Vol. I, No. 3

Eur. J. Epidemiol. 0392-2990 September 1985, p. 193-201

NEUROPHYSIOLOGICAL ASPECTS OF TETANUS TOXIN

EFFECTS

ON

THE

MOTOR

SYSTEM

K. T A K A N O Abteihmg PathoneurophysioIogie, Universitiit GOttingen, Humboldtallee 23, D-3400 G6ttingen, West Germany

Key words: Tetanus toxin - Motor system - Central nervous system - Synaptic transmission.

The action of tetanus toxin on the motor system in experimental tetanus relaLing to the clinical one was reviewed. Special attention was paid to several controversial results in recent years.

INTRODUCTION

The m o s t representative sign of the tetanus intoxication in h u m a n s and animals 'is the hyperactivity of the m o t o r system. Most of 'the patients die, if not relaxed by adequate therapy, due to a n ,apnea, which is caused by the hyperactive spasms of the r e s p i r a t o r y musc'les. L o c k j a w ( t r i s m u s ) a n d / o r so-called, local tetanus ,, ,are frequently the 'first s y m p t o m s ~o,f the disease. The latter is characterized by rigidity or painful m u s c u l a r spasm at the site of injury. It .occurrence is not too unusual, especially in light cases but m a n y of t h e m m a y remain unrecognized as tetanus by physicians, and some patients m a y even not visit 'the clinic. Local tetanus can well be studied experimentally by injection of tetanus toxin into either the muscle, a peripheral nerve o r the spinal cord. Fully developed cl,in~,c:al tetanus is r a t h e r well characterized by gen
muscles, occurring stronger in antigrav,ity muscles, (e.g. in the extensor muscles of the legs) and in the ~masseter (trismus). Experimental general 'tetanus c a n be induced by injecting tetanus toxin in Lravenousty. This review will describe the action of tetanus toxin .on the m o t o r system in experimentM tetanus .and relate it to the state .of clinical tetanus. Special attention will be paid to several controversial results reported in the last ten years. Fo.r recent general reviews see H a b e r m a n n (24), Kryzhanovsky (36), Bizzin,i (4, 5), Takano (62), Mellanby and .Green (44), Wellh6ner ,(73), Matsuda (41). FATE OF THE

TOXIN

IN THE

NERVOUS

SYSTEM

B i n d i n g to c e n t r a l n e r v o u s s y s t e m . - It i.s well k n o w n as Wass,ermann-Takaki p h e n o m e n o n (72), that tetanus to~in has a strong affinity to the tissue of the central nervous system but not to other .organs. The gray m a t t e r is stronger m o r e

Corresponding author. 193

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in ,binding the toxin t h a n the white m a t t e r . Tetanus :toxin shows ,an o p t i m a l i n t e r a c t i o n w i t h l h e gangliosid, es containing c e r e b r o s i d e (27, 28). In s u b f r a c t i o n a t e d gangliosides the s y n a p t o s o m e m e m b r a n e fraction b o u n d ten times the a m o u n t of toxin b o u n d by the synaptic vesicles (45). Dimpfel .et .al. (12) using .cell cultures found that cereb,rosides w e r e not crucial f o r toxin binding. Neuronal transport. - T h e r e are n u m b e r s .of studies showing h o w tetanus toxin reaches its .target o r g a n w h e n it is :injected into the muscle. I t ,appea r s :well e s t a b l i s h e d t h a t the ,toxin is mainly trans p o r t e d retrograd.ely via the alpha m o t o r axons to tbe .central n e r v o u s s y s t e m (35, 51, 25, 74). The ascending axonal ' t r a n s p o r t of the toxin is acceler a t e d w h e n n e r v e activity is .increased (75). G r e e n et al. (22) s h o w e d that the labelled ~2sI,tetanus to.xin co,u~d be ,found only in a l p h a - m o t o r fibres in t h e v e n t r a l ro.ot, b u t not in the gamma-fibres. This finding has been one of the a r g u m e n t s to .claim 'that .gamma h y p e r a c t i v i t y has ,only searce significance in tetanus ( h o w e v e r , ~see 65). Though the .main p a t h w a y of :the tox~in are the m o t o r nerves, t r a n s p o r t is also possible in vagal (26) s o m a t o s e n s o r y (19) nerve fibres in cats, and s y m p a t h e t i c ones in rats (56). Recently K a n d a and Takano. (31, 32) suggested that the p a r t o,f the toxin which acts .on the e x c i t a t o r y postsynapti.c potential ( E P S P ) seems to ascend in sensory fibres b e y o n d the d o r s a l r o o t ganglia. The speed of the toxin m i g r a t i o n ,in the peripheral n e r v e has b e e n e s t i m a t e d in the r a t as 5-10 m m / h ,(24), or 7.5 m m / h (56) in the sciatic ner~e .o.f ~he cat 7-9 m m / h '(66). In the b r a i n ,of the cat it is a b o u t 1 m m / h (6, 9). In the n a t u r a l disease the toxin p r o d u c e d b y Clostridium tetani first spreads f r o m the w o u n d into l y m p h a t i c s y s t e m and then passes into the blood s t r e a m (25, 53). H a b e r m a n n and Di.mpSel (25) have i n j e c t e d i.v. ~2SIdab.ell.ed toxin in rats at doses 1.ower t h a n LD:~0. They o b s e r v e d high radioactivity in the plasm a at the .first d a y a f t e r injection a n d r a p i d decay in the following days, w h e r e a s the radioactivity in the central n e r v o u s s y s t e m reached its m a x i m u m at the second day, foil.owed b y a p.er~.o.d of c o n s t a n t activity. After the 6th day, the .activity declined slowly. I t is usuallv p o s t u l a t e d 't(~day that the toxin first is a b s o r b e d ,at endplates o.f the muscles and enters into the m o t o r nerve. H o w e v e r , there are controversial reports, s o m e showing the toxin within the e n d p l a t e s (76, 50) while o t h e r s have failed to d e m o n s t r a t e this (20, 80). A c u r r e n t view is, that general tetanus is the integration of m u l t i p l e local tetanus .events, due to u p t a k e o.f toxin f r o m the b l o o d by endplates t h r o u g h o u t the body (24, 25, 44, 4). However, this point will be discussed (see ,later).

