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Intact inhibition of the stretch reflex during general tetanus
0041-0101 J91 ß.00 + .00 ® 1991 Pergrmao Prou pk
Toztoon Vol . 29, No . 2, pp . 2(11-209, 1991 . Printed in Great Britain .
INTACT INHIBITION OF THE STRETCH REFLEX DURING GENERAL TETANUS K. TAxnxo,l F. KmcxNERI and M. MIZOTE 2 rAbteilung Pathoneurophysiologie, Universität Gôttingen, Hltmboldtallee 23, D-3400 Gôttingen, Germany, and ~Teikyo University of Technology, 2289 Uruido, Ichihara, Chiba 29U-O1, Japan (Received 25 April 1990; Accepted 18 July 1990)
K. TAxANO, F. Kmcl-n~rEtt and M. MizoTE. Intact inhibition of the stretch reflex during general tetanus . Toxicon 29, 201-209, 1991 .-Tetanus toxin at lethal doses (2-10 x 10 3 mouse minimal lethal doses per kg body weight, mMLD/kg) was injected i.v. into 10 cats under pentobarbital anaesthesia . After the appearance of the first sign of generalized tetanus the animal was anaesthetized by a mixture of urethane and chloralose . Experiments were performed several hr thereafter when the toxin action was anticipated to be optimal. The stretch reflexes were elicited manually, by the contraction of the antagonistic muscles or by a stretch device . In toxin treated animals the spontaneous electromyographic activity was inhibited by strong stretching of the tested muscle or by that of the antagonistic muscle. The stretch reflex of the extensor muscle elicited by a contraction of the flexor muscle was inhibited by electrical stimulation of the flexor afferent fibres . The stretch reflex elicited by a stretch device as well as the electrically elicited monosynaptic reflex were inhibited by conditioning stimulation of the antagonistic nerve. The inhibition curves were almost the same as those of healthy animals. It is concluded that the spinal inhibitions, such as antagonistic group Ia, autogenic group Ib, groups II and III and the presynaptic inhibitions, were kept intact in severe general tetanus. INTRODUCTION THERE are many studies on the pathogenesis of tetanus. In most of these investigations
tetanus toxin was injected into the muscle, the peripheral nerve or into the spinal cord . Consequently the animal showed typical signs of a local tetanus. In such preparations Bxooxs et al. (1957) found a selective block of inhibition of the motoneurone without an alteration of the monosynaptic reflex . Further, CuxTrs et al. (1973) could also demonstrate the reduction of presynaptic inhibition of the spinal cord . In review articles (e .g. HABERMANN, 1978) and text books of neurology (e.g . STRUPPLER, 1987) it has been stated that the hyperactivity of the motor system of tetanus is caused by the general disinhibition of the nervous system or at least by the disinhibition of the motoneurone in the spinal cord . However, this may not be the case in clinical tetanus. Studies on local tetanus from our laboratory (TAKANO, 1976x; TAI{ANO et al., 1983) showed that there was no selective blocking of inhibitory synapses when toxin, at not very high doses, was injected into the muscle of the cat. We injected tetanus toxin into the gastrocnemius muscle of the cat at doses of 0.0005 to 5 cat minimal lethal doses. Even at 201
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0.001 minimal lethal dose we always observed local tetanus of the injected muscle and the inhibition of the monosynaptic reflex was blocked at the earlier period and the monosynaptic reflex itself was blocked at a later stage. This finding was supported by intracellular recording observing the change of the IPSP and the EPSP by KANDA and TAKANO (1983) until five days after the injection of tetanus toxin at the dose of 0.05 of a minimal lethal dose. B11tGEY et al. (1987) found similar effects of tetanus toxin on cultured cells. Recently we reported that even a fragment of tetanus toxin (Fragment [A-B]) blocked both types of synapses