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Enhancement of recurrent inhibition of the spinal monosynaptic reflex by preceding stimulation of the medullary raphe´in rats
Brain Research, 417 (1987) 403-407 Elsevier
403
BRE 22425
Enhancement of recurrent inhibition of the spinal monosynaptic reflex by preceding stimulation of the medullary raphe in rats Tsugio Kaneko, Hideki Ono and Hideomi Fukuda Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The Universityof Tokyo, Tokyo (Japan) (Accepted 5 May 1987)
Key words: Recurrent inhibition; Monosynaptic reflex; Raph6 nucleus; Medulla oblongata; Descending control; Lysergic acid diethylamide (LSD)
Recurrent inhibition of the spinal monosynaptic reflex (MSR) elicited by conditioning stimulation of the ventral root in anesthetized rats was weaker than both the recurrent inhibition of the disynaptic reflex and the inhibition of the MSR elicited by conditioning stimulation of the adjacent dorsal root. Among these 3 inhibitions, the recurrent inhibition of the MSR was enhanced to a markedly greater extent by a preceding stimulation of the medullary raph6 nucleus than were the other inhibitions. The magnitude of the enhancement of the recurrent inhibition of MSR also was much greater when the medullary stimulation was delivered 20 ms prior to the ventral root activation, as compared with a 30-ms interval. Recurrent inhibition of the MSR was enhanced by intravenous iniection of lysergic acid diethylamide (LSD); however, the enhanced effect on recurrent inhibition elicited by stimulation of the raph6 nucleus was not attenuated by the drug. These results suggest that there is a non-serotonergic, descending pathway which is capable of modulating motor output solely by means of recurrent inhibition of the MSR. Electrical stimulation of several regions of the brain, including the cerebral cortex, thalamus, reticular formation 14, capsula interna 13, red nucleus 9 and cerebellum 7 reportedly alter the firing of Renshaw cells, such observations suggesting that these supraspinal structures could modulate the output of the motoneuronal system by means of controlling recurrent inhibition n. On the other hand, descending monoaminergic 2° and especially serotonergic 19 systems have been suggested to inhibit tonically recurrent inhibition of monosynaptic reflexes. Although changes in motor output accompanying changes in Renshaw cell activity have been demonstrated recently 6'18, the functional roles of descending pathways regulating recurrent inhibition in the spinal cord are still obscure. We have been studying the descending pathways which regulate synaptic events in the rat spinal cord 1°,a2 and have reported that stimulation of the medullary raph6 nucleus affects several spinal activities via distinct descending pathways 12. In the ex-
periments reported here, we have examined a possible involvement of recurrent inhibition in changes of spinal activities elicited by stimulation of the raph6 nucleus. Male Wistar rats weighing between 280 and 320 g were anesthetized intraperitoneally with a mixture of urethane (1 g/kg) and a-chloralose (25 mg/kg). Laminectomies were performed in the lumbosacral region. Ventral and dorsal roots below L 4 were cut bilaterally and dorsal and ventral roots of the segments of L4 and L 5 were isolated. A skin pouch was formed at the site of the dissection and the exposed tissues were covered with liquid paraffin kept at 37 + 0.5 °C. Rectal temperature was maintained at 37 + 0.5 °C using a heating pad and the rats were ventilated artificially. The dorsal and ventral roots of segment L 5 were placed on bipolar silver wire electrodes for stimulation (test stimulation) and recording, respectively (stimulation frequency 0.2 Hz, duration 0.05 ms, supramaximal). The ventral root reflex (VRR) potential was amplified with an A C amplifier and the
Correspondence: T. Kaneko. Present address: Department of Medical Physiology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, N.W., Calgary, Alta., Canada T2N 4N1. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
404 response displayed on an oscilloscope. Eight consecutive VRRs were averaged, the resulting analog output of which was recorded. Another pair of silver wire electrodes placed proximally to the recording electrodes was used to deliver a conditioning stimulus (a 0.1-ms single pulse at 0.5 V) to ventral root L 5. Inhibition of the V R R elicited by conditioning stimulation of ventral root L 5 was reduced by an intravenous injection of mecamylamine hydrochloride (3 mg/kg; Merck); accordingly, this process was considered to be a recurrent inhibition which was mediated via motor axon collaterals 5'21. In two experiments, dorsal root L 4 was placed on a pair of silver wire electrodes and was stimulated with a single pulse of 0.5 ms duration at ! V, prior to test stimulation of dorsal root L 5. A concentric bipolar electrode