Intraspinal and transsynaptic migration. After i n t r a m u s c u l a r injection into the gastrocnemius at a dose several times higher t h a n one cat m i n i m a l lethal dose (MLD), the first visible sign (light hobbling of freely moving cat) could be o b s e r v e d a f t e r 20-30 h, depending ,on the size of the cat and tax, in doses. I.n the next 10 h the stiff extension of the leg developed r a t h e r speedy up to its m a x i m a l degree (66). In this stage it is a n a l m o s t p u r e local tetanus and the toxin is localized in the ipsilateral spinal s e g m e n t L7 and S~ (74). Three to fiwe clays a f t e r toxin injection the f.o,relegs a n d the c o n t r a l a t e r a l hind leg showed the t e t a n u s signs, p r o v i d e d the doses w e r e sufficiently high. Is this d e v e l o p m e n t of the *etanus signs d u e to ,a,n intras,pinal m i g r a t i o n ,of the toxin? If so, the ipsilateral m i g r a t i o n velocity in the direction of the b o d y axis seems to b e a l m o s t as high as .in the p e r i p h e r a l nerve while that in the c o n t r a l a t e r a l side of the spinal cord a p p e a r s to be one h u n d r e d times slower. T h e r e are a n u m b e r of ex,p e r i m e n t s since the study of Meyer a n d Ransore (46) which s u p p o r t ,the ,concept of ] n t r a s p i n a i m i g r a t i o n of the toxin ( f o r review see 80, 44). However, it is also. possible that the toxin is dis t r i b u t e d b y the b l o o d circulati,on .due to the leakage f r o m the injection .site, b e c a u s e the onset t i m e in all three legs is roughly the same. No exp e r i m e n t concerning the i.ntraspinal migration is av.ai,lable which employs m o d e r n so,phisticated methods (using a label.led toxin etc.). When toxin was injected 'into a fast flexor muscle, like tibialis anterior, initial strong t e t a n u s signs did not o c c u r in this m u s c l e b u t in the gastrocnemi.us. The onset t i m e was 7-8 h o u r s longer than in the case of injection into the gastrocnemius. T r a n s s e g m e n t a l m i g r a t i o n of the toxin f r o m L 4 t o L 7 (,or L 5 t o S l ) w a s t h e r e f o r e a s s u m e d to be 7-8 h (66). Under e x p e r i m e n t a l conditions tetanus toxin reaches to the r n o t o n e u r o n e s m o s t l y via mo*o.r nerve fibres. The action of the toxin ,is presynaptic. This implies that the toxin can r e a c h the presynaptic side a p a r t f r o m the m.otoneurone. Trans.synapti.c m i g r a t i o n of the toxin into the ~.motoneuro,ne was suggested b y Schwab and Thoenen (55). Thev injected labelled toxin at a very high dose (3.75 t~.g, high purified tox,in) into 'the unilateral deltoideus muscle .of the rat. 7 or 14 h a f t e r the injection the radioactivity could b.e r e c o r d e d at the p r e s y n a p t i c side which was not tbe case in the control .animal. ( T h e y have not investigated the c o n t r a l a t e r a l .as a .control side). They f u r t h e r o b s e r v e d a decrease .of the r o u n d synaptic vesicles which p r o b a b l y are excitatory ones. The n u m b e r of the inhibitory, flattened, vesicles was not changed. They discussed t h a t this .exhaustion of r o u n d vesicles indicated a h y p e r a c t i v i t y of the e x c i t a t o r y synapses due t.o disinhibition.

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Tetanus toxin: neurophysiological aspects

ACTION ON T H E MUSCLE On the s e n s o r y m u s c l e r e c e p t o r s . - There are several .old studies declearing that the toxin acts directly on the sensory recepto.rs in the muscle (e.g. 38, 54; for o t h e r old studies see 80). This interpretation suffers f r o m the lack of m o d e r n knowledge in neurophysiology. ]'he a u t h o r s were no.t aware .of tile efferent innervation .of the ,muscle spindle :by the g a m m a m o t o r cells in the spinal .cord ,(see 65). There 'is no convincing study which indicates direct ,effects of this to~in on the sensory receptors, neither in intra- or e x t r a m u s c u l a r regions. However, a change of the p a t t e r n of the muscle spindle discarges, which is probab,ly ,due to an actio,n on the intrafusal fibres in local ~etanus, was r e p o r t e d by .our group (49). On the endplate. - Though the main feature of tetanus is the hyperactivity of the m o t o r system, there is a n u m b e r of observations that the m a m m a l i a n endplate can be blocked u n d e r some experimental conditions as well as in ,clinical tetanus (10, 48, 35). Duchen and his group (13, 14, 15) f o u n d that after .the functional ~, denervation ,, by tetanus toxin there o c c u r r e d ,a sprouting f r o m moto.r nerve terminals and subsequently the f o r m i n g of new endplates. K r e t z s c h m a r et al. (34) have clearly demonstrated that tetanus toxin blocks the n e u r o m u scular :transmission of the pale fast (white phasic) muscle m o r e strongly than that of the ired stow (tonic) mus.cle. There was a controversial result by Duchen and his group, which was .clarified by our g r o u p (34) as an artifact resulting f r o m the injection of too large volumes .o.f toxin solution. Results similar to those of K r e t z s c h m a r et al. (34), were obtain,ed by o t h e r groups (30, 57, 42, 43). For f u r t h e r discuss.ion see (63). A flaccid paralysis was observed when very large d o s e s of toxin were injected (10, 48, 15, 34, 57). The paralysis was p r e c e d e d by a hyperactivity of the intoxicated muscle (34). D i r e c t and~or s e c o n d a r y a c t i o n on the s t r i a t e d m u s c l e . - The ,early period of clinical as well as

of experimental local tetanus, the main,ta.ined shortening of the muscle ~is caused by a ,tonic activity of the m o t o r units, and therefore a high activity in the e l e c t r o m y o g r a m (EMG) can b e observed. When the muscle nerve i.s ,cut the EMG disappears. In the later period the rigid extension of the hind leg can f u r t h e r be sustained even wi,thout any EMG activity. When the muscle nerve 'is cut, the rigidity persists. This now is a contracture. Old investigators of tetanus were aware o,f this fact (e.g. 46). I n 1928 Ranson (52) showed an increase in stiffness of the intoxi.caved muscle. The tens.ionextension curve of the intoxicated muscle was

studied by (66, 61, 30). Takano and Henatsch (66) have shown that in the passive tension-extension relation the a-value (the slopes of the half-logarithmic graph, t=k.a ~ t tension, k constant, 1 length of stretch) increased in the slow muscle u n d e r toxin action. They did not find any a-value change in the fast muscle (60). A change of the a-value could also b.e observed a f t e r long continuous nerve s~imulation (unpublished data). These findings indicate that the change of muscle mechanics 'can be the ~result of prolcnged high activity of the n e u r o m u s c u l a r units. Ebisawa and M a t s u k u r a (16) e.g., f o u n d .by a u t o p s y of tetanus patients pathological changes in the striated muscles, a b n o r m a l bleeding, loss of stripes, r u p t u r e and degenerative changes. EFFECTS ON N E R V E F I B R E S