of the spinal motoneurone (TAKANO et al., 1989a) . OztrrsuMl et al. (1989) reported a `weakness' produced by injection of Fragment [A-B]. They differentiated `weakness' from flaccid paralysis which is caused by blocking of the endplate . The presynaptic, delayed and prolonged inhibition of the monosynaptic reflex in the cat with local tetanus was observed until the reflex faded and the underlying dorsal root potential was observed even after the monosynaptic reflex was blocked (TAKANO et al., 19896) . Thus the presynaptic inhibition in the spinal cord may be blocked only when the local toxin concentration is abnormally high, unlike the case of the natural disease (for further discussion see TAKAIVO et al., 19896) . A local tetanus also occurs more or less in clinical tetanus, but the most remarkable manifestation of the disease results in the generalized hyperactivity of muscles in the whole body, that is general tetanus. In most cases the first clinical sign of tetanus is lockjaw, regardless of the location of the infected wound. This fact indicates that the toxin produced in the wound by Clostridium tetani is transported by blood circulation into the muscles of the whole body, and is then transported via the nerves into the spinal cord or motor nuclei of the brain. The first symptom appears in the muscles like the masseter, which lie nearest to the central nervous system and/or have many muscle spindles (Tnxnxo and KANO, 1973 ; TAKANO and HENATSCH, 1973). When tetanus toxin is injected i.v. the animal shows a generalized hyperactivity of the muscles in the whole body. Hence, for simulation of clinical tetanus, the animal injected i.v. with the toxin is more relevant . To test the spinal inhibition in general tetanus, tetanus toxin at lethal doses was injected i.v. and the acute electrophysiological experiments were performed during periods of the maximal manifestation of the convulsion and tremor shortly before the death of animals. MATERIALS AND METHODS Cats under light sodium pentobarbital anaesthesia (25-30mg/kg i .p .) received 2000 (0 10,000 mouse minimal lethal doses per kg body weight (mMLD/kg) of tetanus toxin (T~, Hehringwerke, Marburg) by i.v. injection . One cat minimal lethal dose is approximately 2000 mMLD/kg . Extreme care was taken in the treatment of the animâl to avoid any kind of stimulations which may cause the death of the animal . When animals injected with tetanus toxin showed any small tetanus symptoms which are mostly some disquiet in their behaviour, and the slight extension of the forelimbs, a mixture of chloralose (40 mgng) and urethane (400 mgng) was injected i .p. Additional anaesthesia was given if necessary . Electrophysiological experiments began several hr after the beginning of the anaesthesia in expectation of the further spread of the toxin and the development of the disease. All animals diod between 68 and 120 hr after toxin injection . Four types of preparation were used. Type 1 preparation
For this there was no treatment other than insertion of the needle electrodes into the triceps surge muscle (extensor) for recording of the electromyogram (EMG) in the first part of the experiment. In the second part of the experiment the ankle tendons of the extensor (triceps surge) and the flexor muscles (tibialis anterior etc .) were cut for slow manual stretching. Two cats received 10'mMLD/kg of tetanus toxin . (see the scheme in Fig . 1) After a small skin incision a pair of stimulating electrodes were laid on the deep peroneal nerve (to the flexor muscles) . The animal was fixed at the hip and knee joints without surgical invasion . The foot could move freely Type 1 preparation
Intact Inhibition in General Tetanus
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2T
4T
3T
4T Without conditioning
10 ms
FIG. 1 . ARRANGEMENT OF THE EXPERIMENT (TYPE 2) AND THE STRETCH REFLEX RESPONSE THE CAT IN GENERAL TETANUS .