was placed stereotaxically into the medullary raph~ nucleus at P 2.4, L 0.0 and D 1.5 below the interaural line, according to the atlas of Pellegrino et a1.17. This location corresponds with the nucleus raph6 magnus, as presented in the atlas of Paxinos and Watson 16. The electrode was made of stainless-steel tubing having an o.d. of 0.5 mm and was insulated with cashew. The inner wire projected 0.4 mm beyond the termination point of the outer (Unique Medical). The stimuli to the raph6 nucleus consisted of 4 trains of 0.1-ms pulses, 200 Hz at 0.1 mA. After each experiment, the whole brain was isolated and the tissue was fixed in 10% formalin for at least 24 h. The stimulating site was verified macroscopically on each longitudinal section at L 0.0. When the electrode proved to be located at a point outside the raph6 nucleus, results obtained from that preparation were excluded from the analysis. Mecamylamine hydrochloride and lysergic acid diethylamide (LSD) tartrate (Sandoz) were dissolved in physiological saline (0.9% NaC1) and injected through an intravenous (i.v.) cannula inserted into the femoral vein. In order to compare the effects of conditioning stimulation of the ventral root L 5 or the dorsal root L 4 with or without the stimulation of the raph6 nucleus, four kinds of VRR recording were obtained (Fig. 1A); the V R R which was conditioned with segmental stimulation at various times prior to the test stimulation was normalized with respect to that induced by the test stimulation alone. The V R R which was conditioned with segmental stimulation following stimulation of the raph6 nucleus by 20 or 30 ms, was nor-
malized with respect to that induced by test stimulation being preceded by activation of raph6 nucleus at a corresponding temporal interval. Differences between these two normalized values were calculated for their statistical significance according to the Student's two-tailed, paired t-test. Mono- and disynaptic reflex (MSR and DSR, respectively) amplitudes were depressed to a different extent as a result of recurrent inhibition (Fig. 1). The peak suppression ratio of the MSR was 18.7 + 2.2% (mean + S.E.M., n = 4), which was significantly smaller than that exhibited by the DSR, which was suppressed by 54.1 + 6.3% (n = 4) (P < 0.01). The inhibition of the MSR elicited by conditioning stimulation of dorsal root L 4 comprised as much as a 50% suppression (Fig. 2). Recurrent inhibition of the MSR was enhanced by a preceding stimulation of the raph6 nucleus relatively much more than was recurrent inhibition of the DSR (Fig. 1). This enhancement was much greater when the activation of the raph6 nucleus preceded the conditioning stimulation of the ventral root by 20 ms (Fig. 1Ba), than if the interval was allowed to increase to 30 ms (Fig. 1Bb). As described previously 12, stimulation of the raph6 nucleus enhanced the MSR and suppressed the DSR. However, as seen in Fig. 1A, recurrent inhibition of the MSR was greatly enhanced even when the preceding stimulation of the raph6 nucleus showed almost no effect on the MSR. On the other hand, the inhibition elicited by a conditioning stimulation of dorsal r o o t L 4 was not affected by a preceding stimulation of the raph6 nucleus (Fig. 2). Intravenous injection of LSD tartrate 50/~g/kg, as described previously 12, depressed the MSR (not shown). The recurrent inhibition of the MSR was enhanced by this dose of LSD (Fig. 3). The facilitative effect of a preceding stimulation of the raph6 nucleus on the recurrent inhibition was, however, not attenuated by this treatment (Fig. 3). The recurrent inhibition of the DSR and the effect of the preceding stimulation on the recurrent inhibition were unaffected by this dose of LSD. Recurrent inhibition of the MSR was weaker than either that of the DSR or inhibition of the MSR elicited by conditioning of the adjacent dorsal root. While the mechanisms whereby inhibitions of the DSR are modulated remain unknown, there are sug-
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Fig. l. Effects of prior stimulation of the medullary raph6 nucleus (Raph6) on recurrent inhibition elicited by a conditioning stimulation of the ventral root L 5 (L5-VR). A: a typical set of recordings, in which the L5-VR was conditioned (+) 20 ms prior to test stimulation, and Raph6 was stimulated (+) a further 20 ms prior to the conditioning of the L5-VR. Each trace represents the average of 8 consecutive responses. Bars = 2 ms and 0.2 mV. B and C: the inhibitory curves for the MSR and the DSR, respectively, in which normalized amplitudes were plotted against various conditioning-test intervals with (closed) or without (open) preceding stimulation of Raph6, 20 ms (a) or 30 ms (b) prior to conditioning. Each point represents the mean of 4 experiments, with S.E.M. indicated. Where S.E.M. bars are not shown, they lie within the dimensions of the symbols. *P < 0.05, **P < 0.01.