Many w o r k e r s have tried to detect effects of the to~in on the excitable axon m e m b r a n e b u t f o u n d no such signs in vivo (e.g. 77). However, a small (but statistically sign~ificant, p < 0.005) reduc,tion .of the axonal conduction v,elocitv of the m o t o r nerve was found by o u r group (31) which is in accord with the increase .o.f conduction time of 'the m o t o r nerve, observed by Mikhailov and Shvarts (47). In severe cases of h u m a n tetanus, nerve conduction was also f o u n d affected (39). EFFECTS ON MONOSYNAPTIC R E F L E X AND POSTSYNAPTIC I N H I B I T I O N OF T H E SPINAL M,OTONEURONE

Brooks, Curtis and E ccles (7) r e p o r t e d no virtual change in the m o n o s y n a p t i c reflex up to 43 h after toxin injection into the spinal cord (0.4 or 10 mouse MLD) or into the sciatic nerve 7×106 mouse MLD of the cat. They investigated five types of p o s t s y n a p t i c inhibition of the motoneurone and f o u n d that all of them were blocked by tetanus toxin. There are several other studies in s u p p o r t of this statement (e.g. 79, 21). I n these studies the well k n o w n hypothesis was est,ablished t h a t the .clinical picture, namely a Lgeneral hyperactivi.ty .o,f the m o t o r system, reflects a disinhibition within the spinal cord. However, Sverdlov (58) r e p o r t e d depression of the monosynap,tic reflex in the later preriod of intoxication. Similar findings were obtained by Mikhailov and Shvarts (47) with single motoneutones. These russian studies remained unnoticed by some reviewers (.e.g. 44, 5) or were misinterpreted (80). Results of our group (68, 71) clearly showed partial or total depression of the m o n o s y n a p t i c

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Takano K.

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reflex after the injection of toxin into the triceps surae .muscle of the cat at doses of 1 m o u s e MLD/kg to 10,000 m o u s e MLD/kg. At the time when the signs of local tetanus .could be .observed we ,could always see the depression o.f the monosynaptic 'reflex. We could never failed to r e c o r d responses of alpha motoneuro.nes 'in experiments concerned with R e n s h a w cells, w h e n a n t i d r o m i c stimulation of the m o t o r nerve was p e r f o r m e d (1, 33). This fact showed that the blocking effect of the toxin was not directed on the mo.toneuronM soma b u t r a t h e r on its .excitatory synapses. To s u p p o r t these findings K a n d a and Takano r e c o r d e d I a I P S P s and EPSPs f r o m micro.recorded indivfdual alpha m o t o n e u r o n e s (31). Tetanus toxin at the dose o,f 100 m o u s e MLD/kg ( c o r r e s p o n d i n g to 0.0'5 cat MLD/kg) was injected into the medial gastrocnemius of the cat. I,aIPSPs could only be prodtlced up to 30 h after toxin .injection, thereafter they had disappeared. I a E P S P s were depressed not earlier than 4 days after the injection. Bigalke (3) a n d Bergey et al. (2) f o u n d similar .effects of tetanus toxin on 'the cultured cell. They observed also that the blocking of IPSPs was followed by that of the EPSPs. H e t e r o n y m o u s EPSPs .remained n o r m a l when the nerve to the lateral gastrocnemius and soleus muscle was sectioned just before the toxin injection. This finding m i g h t be explained bv suggesting that tetanus toxin acting on the EPSP does not a s c e n d in m o t o r axons but in ,sensory fi,bres. Wiegand and Wellh6ner (78) :as well as K a n d a and Takano ,(31), have investigated m o t o n e u r o n e s in the period of local tetanus, induced by intram u s c u l a r injection o.f small doses of tetanus toxin. While the rigidity was evident, they did not observe any change in the electrical pro,perties of mo,toneurones, such as resting potential, after hyperp.olarisalion, m e m b r a n e resistance. However, these were changed in the cultured cell (11).

S p i n a l i n t e r n e u r o n e s . - The facilitation o.f the polysynaptic reflexes indicates hyperactivity of various interneurones. There are no reports about the facilitation of particular interneurones, except for the Renshaw cell and the Ia inhibitory interneurone .( 1 ). When the micropipette .electrode was iffserted into the spinal .cord to r e c o r d activities o.f motoneur.ones (31, 32) or Renshaw cells (1, 33) a highly increased spontaneous activity of undefined interneurones was frequently observed in the int,oxicated animal, as c o m p a r e d to the nonintoxicated one. First observations about 'the Renshaw cell during tetanus intoxication were made by Brooks et al. (7). They r e p o r t e d that the ,toxin prevented the r e c u r r e n t inhibitory action of the Renshaw cell on the m o t o n e u r o n e , w i t h o u t altering the response of the Kenshaw cells themselves. Curtis and de Groat (8) demons'trated that tetanus toxin reduced the a m o u n t of glycine, released f r o m inhibitory p r e s y n a p t i c terminals. Benecke et al. (1), showed that the response to a n t i d r o m i c m o t o r nerve stimulation as well as the spontaneous activity o.f the Renshaw cell increased during the development of local .tetanus. Kircbn.er and Takano (33) observed .the Renshaw cell activity for longer times after the intramuscular injection of the toxin. The a n t i d r o m i c responses as well as spontaneous activity of the Renshaw cell r e t u r n e d to n o r m a l levels after a while. The m u t u a l inhibition of Renshaw ceils, however, disappeared in the local tetanus. In c o n t r a s t to the fact that the toxin can block the endplate, the .cholinergic syn,apses on the Renshaw cell were always left intact in all studies .on this subject (7, 1, 33). Wellh6ner (73) w r o t e that the activation by tetanus toxin of spinal c o r d functions m a y not be due to an effect .on m o t o n e u r o n e s but on interneurones adjacent to them. If so, one might be able to prevent the spinal ,effects o~f the toxin b y intrathecal ~injection of antitoxin. This has been achieved by E r d m a n n et al..(18).

