, Europ. J. Pharmacol., 6 (1969) 29-37. 31 Von Voigtlander, P. F. and Moore, K. E., Dopamine: release from the brain in vivo by amantadine, Science, 174 (1971) 408-410. 32 Von Voigtlander, P. F. and Moore, K. E., Involvement of nigro-striatal neurons in the vivo release of dopamine by amphetamine, amantadine and tyramine, J. Pharmacol. exp. Ther., 184 (1973) 542-552. 33 Weight, F. F. and Salmoiraghi, G. C., Motoneurone depression by norepinepbrine, Nature (Lond.), 213 (1967) 1229-1230. 34 Westlund, K. N., Bowker, R. M., Ziegler, M. G. and Coulter, J. D., Noradrenergic projections to the spinal cord of the rat, Brain Res., 263 (1983) 15-31. 35 White, S. R. and Neuman, R. S., Facilitation of spinal motoneurone excitability by 5-hydroxytryptarnine and noradrenaline, Brain Res., 188 (1980) 119-127.
31
Elsevier BRD 50173
Effects of Catecholamines on Spinal Motoneurones and Spinal Reflex Discharges in the Isolated Spinal Cord of the Newborn Rat TAKIO KITAZAWA, KOJI SAITO* and AKIRA OHGA
Department of Pharmacology, Facultyof Veterinary Medicine, Hokkaido University, Sapporo 060 (Japan) (Accepted September 17th, 1984)
Key words: isolated spinal cord - - spinal motoneurons - - spinal reflex discharges - - catecholamines - - tyramine - - adamantanamine
The effects of catecholamines on spinal motoneurones and spinal reflex discharges were investigated in the isolated spinal cord of newborn rat. Noradrenaline (NA), adrenaline (Adr), dopamine (DA) and isoproterenol (Iso) caused depolarization of the motoneurones in a dose-dependent manner. The depolarizing action persisted in Ca2+-deficient Krebs solution. The order of potency was Adr >NA>DA~,Iso. The effects of NA and Adr on the monosynaptic reflex discharge varied; depression, potentiation or depression followed by potentiation. The polysynaptic reflex discharge was consistently depressed. DA depressed both the mono- and polysynaptic reflex discharges in all the preparations. Tyramine and adamantanamine induced a response similar to that to DA rather than to NA. Depolarization of the motoneurones and the effects on the spinal reflex discharges induced by all the catecholamines were decreased by phentolamine or phenoxybenzamine but not by propranolol or haloperidol. It is suggested that the endogenous catecholamines, mainly DA, depolarize the motoneurones and depress the mono- and polysynaptic reflex discharges through an a-adrenoceptor in the spinal cord of the newborn rat. INTRODUCTION
tic reflex, which was blocked by phenoxybenzamine2S.
