COMPARATIVE STUDY OF THE EFFECTS OF STRYCHNINE AND MEGIMIDE ON THE REFLEX ACTIVITIES OF IN RABBIT

MASAO MORITA, MOTOHIRO YASUHARA, TOSHIKO KIMURA, CHIEKO ITO AND NORIKO KITAO

Department of Pharmacology, Kansai Medical School,Hirakata, Osaka Received for publication September 10, 1958

Concerning the mechanism of action of strychnine on the ,

Dusser de Barenne (1) and others have made various reports. Recently Bradley et al. (2) and Eccles (3) observing strychnine suppresses the direct inhibitory action and depresses the polysynaptic inhibition in the spinal cord through electrophysiological studies have expressed a new opinion that this is related to the mode of central excitatory action of strychnine.

Morita et al. (4) caught the muscle discharge originating in Golgi's tendon organ. which has been considered to be inhibitory on motoneuron in the spinal cord. Next, Takeuchi

(5), one of our colleagues investigated the effects of ether, barbiturate and myanesin on this discharge and other spinal reflex discharges and Kimura (6) that of chlorpromazine on these muscle discharges. We have been able to observe two or three discharges at the same time when the same methods as described in the previous reports were used. Ac cordingly, we have considered it significant to investigate the mechanism of strychnine action on the spinal cord in the electromyography of rabbit's hindlimb.

It was found by Shaw et al. (7) that megimide (ƒÀ-ƒÀ-ethyl-methyl glutarimide) has an antagonism towards barbiturates, and Cass (8) comparing megimide with picrotoxin or pentylenetetrazol to its potency of antagonistic action against barbiturates, concluded that megimide is more effective barbiturate antagonist than these analeptics. It has generally been said that such medullary stimulants have different loci of action from that of stry chnine and though strychnine produces predominant responses by the functional changes of in the spinal cord, on the contrary, medullary stimulants have strong action at a higher center than the spinal level. Therefore, the authors studying the comparative effect of strychnine and megimide on spinal reflex activities, have tried to discover the difference in spinal action of these two drugs.

METHOD

Adult rabbits were used in these experiments whose spinal cord had been transected between Th 12 and L 1 without any anesthetic agents. These spinal rabbits had their skin cut open at fossa poplitea ; peroneal nerve and tibial nerve were exposed and the latter one resected. In all cases, ipsilateral transaction was made on Achilles tendon to relax gastrocnemius and soleus muscle. Animals were put on experimentation three hours after the transection of their spinal cord. Rabbits were fixed on supine position on the metal instrument designed in our laboratory whose photograph was put in Kimura's paper (6). In the nociceptive reflex experiment, electric stimulation• was given to the central end of the ipsilateral, cut tibial nerve to induce twitch of tibialis anterior muscle. In the pro prioceptive reflex experiment, a certain amount of stretch was given to tibialis anterior muscle by bending the ankle joint dorsally. The strength of electrical stimuli was always kept constant with the frequency of 1.5 c/sec. The lead off electrode was coaxial needle type. An insulated Copper wire of 0.1 mm was sealed as active electrode in a 1/3 hypo dermal needle. The tip of wire was left exposed. The electrode was inserted into the tibialis anterior muscle. Thus, obtained of tibialis anterior muscle was observed by GR coupled cathode ray oscillograph and recorded with a magnetic oscillo graph. In this paper, "after-discharge" and "delayed discharge" are being used as well as "evoked discharge" in electromyogram of nociceptively induced reflex twitch. Evoked discharge was considered to develop through excitation of polysynaptic reflex arcs and after discharge through the reverberating neuron circuit in spinal cord and delayed discharge to originate from proprioceptive reflex arcs. In proprioceptively induced reflex discharges in spinal rabbits, larger discharge (hereafter to be abbreviated as "GIb discharge") is of Golgi's tendon organ origin and smaller one (hereafter to be abbreviated as Gla discharge) originates from . Therefore, the effects of central drugs on these nocicep tively or proprioceptively induced discharges may be presumed to correspond to those on some functions of spinal reflex arcs. Strychnine nitrate as well as megimide were given intravenously in all samples. Megimide was used as 0.5 per cent solution in Ringer's solution.