gestions that descending inhibitory influences exist, which are active tonically on recurrent inhibition of the MSR 6'19'2°. The recurrent inhibition of the MSR observed in this experiment was not so great as that which has been reported in decerebrated cat preparations 3,4,6. It has been reported that there are species differences in the subtypes of receptors for ace-
tylcholine on Renshaw cells s,15, so one might expect that there could be some difference in the nervous lOO 0~0~0
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Fig. 2. A n inhibitory curve for the MSR in a case of conditioning of the dorsal root L 4 with (closed) or without (open) preceding stimulation of the raph6 nucleus, 20 ms prior to conditioning. n = 2.
Fig. 3. Effects of LSD on recurrent inhibition with (closed) or without (open) preceding stimulation of the raph6 nucleus. The ventral root L 5 was conditioned 10 ms prior to test stimulation of the dorsal root L 5 and the raph6 nucleus was stimulated 20 ms prior to conditioning of the ventral root L 5. LSD tartrate (50 ~g/kg) was injected intravenously 1 min prior to time 0. Each point represents the mean of 4 experiments. Statistical procedures were the same as for Fig. 1.
406 system regarding the nature of recurrent inhibition in the spinal cord. However, even in rats, the recurrent inhibition observed in C x transected preparations is greater than that obtained in intact, anesthetized animals (unpublished observation). Accordingly, any differences observed in the magnitudes of these recurrent inhibitions could, in part at least, be ascribed to the difference in the preparation. It was observed in the present experiment that i.v. LSD enhanced recurrent inhibition of the MSR (Fig. 3). Is is possible that this effect could be attributed to the known agonistic action of low doses of LSD at a serotonin autoreceptor, as Aghajanian et al. 1'2 have found that LSD reduces the spontaneous firing of serotonergic neurones of the brainstem. Our observations with systemic administration are therefore quite consistent with the proposal made by Sastry and Sinclair 19, who postulated the existence of a tonically active, inhibitory control of recurrent inhibition, mediated by a descending serotonergic pathway. On the other hand, the stronger segmental inhibition, such as the recurrent inhibition of the DSR or the inhibition of the MSR elicited by conditioning of the adjacent dorsal root, seems to be relatively independent of the descending control. Stimulation of the raph6 nucleus reportedly produces a dual effect on the VRRs, differing in the time course of the action: the points of maximal facilitation of the MSR and of maximal suppression of the DSR are observed within 20 ms and from 30 to 40 ms after the commencement of the medullary stimulation, respectively 12. In the present study, recurrent inhibition of the MSR was enhanced by a preceding stimulation of the raph6 nucleus. This enhancement effect was much greater when the conditioning stimulation of the ventral root followed the raph6 stimulation by an interval of 20 ms rather than of 30 ms (Fig. 1B). Neither the facilitative effects on the MSR 12 nor on the recurrent inhibition (Fig. 3), which were elicited by stimulation of the raph6 nucleus, were attenuated by the injection of LSD. The similarities in time course of these two LSD-insensitive facilitations, as well as the fact of their insensitivity, suggest the employment of a common pathway subserving the two facilitative effects observed in the spinal cord. Enhancement of the recurrent inhibition seen here (Fig. 1) as well as facilitation of the MSR reported earlier 12 both could be accounted for as a re-