The presence of n o r a d r e n e r g i c fibers has been d e m o n s t r a t e d in the spinal cord of rat 3-5,9,23,34, mouse 3,9 and cat 9,19 by using fluorescent histochemical and immunocytochemical techniques. These nerve fibres originate mainly from the locus coeruleUS4,5,23,34, subcoeruleus 34 and medial and lateral para-
There have been inconsistent results concerning the effecl;s of noradrenaline ( N A ) on the spinal motoneurone. N A applied iontophoretically caused hyperpolarization12. 25 and depression of the excitability of the m o t o n e u r o n e in the cat in situ 25,33, while N A a d d e d to a perfusion m e d i u m depolarized the m o t o n e u r o n e in the isolated spinal cord of the rat 1 and iontophoretically applied N A enhanced the excitability of the m o t o n e u r o n e of the rat in situ2, 35. The effects on the spinal reflexes of exogenous N A were also varied; depression of the k n e e - j e r k reflex22; potentiation of the flexor 11 and C-fibre reflexes 21, or no effects on the monosynaptic reflex discharge 8. Thus, the physiological roles of N A in the spinal cord are not clear at present. Recently, the presence of other descending fibres containing d o p a m i n e ( D A ) have been demon-
brachial nuclei in the pons 34. T h e y terminate near a - m o t o n e u r o n e s , interneurones in the ventral horn and interneurones in the superficial layer of the dorsal horn5,19,23,34. Close contact between n o r a d r e n e r gic fibers and a-motoneuronesSA9, 34 suggests functional synapses between them. F r o m these findings, it was p r o p o s e d that descending n o r a d r e n e r g i c fibers from the higher central nervous system play a role in the regulation of the spinal locomotion circuitryl6. In fact, electrical stimulation of the locus coeruleus in the cat caused facilitation of the l u m b a r monosynap-
* Present address: Division of Cell Biology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo 187, Japan. Correspondence: T. Kitazawa. Present address: Department of Veterinary Pharmacology, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori 034, Japan. 0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
32
a
strated 4,6A7 but there are few reports concerning the effects of DA on the motoneurone and spinal reflex activity. Much of the knowledge about the effects of catecholamines in the spinal cord was obtained for the cat in situ where catecholamines were applied iontophoretically or intravenously. In the present experiments, the effects of catecholamines and endogenous catecholamine releasers on the spinal motoneurone and the segmental reflex discharges were investigated in the isolated spinal cord of the newborn rat. In this preparation, drugs can be applied at known concentrations for a required period and the ionic environment of the neurone can be modified easily24.
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MATERIALS AND METHODS
Both sexes of newborn Wistar rats (0-5-days-old) were used. The preparation was made by methods similar to those described previously24. 26. The spinal cord was isolated from a newborn rat anesthetized with ether, and then hemisected sagittally. The isolated preparation was placed in a chamber (0.3 ml) which was perfused with a nutrient medium at 3 ml/min. Krebs solution of the following composition (mM) was used as the nutrient medium: NaC1, 124; KCI, 5; MgSO 4, 1.3; CaC12, 2.4; KH2PO 4, 1.24; NaHCO3, 26 and glucose, 10. The solution was kept at 30 + 1 °C and bubbled with a 95% 0 2 and 5% CO 2 gas mixture. A Ca2+-deficient solution was prepared by increasing MgSO4 to 3.5 mM and decreasing CaC12 to 0.1 mM. Drugs were dissolved in the Krebs solution and applied by means of perfusion. The potential changes generated in the spinal motoneurones were recorded extracellularly from the L3 ventral root with a suction electrode. To evoke the segmental reflex discharges, the corresponding dorsal root was stimulated electrically (400/~s duration and supramaximai intensity).
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2 rain Fig. 1. Effects of catecholamines on the motoneurones. The traces show potential changes in the motoneurones recorded from the L3 ventral root: A, segmental reflex discharge; B, depolarization induced by noradrenaline (5×10 -6 M); C, by adrenaline (5×10 -6 M); D, by dopamine (10 -4 M). Responses in the normal Krebs solution (Ca 2+, 2.4 mM; Mg 2÷, 1.3 mM) are shown in a and in the Ca>-deficient solution (Ca 2÷, 0.1 mM; Mg2+, 3.5 mM) in b. Drugs were added to the bath during the periods indicated by the horizontal bars (30 s).
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RESULTS
Effects of catecholamines on the spinal motoneurones Application of NA (10-7-5 × 10-5 M), adrenaline (Adr, 5×10-8-5x10 -5 M) and DA (10-6-2x 10-4 M) to the perfusing solution for 30 s caused depolarization of the motoneurones (Fig. 1Ba-Da). Many small spike discharges appeared near and at
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Fig. 2. Depolarization of the motoneurones induced by catecholamines. Symbols show responses induced by noradrenaline (O), adrenaline (©), dopamine ([3), isoproterenol (A), tyramine (11) and adamantanamine (&) in the normal Krebs solution (mean + S.E., n = 5-7). The amplitude of the depolarization (ordinate) is expressed as a percentage of that induced by 130 mM K ÷.