RESULTS 1. Effect of Strychnineon the Proprioceptirel y Induced Reflex Discharges Strychnine, in a dose of 0.03 to 0.04 mg/kg given intravenously to spinal rabbit, increased the frequencies and the voltages in both GIb and GIa discharges, in a dose of 0.05 to 0.06 mg/kg gradually decreased GIb discharges and in a dose of more than 0.07 mg/kg eliminated GIb discharges and only GIa discharges remained. When a much larger amount of strychnine is given, the burst of discharges of high voltage may be observed in tibialis anterior muscle (See Fig. 1). On the muscle discharges under strychnine action, we checked the influence in this case when the a Terent nerve impulse is blocked by injection of procaine in the concentration of 2 per cent in Ringer's solution around the tendon of tibialis anterior muscle. After this procedure. we found that when both GIb and (;la discharge-, arc augmenting the GIb discharges was eliminated and ~%-hen(;la discha ge eni obsei,,t'd iindri th( :tt vrhntnr action, procaine did not have any influ ence on the discharge. However, when we apply procaine in the concentration of 2 per cent in Ringer's solution on peroneal nerve, both GIa and GIb discharge were eliminated. It has been found by the authors that proprioceptive reflex dis charges are in general easily affected by procaine and GIb discharges are elimi nated by injection of procaine around the tendon. Then, according to the effects of procaine on the changes in discharges caused by strychnine, it has been clear that the discharges increased by strych nine should be put with GIa and GIb discharges. FIG. 1. Effect of strychnine on proprioceptive reflex discharges from tibialis anterior muscle Next, we investigated the influence of during stretch of ankle joint in spinal rabbit. myanesin on the reflex discharges under A : Control, B : after intravenous injection of 0.03 mg/kg of strychnine, C : after 0.07 the strychnine action. When GIa and mg/kg of strychnine, D : after 0.1 mg/kg of GIb discharge are increasing after stry strychnine, burst of discharges appeared. chnine, 10 to 20 mg/kg of myanesin given Time scale, 60 c/sec. intravenously, depressed these discharges and 50 mg/kg of myanesin suppressed the discharges entirely. When the burst of discharges after strychnine action is observed, 50 mg/kg of myanesin eliminated the burst and yielded the GIa discharge instead of the burst and even 80 mg/kg of myanesin did not eliminate this GIa discharge. But in this situation, by applying procaine in the concentration of 2 per cent in Ringer's solution on peroneal nerve the GIa discharge was easily eliminated. On the central action of myanesin, detailed pharmacological studies have been made by Berger (9), Henneman et al. (10), Kaada (11), King and Unna (12) and it has been found that polysynaptic spinal reflexes are readily inhibited by small doses of myanesin while monosynaptic reflexes are resistant to the acion of the drug, and both supraspinal suppression and facilitation of spinal reflexes are readily abolished . Therefore the burst of discharges after strychnine action may be considered as the different discharges from GIa or GIb discharge, resulting from the excitation of of the spinal cord .