sult of an elevation in motoneuronal activities. With respect to the DSR suppression however, it has been shown that whereas the MSR enhancement is LSD-insensitive, the DSR suppression induced by raph6 stimulation is reversed by LSD 12. The present work has emphasized the separation of the mechanisms responsible for modulation of spinal reflexes by making the additional observation, that the facilitation of recurrent inhibition of the MSR is LSD-insensitve (Fig. 3) - - further, that the enhancement of this suppression by recurrent inhibition is proportionately less for the DSR than for the MSR (Fig. 1). Reductions in Renshaw cell firing 7'9A3'14'18 have been demonstrated through stimulation of various regions of the brain. Descending inhibitory influences on recurrent inhibition of the MSR also have been proposed 6'19'2°. These descending influences ultimately increase the output from the motoneurone pool and lead to a relatively more intense muscular contraction n. Yet activation of descending pathways originating from, and perhaps passing through the medullary raph6 nucleus 12, which were demonstrated in this experiment, would enhance only the initial output of the monosynaptic transmission, the subsequent outputs being attenuated strongly because of the resultant enhanced recurrent inhibition; all the polysynaptic outputs however, which are recipient of the same sensory messages, also would be reduced via a distinct serotonergic descending pathway 12. It might then be postulated that these pathways serve as a selector and as an amplifier for a particular form of output, required of the motoneuronal pool. In the present study, we have demonstrated a modification of the processes of recurrent inhibition of the MSR via a descending pathway which was activated by stimulation of the raph6 nucleus, this resulting in an adjustment of spinal activities. The recurrent inhibition of the MSR is, by itself, much weaker than that of the DSR or the inhibition of the MSR elicited by a conditioning stimulus to the adjacent dorsal root, and the descending pathway responsible for the modulation of this pathway is LSD-insensitive in nature. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful
407 to M e r c k ( J a p a n ) for the supply of m e c a m y l a m i n e h y d r o c h l o r i d e a n d to S a n d o z ( J a p a n ) for t h e supply
T.P. Hicks (The U n i v e r s i t y of Calgary) for his critical r e a d i n g of the m a n u s c r i p t .
of lysergic acid d i e t h y l a m i d e tartrate. W e t h a n k D r .
1 Aghajanian, G.K. and Haigler, H.J., Mode of action of LSD on serotonergic neurons. In E. Costa, G.L. Gessa and M. Sandier (Eds.), Serotonin -- New Vistas, Advances in Biochemical Psychopharmacology, Vol. 10, Raven, New York, 1974, pp. 167-177. 2 Aghajanian, G.K., Haigler, H.J. and Bloom, F.E., Lysergic acid diethylamide and serotonin: direct actions on serotonin-containing neurons in rat brain, Life Sci., 11 (1972) 615-622. 3 Brooks, V.B. and Wilson, V.J., Recurrent inhibition in the cat's spinal cord, J. Physiol. (London), 146 (1959) 380-391. 4 Cullheim, S. and Kellerth, J.-O., Two kinds of recurrent inhibition of cat spinal a-motoneurones as differentiated pharmacologically, J. Physiol. (London), 312 (1981) 209-224. 5 Eccles, J.C., Fatt, P. and Koketsu, K., Cholinergic and inhibitory synapses in a pathway from motor-axon collaterals to motoneurones, J. Physiol. (London), 126 (1954) 524-562. 6 Fung, S.J., Pompeiano, O. and Barnes, C.D., Suppression of the recurrent inhibitory pathway in lumbar cord segments during locus coeruleus stimulation in cats, Brain Research, in press. 7 Granit, R., Haase, J. and Rutledge, L.T., Recurrent inhibition in relation to frequency of firing and limitation of discharge rate of extensor motoneurones, J. Physiol. (London), 154 (1960) 308-328. 8 Headley, P.M., Lodge, D. and Biscoe, T.J., Acetylcholine receptors on Renshaw cells of the rat, Eur. J. PharmacoL, 30 (1975) 252-259. 9 Henatsch, H.-D., Meyer-Lohmann, J., Windhorst, U. and Schmidt, J., Differential effects of stimulation of the cat's red nucleus on lumbar alpha motoneurones and their Renshaw cells, Exp. Brain Res., 62 (1986) 161-174. 10 Hino, M., Ono, H. and Fukuda, H., Supraspinal tonic influence on spinal reflexes and involvement in the effect of chlorpromazine, Gen. Pharmacol., 15 (1984) 155-158.