POLYSYNAPTIC REFLEXES AND SPINAL INTERNEURONES

PRESYNAPTIC

P o I y s y n a p t i c reflex. - A n u m b e r of authors observed great facilitations .of polysynaptic retiexes in the period when the spontaneous activity of the muscle was increased after the intramuscular o r i n t r a n e u r o n . a l injection of the ~toxin (e.g. 7, 10, 79). The potysynaptic reflexes were nociceptive reflexes a n d / o r of undefined nature, since these authors recorded f r o m whole ventral roots consisting of nerve fibres to flexor as well as extensor muscles. We have d e m o n s t r a t e d (64) that the proprioceptive stretch reflex as wel,1 as non-proprio,ceptive reflexes are facilitated during tetanus.

INHIBITION

The depolarisation of p r i m a r y afferent fibres t h r o u g h axo-axonal synapses reduces the amplitude of their terminal action potentials, .resulting in .a decrease .of t r a n s m i t t e r release f r o m the presynaptic terminals on motoneurones. Consequently the excitatory p o s t s y n a p t i c potential of the m o t o n e u r o n e is depressed. This action is k n o w n as presynaptic ,inhibition. The electronic spread of depolarizati,on of the p r i m a r y afferent endings can be recorded f r o m the dorsal root surface of the spinal cord, which is called ,, .dorsal root potential ,,. Presynaptic inhibition ,is .characterized by its ,delayed appearance ,and prolonged duration

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(for f u r t h e r i n f o r m a t i o n see textbooks of neurophysiology). Sverdloaz a n d Aleks.eeva (59) r e p o r t e d that app a r e n t p r e s y n a p t i c inhibition of gastrocnemius ,motoneurones was decreased in local tetanus after toxin injection at doses of 500-1000 mo.use MLD/kg into the g a s t r o c n e m i u s muscle, but that ,the dorsal roo.t potential was not changed. Curtis and his coworkers (9) f o u n d neither dorsal root potentials n o r presynaptic inhibition nine hours after they had injected tetanus toxin at doses .of 6000 m o u s e MLD/kg tinto the lateral aspect of the ventral h o r n at $1-$2 junction of the cat. On the other h a n d K r y z h a n o v s k y and Lutsenko (37) f o u n d an increase of the dorsal root potential after i n t r a m u s c u l a r injecti,on of toxin at the dose of 5 rat MLD. We, too, observed that the presynaptic inhibition was prolonged up to 2000 ms or m o r e u n d e r the toxin action. T,o study presynaptic inhibition, we ,i,njected tetanus toxin ,at doses of 2-2000 m o u s e MLD/kg into the gastrocnemius of the left bind leg of the cat. Acute experiments were p e r f o r m e d at various later times when the intoxicated hind leg was strongly extended. An inhibition of p r e s u m e d pvesynaptic ,type of the m o n o s y n a p t i c reflex of gastrocnemius m o t o n e u r o n e s was elicited by single electrical stimuli to the antagonistic deep peroneal nerve. I n d e p e n d e n t of the doses used, the typical long lasting inhibition (up to 2000 m s ) c o u l d be observed as long as the m o n o s y n a p t i c reflex could be r e c o r d e d (71). Considering the different m e t h o d s o.f toxin application in the experiments of the groups on presynaptic .inhibit:ion, we come to the foil.owing conclusions: Tetanus toxin at a high local concentration blocks or reduces b o t h the p r e s y n a p t i c inhibition a n d the dorsal root potential, p r o b a b l y by depressing the t r a n s m i t t e r release at the axoaxonal Synapses as shown in .the study of Curtis et ,al. (9). When the local c o n c e n t r a t i o n is considerably lower, like in the studies of S~¢erdlov and Alekseeva (59), .of K r y z h a n o v s k y and Lutsenko (37) and of ,ours ,(71), the m e c h a n i s m ,of presynap,tic inh,ib.ition seems to remain intact, or m i g h t even be a u g m e n t e d for so,me time. In h u m a n ~e.tanus intoxication, which comm o n l y shows s y m p t o m s of general tetanus, the toxin c o n c e n t r a t i o n within the spinal segments is far lower than can be achieved in most experiments with local tetanus. Therefore it ~s highly probable that p r e s y n a p t i c inhibition is left intact in clinical tetanus. GENERAL (,OR GENERALIZED) TETANUS AFTER TOXIN INJECTION

Though the experimental generalized tetanus after intravenous injection of tetanus toxin, ,is a

better s.imulation of the clinical tetanus than the experimental local tetanus, there .are only few neurophysiologieal experiments on the m o t o r system after i.v. injection of the toxin. H a b e r m a n n and Dimpfel (25) injected 12sIlabelled tetanus toxin i.v. in rats. After a few hours they could find some radioactivity in the brain stem and spinal cord, but not in the forebrain and cerebellum. Less than 1% of the radioactivity injected was fo.und in the structures of central nervous system. They concluded that the blood-brain b a r r i e r was practically impermeable for tetanus toxin. After m o r e detailed study Erdm a n n and H a b e r m a n n (1.7) pushed f o r w a r d s the hypothesis that generalized tetanus is a multiple local tetanus. When Huck et al. (29) injected tetanus toxin ( 2 x 1 0 s m o u s e LD~0)i.v. in rabbits, a r h y t h m i c electrical activity was recorded in the cerebellum and in the spinal cord. More than 50% of alphaand g a m m a - m o t o n e u r o n e s to the extensor muscle and o,f Renshaw cells in the L 7 spinal segment discharged =in correlation with the :cerebellum waves. Cooling of the surface of cerebellum suppressed the r h y t h m i c activity in the cerebellum as well as in the spinal cord. After spinal transection at T 2 the r h y t h m i c .activity could be r e c o r d e d in the cerebellum but no longer in the spinal cord. F r o m these results we concluded that ~h.e main source of the r h y t h m i c hyperactivity o.f the spinal cord, as observed in the generalized tetanus, lies no,t in the spinal c o r d itself but in the supraspinal structures, possibly in the brain stem. In severe cases o,f general tetanus of the cat we could totally inhibit the ,stretch reflex using severa,1 types .of postsynaptic inhibition .(e.g. an~tagonistic and Ib .inhibition) as well as presynaptic tinhib.ition (69). Also the m u t u a l inhibition o,f Renshaw cells in rabbits could always be observed during this stage in contrast to local tetanus. (Kirchner, unpubl,ished observation). In view o.f these results a critical reconsideration of the hypothesis that the general tetanus is ran :integration of the multiple local tetanus is needed. R e m e m b e r that b o t h types of synapses can be blocked in local tetanus when toxin dose is low.