II. Effect of Strychnine on the NociceptivelyInduced Reflex Discharges

In most cases the nociceptive reflex discharges in tibialis anterior muscle of spinal rabbit consist of evoked discharges and delayed discharges. When strychnine was given intravenously in the amount of 0.05 mg/kg or more, first the voltage of evoked discharges increases and the duration is prolonged, and then delayed discharges augment . Even in the case on which the delayed discharges were not ob served before the application of the drug, the delayed discharges were newly showen. Increasing the dose up to more than 0.1 mg/kg the after-discharges could be observed (See Fig. 2 A, B). Such after-discharges and delayed discharges pro duced by strychnine were scarcely influenced by ap plication of procaine on the tendon or peroneal nerve. However, when 20 mg/kg of myanesin or 5 mg/kg of pentothal sodium is administered intravenously, though the evoked discharges were not influenced, these after as well as delayed discharges were elimi nated (See Fig. 2 C). Therefore it is considered that after-discharges or delayed discharges which became FIG. 2. Effect of strychnine on nocicep active after strychnine are not caused by the activities tive reflex discharges from tibialis of small motor nerve system but are caused as a result anterior muscle following electrical stimulation of the central end of of activities of the structure in the spinal ipsilateral, cut tibial nerve in spinal cord which consists of a chain of many interneurons, rabbit and antagonistic action of for they have a strong susceptibility to myanesin or myanesin against strychnine. A : Control, B : after intravenous pentothal sodium. injection of 0.1 mg/kg of strych nine, C : after intravenous injec III. Effect of Megimide on the Proprioceptively tion of 20 mg/kg of Myanesin in B, D : after intravenous injection Induced Reflex Discharges of 50 mg/kg of myanesin in B. Time scale, 60c/sec. Concerning the influence of megimide upon the activity of small motor nerve system, Soderberg (13) has found that the muscle spindle activity is increased by a subconvulsive dose of megimide. Authors have found that megi mide has two modes of action on proprioceptively induced reflex discharges. One of them, the increase of GIa and GIb discharges by 3 mg/kg of the drug which was not produced in the case of 0.3 mg/kg administration, was observed in six out of eight cases. In the other two cases, GIa and GIb discharges were increased even by 0.3 mg/kg administration and GIb discharge was decreased by 3 mg/kg administration. Then by 10 mg/kg of megi mide in any case, there were induced tonic and clonic convulsions in the forepart of the rabbit's body including its forelimb, but no convulsions in hindlimb as well as hindtrunk. After the administration of the amount of megimide need to produce such convulsions both GIa and GIb discharges were decreased in general. That is, it was observed that proprioceptive reflex activity was being depressed by a large amount of megimide. We have investigated the action of myanesin on proprioceptive reflex patterns which are seen after megimide. In the course of the drug action where GIa and GIb discharges are increasing by 0.3 to 3 mg/kg of megimide, myanesin was given in the amount of 10 to 20 mg/kg intravenously but it was not seriously affected u this case, However, admi nistering 50 mg/kg of myanesin, GIa and GIb discharges were depressed, strongly in later one urder the megimide action. In the cases in which GIa or GIb discharges are depressed by megimide, 10 to 20 mg/kg of myanesin was given but it was not affected while by 50 mg/,kg of myanesin these discharges were depressed still more. It may be therefore said that the antagonistic action of myanesin was strong upon GIa and GIb discharges augmented in the magnitude by strychnine and also upon stry chnine bursts of discharges due to the increased activity of interneuron of spinal cord, but on the contrary upon GIa and GIb discharges affected by megimide the influence of myanesin was weak.