11 Hultborn, H., Lindstr6m, S. and Wigstr6m, H., On the function of recurrent inhibition in the spinal cord, Exp. Brain Res., 37 (1979) 399-403. 12 Kaneko, T., Ono, H. and Fukuda, H., LSD but not methysergide reduces the inhibitory effect of the medullary raph6 stimulation on the spinal reflex in rats, Gen. Pharmacol., 15 (1984) 79-83. 13 Koehler, W., Windhorst, U., Schmidt, J., Meyer-Lobmann, J. and Henatsch, H.-D., Diverging influences on Renshaw cell responses and monosynaptic reflexes from stimulation of capsula interna, Neurosci. Lett., 8 (1978) 35-39. 14 MacLean, J.B. and Leffman, H., Supraspinal control of Renshaw cells, Exp. NeuroL, 18 (1967) 94-104. 15 McLennan, H. and Hicks, T.P., Pharmacological characterization of the excitatory cholinergic receptors of rat central neurones, Neuropharmacology, 17 (1978) 329-334. 16 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. 17 Pellegrino, L.J., Pellegrino, A.S. and Cushman, A.J., A Stereotaxic Atlas of the Rat Brain, 2nd edn., Plenum, New York, 1979. 18 Sakamoto, T., Atsuta, Y. and Mori, S., Long-lasting excitability change of soleus a-motoneuron induced by midpontine stimulation in decerebrate, standing cat, J. Neurophysiol., 55 (1986) 449-468. 19 Sastry, B.S.R. and Sinclair, J.G., Tonic inhibitory influence of a supraspinal monoaminergic system on recurrent inhibition of an extensor monosynaptic reflex, Brain Research, 117 (1976) 69-76. 20 Sinclair, J.G. and Sastry, B.S.R., Supraspinal monoaminergie involvement in the blockade of recurrent inhibition of the monosynaptic reflex, Neuropharmacology, 13 (1974) 741-747. 21 Ueki, S., Koketsu, K. and Domino, E.F., Effects of mecamylamine on the Golgi recurrent collateral - - Renshaw-cell synapse in the spinal cord, Exp. Neurol., 3 (1961) 141-148.
403
BRE 22425
Enhancement of recurrent inhibition of the spinal monosynaptic reflex by preceding stimulation of the medullary raphe in rats Tsugio Kaneko, Hideki Ono and Hideomi Fukuda Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The Universityof Tokyo, Tokyo (Japan) (Accepted 5 May 1987)
Key words: Recurrent inhibition; Monosynaptic reflex; Raph6 nucleus; Medulla oblongata; Descending control; Lysergic acid diethylamide (LSD)
Recurrent inhibition of the spinal monosynaptic reflex (MSR) elicited by conditioning stimulation of the ventral root in anesthetized rats was weaker than both the recurrent inhibition of the disynaptic reflex and the inhibition of the MSR elicited by conditioning stimulation of the adjacent dorsal root. Among these 3 inhibitions, the recurrent inhibition of the MSR was enhanced to a markedly greater extent by a preceding stimulation of the medullary raph6 nucleus than were the other inhibitions. The magnitude of the enhancement of the recurrent inhibition of MSR also was much greater when the medullary stimulation was delivered 20 ms prior to the ventral root activation, as compared with a 30-ms interval. Recurrent inhibition of the MSR was enhanced by intravenous iniection of lysergic acid diethylamide (LSD); however, the enhanced effect on recurrent inhibition elicited by stimulation of the raph6 nucleus was not attenuated by the drug. These results suggest that there is a non-serotonergic, descending pathway which is capable of modulating motor output solely by means of recurrent inhibition of the MSR. Electrical stimulation of several regions of the brain, including the cerebral cortex, thalamus, reticular formation 14, capsula interna 13, red nucleus 9 and cerebellum 7 reportedly alter the firing of Renshaw cells, such observations suggesting that these supraspinal structures could modulate the output of the motoneuronal system by means of controlling recurrent inhibition n. On the other hand, descending monoaminergic 2° and especially serotonergic 19 systems have been suggested to inhibit tonically recurrent inhibition of monosynaptic reflexes. Although changes in motor output accompanying changes in Renshaw cell activity have been demonstrated recently 6'18, the functional roles of descending pathways regulating recurrent inhibition in the spinal cord are still obscure. We have been studying the descending pathways which regulate synaptic events in the rat spinal cord 1°,a2 and have reported that stimulation of the medullary raph6 nucleus affects several spinal activities via distinct descending pathways 12. In the ex-