GAMMA MOTOR SYSTEM

The ~importance of the g a m m a m o t o r system in the m o t o r control functions is well establ, ish.ed (see text books of neurop.hysio!ogy). The possible contri,bution of the g a m m a m o t o r ,system ,in tetanus :disease was discussed by m a n y reviewers. My point of view, based on a series of specific experiments, is that the g a m m a m o t o r system plays an ,important role in clinical as well as in refevant experimental tetanus. One m a y be remin-

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ded that the first t h e r a p e u t i c a l choice of m o s t physicians in .cases of tetanus is the a p p l i c a t i o n of d i a z e p a m or o t h e r b.enzodiazepines (.e.g. 40) which act p a r t i c u l a r l y on the g a m m a s y s t e m (70). For f u r t h e r details see a s e p a r a t e review special on that subject which a p p e a r s e l s e w h e r e (65).

ACTION OF TETANUS TOXIN INJECTED INTO THE BRAIN

Many a u t h o r s have injected the toxin directly at definite sites of the brain. I n m o s t o.f these studies a b n o r m a l m o t o r activities like seizures and forced turning m o v e m e n t s could be observed. WellhSn.er (73) has p r e s e n t e d an extensive table of the effects o b t a i n e d in such studies. PRESYNAPTIC ACTION OF TETANUS TOXIN

Many a u t h o r s have suggested or d e m o n s t r a ted that the action of tetanus toxin .on the endplate is located p r e s y n a p t i c a l l y (see for review 73). T e t a n u s ,toxin can b l o c k the n e u r o m u s c u l a r transmission b u t does not block the r e c e p t o r s for the t r a n s m i t * e r at the .postsynaptic .membrane. The thesis of p r e s y n a p t i c action of the toxin on the e n d p l a t e was s u p p o r t e d by m o r p h o l o g i c a l studies (50, 76). Curtis a n d de G r o a t (8) f o u n d t h a t tetanus toxin blocks the synaptic inhibition by the Renshaw cell w i t h o u t affecting the inhibitory action of glycine. Glycine is a s s u m e d to be the t r a n s m i t ter o,f the inhibitory synapses f o r m e d b y the Rens h a w cell at m o t o n e u r o n e s as well .as in o t h e r types of p o s t s y n a p t i c ,inhibition. They suggested t h a t t e t a n u s toxin exerts its action by reducing the a m o u n t of glycine released f r o m the inhibitory terminals. F u r t h e r Curtis et al. (9) d e m o n s ' t r a t e d t h a t the inhibitory action of the P u r k i n j e cell was totally d e p r e s s e d by tetanus toxin. The P u r k i n j e .cell could be d e p r e s s e d by electrophoretic application .of GABA. This s t u d y also suggests that the toxin dimin, ished the synaptic release o.f GABA r a t h e r t h a n 'its p o s t s y n a p t i c .effect. The s a m e conclusion was m a d e by Gushkin et al. (23), w i t h respect to the m o t o n e u r o n e .

to elucidate the pathogenesis and t h e r a p y of tetanus. In m o s t cases of m o d e r n .animal experiments, the local c o n c e n t r a t i o n of tetanus tox,in at the investigated site was far higher t h a n in the case o.f the .clinical disease. Let us r e t u r n to the principal question: W h a t is the ,origine o.f the h y p e r a c t i v i t y of the m o t o r s y s t e m in tetanus a p p e a r i n g as convulsions and rigidi.ty? The e x p e r i m e n t a l finding t h a t the moton e u r o n e s of the spinal c o r d .or o t h e r nerve cells in the central nervous s y s t e m are ,disinhibited in the early period a f t e r local injection of toxin, was e x t r a p o l a t e d to the situation in clinical tetanus. Under the .assumption that the general tetanus m i g h t be a multiple local tetanus, one t r i e d to explain the prevailing h y p e r a c t i v i t y b y an overall general disinhibition of all p a r t s of m o t o r system. However, we have seen t h a t in the later period of 1.ocal tetanus the excitatory .transmission to mo.toneurones is also d e p r e s s e d at low toxin doses, the endplate can be blocked and the muscle itself r e m a i n s in a rigid state w i t h o u t any electrical ,activity .(contracture). Therefore, the disinhibition of the spinal m o t o n e u r 0 n e s ~is obviously p r e d o m i n a n t only in the early d e v e l o p m e n t of ,the iocM tetanus as already described. We ,couM also .observe the intact functioning .many types of inhibition in the spinal ,cord u n d e r general tetanus. T,o m a i n t a i n the state which causes co,n~vulsions a n d rigidity, excitation as well ,as inhibition of the m o t o n e u r o n e s m u s t be r a t h e r intact. I t could be suggested t h a t the .origin of general m o t o r h y p e r a c t i v i t y lies in higher central n e r v o u s structures, including s u p r a s p i n a l g a m m a mo.tor ,activation, and not in the spinal cord. I t is an open question w h e t h e r such higher centres a r e solely responsible for the state of general tetanus, or w e t h e r ~it also needs s o m e s u p p l e m e n t a r y actions of the toxin at the spinal m o t o r level.

GENERAL DISCUSSION

Discrepancies of the results of various investigations r e p o r t e d ,above, seem to be due m o s t l y to different toxin applications and o t h e r variations of e x p e r i m e n t a l conditions (e.g. animal species, t i m e a f t e r injection). H a b e r m a n n (24), as well as o u r group (67), have distinguished two types .of ,experimentation: one a i m e d at the m o d e .of action of the toxin w i t h o u t being i n t e r e s t e d in the clinical relevance, the o t h e r was designed

~eknowledgement This study is dedicated to Prof. Dr. H.D. Henatsch on occasion of his 65th aniversary. The author thanks Prof Dr. H.D. Henatsch and Dr. F. Kirchner for reading the manuscript. 198

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16. Ebisawa I. and Matsukura M. (1968): P u l m o n a r y and m u s c u l a r changes in tetanus. - Jpn. J. Exp. Med. 38, 27-36.