33 the peak of the depolarization when the response was large. These depolarizations were reproducible on 10 min interval application. The relationships between the amplitude of depolarization and the concentration of the drugs are shown in Fig. 2. Each catecholamine caused depolarization in a concentration dependent manner to induce the maximum response. The EDs0 values were 3x 10-6 M for NA, 10 -6 M for Adr and 5 x 10-5 M for DA. Isoproterenol (Iso) caused only slight depolarization. The order of potency for depolarization of the motoneurones was A d r > N A > D A - > I s o . The time courses of depolarization induced by the catecholamines were compared by employing concentrations at which about the same amplitude of depolarization occurred with each amine. The times (s) between the half maximum response were 55.7 + 1.7 (mean + S.E., n = 7) for NA (5×10 -6 M), 83.0 ___4.4 (n = 5) for Adr (10 -6 M) and 97.2 + 4.8 (n = 5) for D A (5 x 10-5 M). In the Ca2+-deficient solution (Ca 2+, 0.1 mM; Mg 2÷, 3.5 mM), segmental reflex discharges recorded from the L3 ventral root were almost completely abolished (Fig. l a b ) . In this condition, however, the amplitude of the depolarization of the motoneurones induced by NA, Adr and D A was nearly completely unaffected (Fig. 1Bb-Db). It was reported that ~-aminobutyric acid (GABA) acted directly on the isolated dorsal root of the frog and generated potential changes TM. This possibility was examined in the ventral root with catecholamines. The depolarization induced by the addition of 5 mM K ÷ was observed in the ordinary way, and then the ventral root was cut off close to the spinal cord. After this, the K+-induced depolarization in the cut off root remained at more than 70% of the control level. On the other hand, an appreciable potential change was not induced by NA, Adr or DA.
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Fig. 3. Effects of noradrenaline and dopamine on the monosynaptic reflex discharges (L3). Noradrenaline (5x10-6M) caused depression (A), depression followed by potentiation (B) and potentiation (C) of the monosynaptic reflex discharges ((3). The polysynaptic reflex discharges (0) were depressed (A-C) in all the preparations. Dopamine (5 x 10-5 M) also depressed monosynaptic reflex discharges (D,©). This effect was antagonized by phentolamine (2.7x 10-5 M) (D,II). Noradrenaline and dopamine were added to the bath during the periods indicated by the horizontal bars (3 min).
preparation. Polysynaptic reflex discharges were decreased in all the preparations examined (Fig. 3A-C). D A (10-6-5×10 -4 M) depressed both mono- and polysynaptic reflex discharges to almost the same extent and potentiation was never seen (Fig. 3D). Iso slightly depressed the mono- and polysynaptic reflex discharges at high concentrations (10-4-10 -3 M). Fig. 4 is a summary of the depressant effects of the catecholamines on the monosynaptic ioo
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Effects of catecholamines on the segmental reflex discharges NA (5×10-7-10 -4 M) and Adr (10-7-10 -4 M) had various effects on the amplitude of the monosynaptic reflex discharge; depression (NA, 18/27 preparations; Adr, 20/28), potentiation (NA, 2/27; Adr, 2/28) or depression followed by potentiation (NA, 7/27; Adr, 6/28) (Fig. 3 A - C ) . The response was not altered to one of the other types with time in the same
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Fig. 4. Depression of the monosynaptic reflex discharges by catecholamines. The symbolsshow the effects of noradrenaline (O), adrenaline (C)), dopamine (D), isoproterenol (A), tyramine (11) and adamantanamine (A) in the normal Krebs solution (mean _+S.E., n = 3-5). Only the inhibitory response concerning adrenaline and noradrenaline was summarized (see the text).