IV. Effect of Megimide on the NociceptivelyInduced Reflex Discharges When 0.3 to 0.8 mg/kg of megimide was given intravenously, in four out of eight cases tested, the nociceptively induced reflex discharges were suppressed and delayed discharges were eliminated (See Fig. 3). Then, an additional dose of 2 to 8 mg/kg of drug was ad ministered in the cases where nociceptively induced reflex discharges were suppressed, the reflex discharges began to increase. Its evoked discharges showed an increase in voltage and duration and in delayed dis charges an increase in its frequency. In some cases, after-discharges were newly ap peared. Such facilitatory effect of megimide on nociceptively induced reflex discharges was observed already even by 0.3 mg/kg in cases where there is no inhibitory effect in a small amount of megimide, then by 5 mg/kg this facilitatory effect became much FIG. 3. Effect of megimide on nociceptive reflex stronger. However, the after-discharges discharges from tibialis anterior muscle follow which appeared after strychnine action ing electrical stimulation of the central end of ipsilateral, cut tibial nerve in spinal rabbit. mentioned above, were not observed even A : Control, B : after intravenous injection while megimide was producing the facili of 0.3 mg/kg of megimide, C : after 2 mg/kg tatory effect. of megimide, D : after 8 mg/kg of megimide. Time scale, CO c/sec. After the administration of megimide in a dose of 8 to 10 mLy/kLy.tonic and clonic convulsions were produced in the forepart of the rabbit's body and the reflex discharges were gradually depressed. The inhibitory effects which are seen under the action of a large dose of megimide are observed running parallel with evoked and delayed discharges in many caces, but in some cases the delayed discharges were more strongly affected . As we saw above it has been investigated that, upon nociceptively induced reflex dis charges, a small amount of megimide had inhibitory effects and increasing its dose it changed into facilitatory effects then with convulsant dose into depressive effects, that is it seems that these effects resemble those shown in the case of proprioceptively induced reflex discharges. However, in both cases where GIb discharge was depressed by megimide but without any change of GIa discharge and where both discharges were only increased, the doses were equivalent to that which showed facilitatory effect on nociceptively induced reflex discharges. Therefore, we cannot explain the effect of megimide upon noci ceptive reflex arcs taking our stand on the action upon proprioceptive reflex arcs. Only after a convulsive dose of megimide, pro prioceptive and nociceptive reflex activities were depressed simultaneously in the same degree. Next, the effects of myanesin and bar biturate on ncciceptive reflex discharges seen after megimide were investigated. This investigation was tried with rabbits in which inhibitory effect by small dose of megimide has not been manifested, by 50 mg/kg of myanesin evoked and delayed discharges were depressed and by 80 mg/kg delayed discharges totally disappeared (See Fig. 4 D, E). Administering more than 5 mg/kg of pentothal sodium evoked discharges were FIG. 4. Effect of megimide on nociceptive reflex depressed and delayed discharges disap discharges from tibialis anterior muscle and peared. antagonistic action of myanesin against megi mide. It was found that pentothal sodium can A : Control, B : after intravenous injection be regarded as antagonistic towards both of 0.3 mg/k of megimide, C : after 8 mg/kg strychnine and megimide but myanesin is of megimide, D : after intravenous injection of 50 mg/kg of myanesin in C, E : after 80 much more antagonistic towards strychnine mg/kg of myanesin in C. Time scale, 60 than megimide. c/sec.

DISCUSSION

The actions of strychnine upon the reflex discharges in the tibialis anterior muscle are diagrammatized in Fig. 5. It has been shown in this figure that in a very small dose strychnine exerts the effect upon proprioceptive reflex discharges, increases in the magnitude of these discharges then induces the augmentation of nociceptive reflex discharges and final ly causes proprioceptive bursts of discharges or nociceptively induced after-discharges which are considered to be developed by the increased activity of nerve structure including FIG. 5. Diagrammatic representation of the effects of strychnine on reflex discharges from tibialis anterior muscle in spinal rabbit. Nociceptive reflex discharges-evoked discharge, after discharge and delayed dis charge-developed following 1.5 c/sec electrical stimulation of the central end of ipsilateral, cut tibial nerve, and proprioceptive reflex discharges-monosynaptic GIa discharge as well as disynaptic GIb discharge-developed during stretch of ankle joint in spinal rabbit.