R E F E R E N C E S

1. Benecke R., Takano K., Schmidt J. and Henatsch H.D. (1977): Tetanus toxin induced actions on spi-

17. Erdman,z G. and Itabermann E. (1977): autoradiograp,hy of central nervous system with generalized tetanus due to Lzq-toxin. nyn-Schmiedeberg's Arch. Pharmacol. 301,

nal Renshaw cells and Ia-inhibitory i n t e r n e u r o n e s during d e v e l o p m e n t of local tetanus in the c a t . Exp. Brain Res. 27, 271-286.

2. Bergey G., Bigalke H. and Nelson P. (1982): Diffe-

18. E r d m a n n G., Hanauske A. and Wellh6ner H.H. (1981): Intraspinal distribution and reaction in the grey m a t t e r with tetanus toxin of intracisternally injected anti-tetanus-toxoid F(ab)2 fragments. - Brain Res. 211, 367-377.

rential effects of tetanus toxin on inhibitory and excitatory synaptic t r a n s m i s s i o n in m a m m a l i a n spinal cord neurons in culture: A p r a e s y n a p t i c locus of action. - Neurology 32, A72.

3. Bigalke H. (1984): Tetanus.. und Botulinum-A-Neu,

19. E r d m a n n G., Wiegand H. and Wellh6ner H.H. (1975): intcaaxonal and extraaxonal t r a n s p o r t of ~25I-tetanus toxin in early local tetanus. - NaunynS c h m i e d e b e r g ' s Arch. Pharmacol. 290, 357-373.

rotoxin: Wirkungen an inhibitorischen und excitatorischen T r a n s m i t t e r s y s t e m e n Habilitationsschrift H u m a n m e d i z i n , Justus-Liebig-Universitgt, Giessen.

4. Bizzini B. (1979): Tetanus toxin, - Microbiol, Rev. 43, 224-240. 5. Bizzini B. (1976): Tetanus toxin s t r u c t u r e as a basic for elucidating its i m m u n o l o g i c a l and neuropharmacological activities. In: The Specificity and Action of Animal, Bacterial and Plant Toxins (Re,ceptors and Recognition Ser. B, Vol. 1), P. Cuatrecasas (Ed.), C h a p m a n n and Hall, London, 177-218.

6. Brooks V.B. and Asanuma H. (19,62): Action of tetanus toxin in the cerebral cortex. - Science 137, 674-676.

7. Brooks V.B., Curtis D.R. and Eccles J.C. (1957): The action of tetanus toxin on the inhibition of motoneurones. - J. Physiol. (Lond.) 135, 655-672.

8. Curtis D.R. and De Groat W.C. (1968): Tetanus toxin and spinal inhibit:ion. - Brain Res. 10, 208-212. 9. Curtis D.R., Felix D., Game C.J.A. and Mc Culloch R.M. (1973): Tetanus toxin and the synaptic release of GABA. - Brain Res. 51, 358-362. 10. Davies J.R., Morgan R,S., Wrigth E.A. and Wright G.P. (1954): Effect of local tetanus intoxication on the hind l~imb reflexes of the rabbit. - Arch. int. Physiol. 62, 248-263.

Histoin rats - Nau135-138.

20. Fedinec A.A. (1967): Absorption and distribution of tetanus toxin in e x p e r i m e n t a l animals. In: Principles of Tetanus. L. E c k m a n n (Ed.), ttuber, Bern, 169-176. 21. Geinisman

Y.Y., D'yakanova V.N. and Kryzhanovsky G.N. (1967): Effect of tetanus toxin on

morphological and functional state of synapses on spinal m o t o n e u r o n s . - Bull. Exp. Biol. Med. 64, 1203-1206. 22. Green J., E r d m a n n G. and Wellh6ner H.It. (1977): Is there r e t r o g r a d e axonal t r a n s p o r t of tetanus toxin in b o t h alpha and g a m m a fibers? - N a t u r e 265, 370. 23. Gushchin I.S., Kozhechkin S.N. and Sverdlov Y.S. (1969): On the presynaptic n a t u r e of the suppression of postsynaptic inhibition by tetanic toxin. Dokl. Biol. Sci. Proc. Nat. Acad. Sci. 187, 685-688. 24. Habermann E. (1978): Tetanus. In: Infections of the nervous system. Part I. P.J. Vinken and G.W. Bruyn (Eds.), ( H a n d b o o k of clinical neurology. Vol. 33), N o r t h Holland Publ. A m s t e r d a m , New York, Oxford, 491-547.

25. Habermann E. and Dimp[el W. (1973): Distribution of 12q-tetanus toxin and *esI-tetanus toxoid in rats with generalized tetanus, as influenced by antitoxin. - Naunyn-Sohmiedeberg's Arch. Pharmacol. 276, 361-373.

i1. Dimpfel W. (1980): Rat nerve cultures in pharmacology and t0xicolog-y. - Arch. Toxicol. 44, 55-62. 12. Dimpfel W., Huang R.T.C. and Habermann E. (1977): Gangliosides tin nervous tissue cultures and binding of laSI-labeled tetanus toxin, a neuronal marker. - J. N e u r o c h e m . 29, 329-334.

26. Hensel B., Seib U.C. and WellhSner H.H. (1973): Vagal ascent and distribution of 12"~I-tetanus toxin after injection into the a n t e r i o r wall of the stomach. - Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 396-402.

13. Duchen L.W. (1973): The effects of tetanus toxin on t,he m o t o r end-plates of the mouse. An electron m i c r o s c o p y study. -, J. neurol. Sci. 19, 153-167.

27. Heyningen van W.E. (1961): The fixation of tetanus toxin by ganglioside. - J. Gen. Microbiol. 24, 107-119.

14. Duchen L.W. (1973): The local effects of tetanus toxin on the electron m~croscopic s t r u c t u r e of skeletal muscle fibres of the mouse. - J. neurol. Sci. 19, 169-177.