periments reported here, we have examined a possible involvement of recurrent inhibition in changes of spinal activities elicited by stimulation of the raph6 nucleus. Male Wistar rats weighing between 280 and 320 g were anesthetized intraperitoneally with a mixture of urethane (1 g/kg) and a-chloralose (25 mg/kg). Laminectomies were performed in the lumbosacral region. Ventral and dorsal roots below L 4 were cut bilaterally and dorsal and ventral roots of the segments of L4 and L 5 were isolated. A skin pouch was formed at the site of the dissection and the exposed tissues were covered with liquid paraffin kept at 37 + 0.5 °C. Rectal temperature was maintained at 37 + 0.5 °C using a heating pad and the rats were ventilated artificially. The dorsal and ventral roots of segment L 5 were placed on bipolar silver wire electrodes for stimulation (test stimulation) and recording, respectively (stimulation frequency 0.2 Hz, duration 0.05 ms, supramaximal). The ventral root reflex (VRR) potential was amplified with an A C amplifier and the
Correspondence: T. Kaneko. Present address: Department of Medical Physiology, Faculty of Medicine, The University of Calgary, 3330 Hospital Drive, N.W., Calgary, Alta., Canada T2N 4N1. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
404 response displayed on an oscilloscope. Eight consecutive VRRs were averaged, the resulting analog output of which was recorded. Another pair of silver wire electrodes placed proximally to the recording electrodes was used to deliver a conditioning stimulus (a 0.1-ms single pulse at 0.5 V) to ventral root L 5. Inhibition of the V R R elicited by conditioning stimulation of ventral root L 5 was reduced by an intravenous injection of mecamylamine hydrochloride (3 mg/kg; Merck); accordingly, this process was considered to be a recurrent inhibition which was mediated via motor axon collaterals 5'21. In two experiments, dorsal root L 4 was placed on a pair of silver wire electrodes and was stimulated with a single pulse of 0.5 ms duration at ! V, prior to test stimulation of dorsal root L 5. A concentric bipolar electrode was placed stereotaxically into the medullary raph~ nucleus at P 2.4, L 0.0 and D 1.5 below the interaural line, according to the atlas of Pellegrino et a1.17. This location corresponds with the nucleus raph6 magnus, as presented in the atlas of Paxinos and Watson 16. The electrode was made of stainless-steel tubing having an o.d. of 0.5 mm and was insulated with cashew. The inner wire projected 0.4 mm beyond the termination point of the outer (Unique Medical). The stimuli to the raph6 nucleus consisted of 4 trains of 0.1-ms pulses, 200 Hz at 0.1 mA. After each experiment, the whole brain was isolated and the tissue was fixed in 10% formalin for at least 24 h. The stimulating site was verified macroscopically on each longitudinal section at L 0.0. When the electrode proved to be located at a point outside the raph6 nucleus, results obtained from that preparation were excluded from the analysis. Mecamylamine hydrochloride and lysergic acid diethylamide (LSD) tartrate (Sandoz) were dissolved in physiological saline (0.9% NaC1) and injected through an intravenous (i.v.) cannula inserted into the femoral vein. In order to compare the effects of conditioning stimulation of the ventral root L 5 or the dorsal root L 4 with or without the stimulation of the raph6 nucleus, four kinds of VRR recording were obtained (Fig. 1A); the V R R which was conditioned with segmental stimulation at various times prior to the test stimulation was normalized with respect to that induced by the test stimulation alone. The V R R which was conditioned with segmental stimulation following stimulation of the raph6 nucleus by 20 or 30 ms, was nor-