34 reflex discharge. The effects of NA and Adr were maximal at 10-5-10 -4 M, at which the discharge was reduced to about half of the control level. D A inhibited the discharge more extensively than Adr or NA and depressed it almost completely at 5 x 10-a M. Strychnine (10-6g/ml) and picrotoxin (5×10 -6 g/ml) did not reduce the depressant effect of the catecholamines.
Effects of receptor blocking agents Phentolamine (2.7x10-6-2.7x10-SM) or phenoxybenzamine (5 x 10-6 M), which did not produce any appreciable potential changes in the motoneurones, decreased the depolarization induced by NA (5x10 -6 M), Adr (5x10 -6 M), D A (5x10-5 M) and Iso (10 .3 M). Depolarization induced by substance P (2x10 -7 M) or serotonin (5x10 -6 M) was not affected by phentolamine (2.7x10-6 M) (Fig. 5). Propranolol (5 x 10-6 M) did not affect all the depolarizations. The inhibitory effects of catecholamines on the monosynaptic reflex discharges were also decreased by phentolamine (2.7x 10-5 M) but not by propranolol (5 x 10-6 M). Fig. 3D shows the effect of phentolamine on the depressant effect of DA. Both receptor blocking agents did not influence the inhibition induced by serotonin (5x10 -6 M) or the reflex discharge itself at the concentrations employed. The potentiation of the monosynaptic reflex discharges induced by NA and Adr in some preparations was also blocked by phentolamine.
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Fig. 5. Effects of phentolamine (2.7x 10-6 M) on the depolarization of the motoneurones induced by catecholamines. The traces show the depolarization of the motoneurones recorded from the L3 ventral root induced by A, noradrenaline (5X10 -6 M); B, adrenaline (5x10 -6 M); C, dopamine (5 x 10-5 M); D, serotonin (5 x 10-6 M). Depolarizations in the normal Krebs solution are shown in a and in the Krebs solution containing phentotamine (2.7x10-6 M) in b. Drugs were added to the bath during the periods indicated by the horizontal bars (30 s).
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Fig. 6. Effects of tyramine and adamantanamine on the spinal motoneurones. The traces show potential changes in the motoneurones recorded from the L3 ventral root; A, depolarization induced by tyramine (5x10 -5 M) and B, by adamantanamine (5×10-3 M). Responses in the normal Krebs solution arc shown in a, in the Ca2+-deficient Krebs solution (Ca2", 0.1 mM; Mg2÷, 3.5 mM) in b, and in the Krebs solution containing phentolamine (2.7x 10-6 M) in c. The effect of adamantanamine in the Ca2+-deficientsolution was restored in the normal solution before adding phentolamine (not shown). Tyramine and adamantanamine were added to the bath during the periods indicated by the horizontal bars (30 s).
Haloperidol (10-7-10 -5 M) did not decrease the depolarization of the motoneurones or the inhibition of the reflex discharges evoked by DA. At higher concentrations than 10-5 M, haloperidol itself depressed the reflex discharges.