28. Heyningen van W.E. and Mellanby J. (1968): The

15. Duchen L.W. and Tonge D.A. (1973): The effects of tetanus toxin on n e u r o m u s c u l a r t r a n s m i s s i o n and on the mo.rphology of m o t o r end-plates in slow and fast skeletal muscle of the mouse. J. Physiol. (Lond.) 228, 157-172.

effect of cerebroside and other lipids on the fixation of tetanus toxin by gangli,osides. - J. Gen. Microbiol. 52, 447-454.

29. Huck S., Kirchner F. and Takano K. (1981): Rhyt-

199

mic activity in the c e r e b e l l u m and spinal cord of rabbits receiving tetanus toxin intramuscu-

Takano K.

Eur. J. Epidemiol.

larly. - Naunyn-Schmiedeberg's Aroh. Pharmacol.

317, 51-53. 30. Huet de la Four E., Tardieu C., Tabary J.C. and Tabary C. (1979): Decrease of muscle extensibi!ity

31.

32.

33.

34.

and reduction of sacomere n u m b e r in so lues muscle following a local injection of tetanus toxin. J. Neurol. Sci. 40, 123-131. Kanda K. and Takano K. (1983): Effect of tetanus toxin o n the excitatory and inhibitory post-synaptic-pobentials i n the cat motoneurones. - J. Physiol. (Lond.) 335, 319-333. Kanda K. and Takano K. (1984): An evidence for anterograde t r a n s p o r t of t e t a n u s toxin. In: Abstr. of the 7th I n t e r n a t . Conf. o n Tetanus, G. Nistico, M.C. Strongoli (Eds.), Copanello, 79. Kirchner F. and Takano K. (1977): The activity of the Renshaw cell in the later period of local tetanus. In: Abstr. X X V I I t h I n t e r n a t . Cong. Physiol. S ci. 8, 388. Kretzschmar H., Kirchner F. and Takano K. (1980): Relations between the effect of tetanus toxin on the n e u r o m u s c u l a r t r a n s m i s s i o n and histological funct,ional properties of various muscles of the rat. - Exp. Brain Res. 38, 181-187.

35. Kryzhanovsky G.N. (1967): The n e u r a l p a t h w a y of toxin: its t r a n s p o r t to the central nervous system and the state of the spinal reflex a p p a r a t u s in tetanus intoxication. In: Principles of tetanus. L. E c k m a n n , (Ed.), H. Huber, Bern, Stuttgart, 155~168. 36. Ktyzhanovsky G.N. (1981): Pathophysiology. In: Tetanus New Concepts. R. Veronesi (E,d.), Excerpta Medica, Amsterdam-Oxford-Princeton, 109-182. 37. Kryzhanovsky G.N. and Lutsenko V.K. (1969): Dorsal root potential of the spinal cord in rats wi,th convulsions due to ascending tetanus. - Bull. exp. Biol. Med. 67, 16-19. 38. Liljestrand G. and Magnus R. (19'19): L~ber die Wirkung des Novokains auf den n o r m a l e n u n d den t e t a n u s s t a r r e n Skelettmuskel u n d tiber die E n t s t e h u n g der lokalen MuskelstaFre beim Wunds t a r r k r a m p f . - Pfl/_igers Arch. 176, 168-208. 39. Malek P., Kole J. and Zak F. (1957): Zur Pathogenese u n d der experimentellen Therapie des Tetanus: /.iber die M6gI,ichkeit der ~ specifischen Blockade ,, des l y m p h a t i s c h e n System. - Zbl. Ba,kt. I. Abt. Orig. 69, 233. 40. Manik Shahani F.D., Dastur D.H. and Dastoor V.P. (1979): Neuropathy in tetanus: - J. Neurol. Sci., 43, 183. 41. Matsuda M. (1984): Tetanustoxin. In: Protein T ox,in. Ishiyaku-Shuppan, Tokyo, 233-265 (in Japanese). 42. Matsuda M., Sugimoto N., Ozutsumi K. and Hirai T. (1982): Acute botulin,um-like intoxication by tetanus n e u r o t o x i n in mice. - Biochem. Biophys, Res. C o m m u n . 104, 799-805.

43. Matsuda M., Sugimoto N. and Ozutsumi K. (1982): Acute b o t u l i n u m like intoxication by tetanus in mice and the localization of the acute toxicity in the N-terminal papain f r a g m e n t of the toxin. In: 6th I n t e r n a t . Conf. on Tetanus. F o u n d a t i o n Marcel Merieux, 21-32. 44. MelIanby J. and Green J. (1981): Commentary. How does tetanus act? - Neudosci. 6, 281-300. 45. Mellanby J. and Whittaker V.P. (1968): The fixation of tetanus toxin by synaptic m e m b r a n e s . J. Neurochem. 15, 205-208. 46. Meyer H. and R a n s o m F. (1903): U n t e r s u c h u n g e n tiber den T e t a n u s . - Naunyn-Schm~edeberg's Arch. Pharmacol. 49, 369-416. 47. Mikhailov V.V. and Shvarts I.L. (1969): M,icrophysiological analysis of electrical activity of spinal n e u r o n s of various types in experimental tetanus. Bull. exp. Biol. Med. 68, 1340-1342. 48. Miyasaki S., Okada K., Muto S., I tokazu T., Matsui M., Ebisawa I., Kabage K. and Kimuro T. (1967): On the mode of action of tetanus toxin i n rabbit. I. D i s t r i b u t i o n of tetanus toxin i n vivo and devel o p m e n t of paralytic signs u n d e r some conditions. - Jpn. J. Exp. Med. 37, 217-225. 49. Mizote M. and Takano K. (1984): Two kinds of discharge p a t t e r n of p r i m a r y endings to FM muscle vibration in the t etan~c cat. In: Abstr. Sympos. ,, The m a m m a l i a n muscle spindle ,,, Glasgow, C 44. 50. Price D.L., Griffin J.W. and Peck K. (1977): Tetanus evidence for b i n d i n g at presynaptic nerve endings. Brain Res. 121, 379-384. 51. Price D.L., Griffin J.W., Young A., Peck K. and Stocks A. (1975): Tetanus toxin: direct evidence for retrograde intraaxonal transport. - Science 188, 945-947. 52. Ranson S W . (1928): Local tetanus - a study of muscle tonus and contracture. - Arch. Neurol. Phychiat. 20, 663-70,1. 53. R a n s o m F. (1901): D~e Verteilung yon Tetanusgift u n d T e t a n u s a n t i t o x i n im lebenden thierischen KSrp e r . - Berl. Klin. Wschr. 38, 337. 54. Schaefer H. (1944): Weitere U n t e r s u c h u n g e n zum Mechanismus u n d zur Therapie des Wundstarrkrampfes. - Arch. exp. Path. Pharmak. 203, 59-84. -