malized with respect to that induced by test stimulation being preceded by activation of raph6 nucleus at a corresponding temporal interval. Differences between these two normalized values were calculated for their statistical significance according to the Student's two-tailed, paired t-test. Mono- and disynaptic reflex (MSR and DSR, respectively) amplitudes were depressed to a different extent as a result of recurrent inhibition (Fig. 1). The peak suppression ratio of the MSR was 18.7 + 2.2% (mean + S.E.M., n = 4), which was significantly smaller than that exhibited by the DSR, which was suppressed by 54.1 + 6.3% (n = 4) (P < 0.01). The inhibition of the MSR elicited by conditioning stimulation of dorsal root L 4 comprised as much as a 50% suppression (Fig. 2). Recurrent inhibition of the MSR was enhanced by a preceding stimulation of the raph6 nucleus relatively much more than was recurrent inhibition of the DSR (Fig. 1). This enhancement was much greater when the activation of the raph6 nucleus preceded the conditioning stimulation of the ventral root by 20 ms (Fig. 1Ba), than if the interval was allowed to increase to 30 ms (Fig. 1Bb). As described previously 12, stimulation of the raph6 nucleus enhanced the MSR and suppressed the DSR. However, as seen in Fig. 1A, recurrent inhibition of the MSR was greatly enhanced even when the preceding stimulation of the raph6 nucleus showed almost no effect on the MSR. On the other hand, the inhibition elicited by a conditioning stimulation of dorsal r o o t L 4 was not affected by a preceding stimulation of the raph6 nucleus (Fig. 2). Intravenous injection of LSD tartrate 50/~g/kg, as described previously 12, depressed the MSR (not shown). The recurrent inhibition of the MSR was enhanced by this dose of LSD (Fig. 3). The facilitative effect of a preceding stimulation of the raph6 nucleus on the recurrent inhibition was, however, not attenuated by this treatment (Fig. 3). The recurrent inhibition of the DSR and the effect of the preceding stimulation on the recurrent inhibition were unaffected by this dose of LSD. Recurrent inhibition of the MSR was weaker than either that of the DSR or inhibition of the MSR elicited by conditioning of the adjacent dorsal root. While the mechanisms whereby inhibitions of the DSR are modulated remain unknown, there are sug-
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Fig. l. Effects of prior stimulation of the medullary raph6 nucleus (Raph6) on recurrent inhibition elicited by a conditioning stimulation of the ventral root L 5 (L5-VR). A: a typical set of recordings, in which the L5-VR was conditioned (+) 20 ms prior to test stimulation, and Raph6 was stimulated (+) a further 20 ms prior to the conditioning of the L5-VR. Each trace represents the average of 8 consecutive responses. Bars = 2 ms and 0.2 mV. B and C: the inhibitory curves for the MSR and the DSR, respectively, in which normalized amplitudes were plotted against various conditioning-test intervals with (closed) or without (open) preceding stimulation of Raph6, 20 ms (a) or 30 ms (b) prior to conditioning. Each point represents the mean of 4 experiments, with S.E.M. indicated. Where S.E.M. bars are not shown, they lie within the dimensions of the symbols. *P < 0.05, **P < 0.01.
gestions that descending inhibitory influences exist, which are active tonically on recurrent inhibition of the MSR 6'19'2°. The recurrent inhibition of the MSR observed in this experiment was not so great as that which has been reported in decerebrated cat preparations 3,4,6. It has been reported that there are species differences in the subtypes of receptors for ace-
tylcholine on Renshaw cells s,15, so one might expect that there could be some difference in the nervous lOO 0~0~0
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Fig. 2. A n inhibitory curve for the MSR in a case of conditioning of the dorsal root L 4 with (closed) or without (open) preceding stimulation of the raph6 nucleus, 20 ms prior to conditioning. n = 2.
Fig. 3. Effects of LSD on recurrent inhibition with (closed) or without (open) preceding stimulation of the raph6 nucleus. The ventral root L 5 was conditioned 10 ms prior to test stimulation of the dorsal root L 5 and the raph6 nucleus was stimulated 20 ms prior to conditioning of the ventral root L 5. LSD tartrate (50 ~g/kg) was injected intravenously 1 min prior to time 0. Each point represents the mean of 4 experiments. Statistical procedures were the same as for Fig. 1.