numerous interneurons in the spinal cord. Accordingly as the general tendency of the action of strychnine on spinal reflex activities, it is much more effective with smaller dose on proprioceptive reflex arcs than on nociceptive reflex arcs. Nam (14), Brooks and Ko izumi (15) and Bernhard el a[. (16) published papers on the mode of actions of strychnine on the spinal cord and it has been made clear that the subtetanic doses of strychnine potentiate both the monosynaptic and polysynaptic reflex responses. Applying these results of our predecesssors' investigations to author's result, it may be possible to consider that GIa discharge and nociceptive evoked discharge should be augmented in its magnitude. In fact, the increase of these two kinds of discharge was observed by subtetanic doses, the result of the author's investigation completely coincided with that of our predecessor's study. Furthermore, we have newly revealed the following two facts: firstly, that GIa discharge and nociceptive evoked discharge are augmenting under strychnine action while GIb discharge is gradually depressed, secondly, the amount of strychnine need to produce an excitation of spinal interneuron circuits is much larger than that to produce the aug mentation in the magnitude of GIa discharge and nociceptive evoked discharge. 'Me GIb discharge may be to correspond with the function of Golgi's tendon organ and it is sup posed that the increased activit, of this organ results in depression of motoneuron in the :ppnai cord !hi is ailed autogens iic ii hihiii l h, tan that (,It, discharge was depressed by a moderate dose of strychnine suggests that spinal excitatory action of strychnine may appear partly by its blocking action on the inhibitory in spinal reflex arcs in the intact animal. Purpura and Grundfest (17, 18) and Eccles et al. (3) reported that strych nine depressing inhibitory in central nervous system, yields a facilitation of exci tatory synapses. Then it is quite natural to think about the elimination of inhibitory brakes concerning the change of spinal reflex activity by the drug , and moreover we could find this blocking action surely manifests during a certain period of strychnine action through the effects of the drug on various reflex discharges of tibialis anterior muscle. But stry chnine convulsion does not appear until much larger doses are administered than those which exert blocking -action on inhibitory synapses. We could also observe the develop ment of strychnine bursts of discharges and nociceptive after-discharges which are consi dered to be based on the excitation of interneuron circuits , when much larger doses are given than that which was sufficient to cause an augmentation of nociceptive evoked dis charges. It may be, therefore, considered that strychnine convulsion should be attributed to the excitatory effect of strychnine on the activity of interneuron circuit in the spinal cord. Now let us summarize our discussion above. As a result of our experiment , it is revealed that strychnine, in the spinal cord, acts first on the proprioceptive reflex arcs and raises the excitability of motoneuron, and after that acting on the interneuron induces convulsions. Next, concerning the effect of megimide, it is considered that its mode of action on the spinal cord is not the same as that of strychnine. The small dose of megimide influences nociceptive evoked discharges as well as delayed discharges. On the evoked and delayed discharges megimide had both depressive and augmentative action . As this depressive action was, however, eliminated by small additional doses of megimide , the augmentative action of megimide may be to become direct action on the nociceptive reflex arcs . And the depressive action is regarded as an indirect and temporary inhibition which was induced by an excitation of spinal nerve structure whose function resembles the stem reticular formation. The actions of megimide on nociceptive spinal reflex arcs were shown in Fig . 6. Concerning the strychnine action, we have referred to, that the affected activity of GIb system , is an important factor for the augmentation of nociceptive evoked discharges, in the case of megimide such cases where GIb discharge decreases while GIa discharge is increasing as under strychnine action, were only two out .of eight cases tested. And it is thought that the augmentation of evoked discharge by megimide is practically irrelevant to its action against GIb system because we had good many cases in which we could observe the augmentation of nociceptive evoked discharges by smaller doses than that need to decrease GIb discharges. It may be presumed, on the effects of megimide, as follows : in small doses megimide has a depressive action on the spinal reflex arcs in the minority of cases, if the doses are increased it becomes excitatory action, and moreover the augmenta tion was observed upon proprioceptive as well as nociceptive reflex discharges when megi Inide action is regarded as stimulation which affects on the synapse that is common both FIG. 6. Effect of megimide on nociceptive reflex discharges in spinal rabbit . Individual value of percentage change in magnitude of reflex discharges in tibialis anterior muscle is plotted for eight cases in which megimide was given intravenously. White circles : cases in which nociceptively induced reflex discharges were depressed by intravenous application of a small amoint of megimide. Black circles : cases in which no depressive effects were seen in a small dosage of megimide on nociceptively induced reflex discharges.

to proprioceptive and to nociceptive reflex arcs, for example, on sensory afferent synapse. While the inhibition or depression of reflex activity by a small dose of megimide may be an indirect effect which was induced by the excitation of reticular structure in the spinal cord, it has been clear that strychnine and megimide have quite a different mode of action

from each other and this is also seen in antagonism of myanesin towards both these drugs . Finally, -because megimide convulsion in spinal rabbit was only seen on the forepart of the body, the action of megimide upon the spinal cord is irrelevant to the occurrence of the convulsion but megimide convulsion should be produced through the action on the supraspinal nerve structure.