55. Schwab M.E. and Thoenen H. (1976): Electron microscopic evidence for a trans-synaptic migration of t e t a n u s toxin in spinal cord m o t o n e u r o n s : an a u t o r a d i o g r a p h i c and m o r p h o m e t r i c study. Brain Res. 105, 213-227. 56. St6ckeI K., Schwab M. and Thoenen H. (1975): Comparison between the retrogarde axonal transport of nerve growth factor and tetanus toxin i n motor, sensory and adrenergic neurons. - Brain Res. 99, 1-16. 57. Sugimoto N., Matsuda M., Ohnuki Y., Nakayama T. and Imai K. (1982): N e u r o m u s c u l a r blocking in acutely tetanus intoxicated mice. - Biken J, 25, 21-28.

200

Vol. 1, 1985

Tetanus toxin: neurophysiological aspects

58. Sverdlov Y.S. (1960): The spinal cord reflex activity u n d e r local tetanus (electrophysiological study). - J. Physiol. USSR 46, 941-947.

m o t o n e u r o n e of the spinal cord. In: 6th Internat. Conf. on Tetanus. F o u n d a t i o n Marcel Merieux, 83-89.

59. Sverdlov Y.S. and AIe.~seeva V.I. (1966): Effect of tetanus toxin on presynaptic inhibition in the spinal c o r d . - Fed. Proc. (Transl. Suppl.) 25, 931-936. 60. Takano K. (1976): The effects of tetanus toxin on the extensor and flexor muscles of the leg of the cat. In: Animal, P l a n t and Microbial Toxins. A. Ohsaka, K. Hayashi and Y. Sawai (Eds.), Plenum Press, New York, 363-378. 61. Takano K. (1976): Local t e t a n i s m a tool for understanding the stretch reflex. In: Progress in Brain Res. 44, <, Understanding the stretct reflex ,,, S. H o m m a (Ed.), Elsevier A m s t e r d a m , 491-502.

62. Tal
K.

(1980): Tetanus toxin. Advances in Neurolog. Sci. 24, 919-931 (in Japanese).

70. Takano K. and S t u d e n t J.C. (1978): Effect of diazepam on the g a m m a m o t o r system indicated by responses of the muscle spindle of the triceps surae muscle of the d e c e r e b r a t e .cat to the muscle stretch. - Naunyn-Schmiedeberg's Arch. Pharmacol. 302, 91-10'1. 71. Tiebert B., Terhaar P., Kirchner F. and Takano K. (!977) Presynaptic inhibition during local tetanus- Pfltigers Arch. 368, R 36. 72. W a s s e r m a n n A. arid T a k a k i I. (1898): Ober tetanusantitoxische Eigenschaften des n o r m a l e n Centralnervensystem. - Berl. klin. Wschr. 35, 5-6. 73. Wellh#ner H.H. (1982): Tetanus Neurotoxin. - Rev. Physiol. Biochem. Pharmacol. 93, 1-68.

63. 7akano

74. WellhiSner H.H., Hensel B. and Seib U.D. (1973): Local tetanus in cats: N e u r o p h a r m a c o g i n e t i c s of 12~I-tetanus toxin. - Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 375-386.

64. Takano K. (1984): An evidence for the polysynaptic p r o p r i o c e p t i v e reflex. In: Abstr. of the 7th Internat. Conf. on Tetanus, G. Nistico, M.C. Strongoli (Eds.), Copanello, 81.

75. WeIlh6ner H.H., Seib U.C. and Hensel B. (1973): Local tetanus in cats: The influence of neuromuscular activity on spinal d i s t r i b u t i o n of ~2aI-labelled tetanus toxin. - N a u n y m - S c h m i e d e b e r g ' s Arch. Pharmacol. 276, 387-394.

65. Takano K. (1985): Central and p e r i p h e r a l effects of tetanus toxin on the g a m m a m o t o r system - a review, in: 7th Internat. Conf. on Tetanus (in the press).

76. Wernig A., S t g v e r H. and Tonge D. (1977): The labelling of m o t o r end-plates in skeletal muscle of mice with a2~I-tetanus toMn. - Naunyn-Schmiedeberg's Arch. Pharmacol. 298, 37-42.

66. Takano K. and Henatsch H.-D. (1973): Tensionextension d i a g r a m of the tetanus intoxicated muscle of the c a r . - Naunyn-Schmiedeberg's Arch. Pharmacol. 276~ 421-436.

77. W e s t h u e s M. (1964): Untersuc'hungen an m a r k l o s e n N e r v e n f a s e r n tiber den Wirkungsort yon Botulinusund Tetan, ustoxin. - Naunyn-Schmiedebergs Arch. Pharmacol. 246, 309-315.

67. Takano K. and Kgrchner F. (1978): Discussion on the m e t h o d s of neurophysiological and neuropharmaco,logical survey in tetanus .- Abstr. 5th. Internat. Conf. on Tetanus, Ronneby.

78. Wiegand H. and WellhiSner H.H. (1979): Electrical excitability of m o t o n e u r o n e s in early local tetanus. - Naunyn-Schmiedeberg's Arch. Pharmacol. 308, 71-76.

68. Takano K., Kirchner F , Terhaar P. and Tiebert B. (1983): Effect of tetanus toxin o n the monosynaptic reflex. - Naunyn-Schmiedeberg's Arch. Pharmacol. 323, 217-220.

79. Wilson V.J., Diecke F.P.J. and Talbot W.H. (1960): Action of tetanus toxin on conditioning of spinal motoneurones. - J. Neurophysiol. 23, 65%666.

K. (1982): Commenta,ry: Red slow and pale fast muscles in tetanus. In: 6th Internat. Conf. on Tetanus. Foundation Marcel Merieux, 91-96.

69. Takano

K., Kanda K., Kirchner F., Terhaar P., Tiebert B. and Mizote M. (1982): Blocking effect of tetanus toxin on the excitatory synapse on the

80. Zacks S.L and S h e l f M.F. (1970): Tetanism: Pathological aspects of the action of tetanal toxin in the nervous system and skeletal muscle. - Neurosci Res. 3. 209-287.

201