Effects of endogenous catecholamine releasers Tyramine, a releaser of endogenous NA 29,3°, depolarized the motoneurones in a concentration dependent manner (5×10-6-5×10 -4 M) (Fig. 2, II). Adamantanamine (2x10-4-10 -2 M), an endogenous D A releaser 31,32, also depolarized the motoneurones (Fig. 2, A). Fig. 6 shows the effects of tyramine (5x10-5 M) and adamantanamine (5x10 -3 M). In the Ca2+-deficient solution, tyramine-induced depolarization was scarcely affected but depolarization induced by adamantanamine was abolished almost completely, suggesting that adamantanamine depolarized the motoneurones trans-synapticalty. The effect of adamantanamine or tyramine was decreased by phentolamine (2.7x 10-6 M). Tyramine (2x 10-6-5x 10-4 M) and adamantanamine (2x10-4-10 -2 M) decreased the amplitude of the mono- and polysynaptic reflex discharges. The inhibition of the monosynaptic reflex discharge was almost complete (Fig. 4). The depressant effects of tyramine and adamantanamine were antagonized by phentolamine (2.7x 10-5 M) but not by propranolol (5 x 10-6 M) or haloperidol (10 -s M). Potentiation of
35 the monosynaptic reflex discharge was observed with neither of the drugs as in the case of NA or Adr. These effects of tyramine and adamantanamine on the monosynaptic reflex were thus similar to those of DA. DISCUSSION
Effects of catecholamines on motoneurones The present experiments showed that catecholamines directly depolarized the spinal motoneurones. As the axons of the motoneurones (ventral root) showed negligible response to catecholaminesl the sites of action of catecholamines may be on the cell body or dendrites of the motoneurone. Previous histological studies showed that fluorescent varicosities of NA surrounded the perikaryon and dendrites of the a-motoneurones 5A9,34. Radioligand binding studies demonstrated that there were many a-adrenoceptors and few fl-adrenoceptors in the cat 7 and rat TMspinal cord. The present results indicated that NA, Adr and even Iso exerted their effects through a-adrenoceptor activation. Therefore, a-adrenoceptors may be dominant in the rat spinal cord. In the cat ~5 and rat 2 spinal cord, responses induced by D A were reported to be mediated by a D A receptor which was separate from a- or fl-adrenoceptors. In radioligand binding studies, however, the presence 10 and absence 7 of D A receptors have been reported in the spinal cord of rat and cat, respectively. The present experiments showed that the effects of DA were decreased by phentolamine but not by haloperidol, suggesting that the functional D A receptor was not involved in the effects of D A in the spinal cord of the newborn rat.
Effects on segmental reflex discharges It has been reported that NA potentiates11, 21 or depresses 22 the spinal reflexes. NA and Adr not only inhibited but also potentiated the monosynaptic reflex discharge in the present work. Because of this potentiating effect, NA and Adr may not depress the monosynaptic reflex discharge completely. Although the potentiation induced by NA and Adr, as well as the depression, was shown to result from a-adrenoceptor activation, we could not obtain any further information accounting for the mechanism of the potentiating effects.
At some synapses in the peripheral and the central nervous system, a-adrenoceptors on the presynaptic nerve terminals (a2-adrenoceptors) decrease the release of the neurotransmitter and depress the synaptic transmission 20. Clonidine, a potent a2-adrenoceptor agonist20, 27, only slightly depressed the mono-and polysynaptic reflex discharges even at fairly high concentrations (4% and 35% inhibition at 10-4 and 10 -3 M, respectively), suggesting that NA, Adr and D A did not act through such a presynaptic a2-adrenoceptor. Depression of the reflex discharges may not result from depolarization of the motoneurone, since D A still evoked depression of the reflex discharges without further potential changes of the motoneurone when the motoneurone was continuously depolarized to some extent by such as glutamate. As the excitability of the spinal interneurones was decreased by iontophoretically applied NA 13,19, inhibition of the polysynaptic reflex discharge in the present experiment may have resulted from the depression of these interneurones.
Effects of catecholamine releasers In the present experiments, adamantanamine-induced depolarization of the motoneurones disappeared in the absence of Ca 2÷, indicating the indirect action on the motoneurone. Adamantanamine had effects similar to those of DA, that is, D A and adamantanamine depressed the segmental reflex discharges almost completely through a-adrenoceptors and did not potentiate the discharges as NA or Adr did. Therefore, it is possible that adamantanamine releases the endogenous D A in the spinal cord of the newborn rat. The depolarizing effect of tyramine on the motoneurones remained in the Ca2+-deficient solution. As tyramine was reported to release the endogenous NA without requiring the extracellular Ca 2÷ ions 30, it may be that tyramine acted indirectly on the motoneurone. The effect of tyramine on the monosynaptic reflex discharge was similar to the effect of D A rather than NA or Adr in that tyramine only depressed the mono- and polysynaptic reflex discharges almost completely. This observation is consistent with the report that tyramine releases not only NA but also DA32.
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