SUMMARY

Electromyographical studies were made of effects of strychnine and megimide on pro prioceptively as well as nociceptively induced reflex discharges which are seen in tibialis anterior muscle of spinal rabbit, and the spinal actions of these drugs were compared, in special reference to the mode of spinal excitatory action. 1. Towards proprioceptive reflex discharges strychnine augments in the magnitude of GIa and GIb discharges when given in small dose and according to the increase in the dose of drug it gradually suppresses GIb discharges and finally makes bursts of discharges to appear. 2. Towards nociceptive reflex discharges strychnine augments in the magnitude of evoked discharges first, then augments in delayed discharges and by a larger dose makes the after-discharges to appear. 3. Megimide augments in the magnitude of proprioceptive reflex discharge, at the most, by a small to a moderate dose and a large dose causes convulsions to occur in the forepart of the body, it. depresses the reflex discharges in many cases tested. 4. Megimide, in small doses, depressed nociceptive reflex discharges in half the cases among samples. According to the increase in doses it augments in the magnitude of evoked discharges and of delayed discharges. When convulsive doses are administered they are depressed. 5. As a result of our investigations mentioned above, it may be concluded as follows : strychnine, in small doses, first affects proprioceptive reflex arcs and raises the excitability of motoneuron, then affects the interneuron and makes convulsions occur. While megimide stimulates the synapse which is common both to proprioceptive reflex arcs and to noci ceptive reflex arcs and also the regulatory structure in spinal cord, it also causes convul sions in larger doses by the stimulation upon the higher center.

REFERENCES

1) DUSSER DE BARENNE, J.G.: Physiol. Rev. 13, 325 (1933)

2) BRADLEY, K., EASTON, D.M. AND ECCLES, J.C. : J. Physiol. 122, 474 (1953)

3) ECCLES, J.C.: The Physiology of Nerve Colls p. 153, The Johns Hopkins Press, Baltimore (1957)

4) MORITA, M., YASUHARA, M., TAKEUCHI, Y., KIMURA, T. AND ITO,, CH.: Folia pharmacol. japon.

52, 167•˜ (1956) (Japanese)

5) TAKEUCHI, Y.: Ibid. 53, 177 (1957) (Japanese)

6) KIMURA, T.: Jap. J. Pharmacol. 6, 162 (1957)

7) SHAW, F.H., SIMON, S.E., CASS, N.M., SHULMAN, A., AUSTEE, J.R. AND NELSON, E.R.: Nature

174, 402 (1954)

8) CASS, N.M.: Brit. J.Anesth. 28, 324 (1956)

9) BERGER, F.M.: Pharmacol. Rev. 1, 243 (1949)

10) HENNEMAN, E., KAPLAN,A. AND UNNA, K.: J. Pharmacol. 97, 331 (1949)

11) KAADA, B.R. : J. Neurophysiol. 13, 89 (1950)

12) KING, E.E. AND UNNA, K.R. : J. Pharmacol. 111, 293 (1954)

13) SODERBERG, U. : Arch. Neural. Psychiat. 79, 239 (1958)

14) NAESS, K. : Acta physiol. scand. 21, 34 (1950)

15) BROOKS, C.MC. AND KOIZUMI, K. : Ciba Symposium, The Spinal Cord p.63 (1953)

16) BERNHARD, C.G., TAVERNER, D. AND WIDEN, L.: Brit. J. Pharmacol. 6, 551 (1951)

17) PURPURA, D.P. AND GRUNDFEST, H.: Fed. Proc. 15, 146 (1956)

18) PURPURA, D.P. AND GRUNDFEST, H.: J. Neurophysiol. 20, 494 (1957)