406 system regarding the nature of recurrent inhibition in the spinal cord. However, even in rats, the recurrent inhibition observed in C x transected preparations is greater than that obtained in intact, anesthetized animals (unpublished observation). Accordingly, any differences observed in the magnitudes of these recurrent inhibitions could, in part at least, be ascribed to the difference in the preparation. It was observed in the present experiment that i.v. LSD enhanced recurrent inhibition of the MSR (Fig. 3). Is is possible that this effect could be attributed to the known agonistic action of low doses of LSD at a serotonin autoreceptor, as Aghajanian et al. 1'2 have found that LSD reduces the spontaneous firing of serotonergic neurones of the brainstem. Our observations with systemic administration are therefore quite consistent with the proposal made by Sastry and Sinclair 19, who postulated the existence of a tonically active, inhibitory control of recurrent inhibition, mediated by a descending serotonergic pathway. On the other hand, the stronger segmental inhibition, such as the recurrent inhibition of the DSR or the inhibition of the MSR elicited by conditioning of the adjacent dorsal root, seems to be relatively independent of the descending control. Stimulation of the raph6 nucleus reportedly produces a dual effect on the VRRs, differing in the time course of the action: the points of maximal facilitation of the MSR and of maximal suppression of the DSR are observed within 20 ms and from 30 to 40 ms after the commencement of the medullary stimulation, respectively 12. In the present study, recurrent inhibition of the MSR was enhanced by a preceding stimulation of the raph6 nucleus. This enhancement effect was much greater when the conditioning stimulation of the ventral root followed the raph6 stimulation by an interval of 20 ms rather than of 30 ms (Fig. 1B). Neither the facilitative effects on the MSR 12 nor on the recurrent inhibition (Fig. 3), which were elicited by stimulation of the raph6 nucleus, were attenuated by the injection of LSD. The similarities in time course of these two LSD-insensitive facilitations, as well as the fact of their insensitivity, suggest the employment of a common pathway subserving the two facilitative effects observed in the spinal cord. Enhancement of the recurrent inhibition seen here (Fig. 1) as well as facilitation of the MSR reported earlier 12 both could be accounted for as a re-
sult of an elevation in motoneuronal activities. With respect to the DSR suppression however, it has been shown that whereas the MSR enhancement is LSD-insensitive, the DSR suppression induced by raph6 stimulation is reversed by LSD 12. The present work has emphasized the separation of the mechanisms responsible for modulation of spinal reflexes by making the additional observation, that the facilitation of recurrent inhibition of the MSR is LSD-insensitve (Fig. 3) - - further, that the enhancement of this suppression by recurrent inhibition is proportionately less for the DSR than for the MSR (Fig. 1). Reductions in Renshaw cell firing 7'9A3'14'18 have been demonstrated through stimulation of various regions of the brain. Descending inhibitory influences on recurrent inhibition of the MSR also have been proposed 6'19'2°. These descending influences ultimately increase the output from the motoneurone pool and lead to a relatively more intense muscular contraction n. Yet activation of descending pathways originating from, and perhaps passing through the medullary raph6 nucleus 12, which were demonstrated in this experiment, would enhance only the initial output of the monosynaptic transmission, the subsequent outputs being attenuated strongly because of the resultant enhanced recurrent inhibition; all the polysynaptic outputs however, which are recipient of the same sensory messages, also would be reduced via a distinct serotonergic descending pathway 12. It might then be postulated that these pathways serve as a selector and as an amplifier for a particular form of output, required of the motoneuronal pool. In the present study, we have demonstrated a modification of the processes of recurrent inhibition of the MSR via a descending pathway which was activated by stimulation of the raph6 nucleus, this resulting in an adjustment of spinal activities. The recurrent inhibition of the MSR is, by itself, much weaker than that of the DSR or the inhibition of the MSR elicited by a conditioning stimulus to the adjacent dorsal root, and the descending pathway responsible for the modulation of this pathway is LSD-insensitive in nature. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. We are grateful
407 to M e r c k ( J a p a n ) for the supply of m e c a m y l a m i n e h y d r o c h l o r i d e a n d to S a n d o z ( J a p a n ) for t h e supply
T.P. Hicks (The U n i v e r s i t y of Calgary) for his critical r e a d i n g of the m a n u s c r i p t .
of lysergic acid d i e t h y l a m i d e tartrate. W e t h a n k D r .
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