(EMG) OF
The triceps surge muscle was stretched by the contraction of the flexor muscle which was elicited by the electrical stimulation of the peroneal nerve at the popliteal fossa. The afferent impulses conditioned the motoneurones of the triceps surge muscle . 2T, 3T, 4T are stimuli strength two, three and four times the threshold of the motor nerve. At 2T and 3T the stretch reflex was depressed and at 4T the reflex was totally blocked in general tetanus. At the end of the experiment, the peroneal nerve was cut at the central side of the stimulation as indicated by the arrow in the scheme (4T without conditioning). Because of the slow rise time of the muscle contraction the group Ia input of the stretch reflex arrives at the motoneurone not in the effective period of the antagonistic Ia inhibition but only later. Therefore, the slight inhibition in `2T' does not mean that the inhibition was depressed. Cat, 40 hr after i.v. injection of tetanus toxin at lO~mMLD/kg . when the nerve to the muscle was stimulated . When the peroneal nerve was electrically stimulated (rectangular pulse with a duration of 0.2 rllsec and a stimulus strength two to four times of threshold of the motor nerve, 2T4T) the flexor muscles contracted and the extensor muscles were stretched by the flexor (ascent time ca 25 cosec according to the contraction time of the flexor muscles). The reflex response was recorded by needle electrodes inserted into the triceps surge muscle as in the Type 1 preparation (the same in Types 3 and 4) . Two cats received l0` or 5 x 10 3 mMLD/kg of toxin. Type 3 preparation (see the scheme in Fig. 2) In addition to the operation of the Type 2 preparation the tendon to the triceps surge muscle was cut. The muscle was freed from the surrounding tissue and connected with an electromagnetic stretch device . The zerolength of the triceps surge muscle was adjusted by extending it to a point, where the tension was 1 N. The muscle was stretched several mm from the zero-length with a triangular pulse (ascent time 2.5 cosec) . The peroneal nerve was cut and the central end was stimulated for the conditioning . The stretch apparatus was triggered by a delay circuit to stretch the muscle with variable delay time after the conditioning electrical stimulation of the peroneal nerve (rectangular pulse with a duration of 0.2 cosec at a stimulus strength 4T). Three cats received 10~, 5 x 10 3 or 2 x l03 mMLD/kg of toxin. Type 4 preparation With a deep anaesthesia (brief ether anaesthesia was used additionally if necessary) a laminectomy was performed. The monosynaptic reflex was recorded from a part of the central cut end of the ventral root of ~ and/or S, . The conditioning stimulus was given to the peroneal nerve and the testing stimulus for the
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,
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ARRANGEANT OF THE aYVme~AtFrrr (TYFE 3) AND THE STRETCH REFLEX RESPONSE THE CAT IN GENERAL TETANUS .
(EMG) OF
The triceps some muscle was stretched by a stretch device (rise time, 2.5 msec) triggered by the test pulse fT) at different times after electrical stimulation of the antagonistic peroneal nerve (conditioning stimulus, C). Responses of the stretch reflex (EMG) with and without conditioning stimulus were shown. The number between C and T shows the interval between conditioning stimulus and test pulse in cosec. Note the different time scales in a, b, c, d, and that the motoneurone is already inhibited by the electrical conditioning stimulus when the Ia reflex impulse elicited by the muscle stretch without delay (COT) arrives at the motoneurone. Cat, 70hr after i.v . injection of tetanus toxin at 2 x lO;mMLD/kg. monosynaptic reflex was applied to the tibial nerve (for further information about this type of preparation, see TAKANO et al., 1983) . Two cats received 5 x 10' or 2 x 10' mMLD/kg of toxin. For further details of the stimulation in each type of the experiments, see results . RESULTS
Inhibition of the continuous activity of the muscle
The Type 1 preparation was used for this experiment (see methods) . Acute experiments were performed two days after the injection of tetanus toxin at 10°mMLD/kg. The
Intact Inhibition in General Tetanus
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"
" "
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2 5 10 10 50 100 200 interrel between conditioning :tinulu: and test stretch ( ms ~
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FIG. 3 . INHIBITION CURVE OF 3TRETCH REFLIX DURING GENERAL TETANUS.
The stretch reflex was conditioned at different times before the test stimulation (abscissa in the logarithmic scale). The same ezperimcnt as in Fig. 2, but from another animal . Each point is the averaged amplitude of reflex responses of 10 trials . The amplitude of reflex responses without inhibition is 100% . Cat, SOhr after i.v . injection of tetanus toxin at 5 x 10'mMLD/kg .
extensor muscles but not the flexor in the hind leg (as in local tetanus; TAKANO, 1976b; Kx>rrrzscxMAx et al., 1980) showed a high amount of spontaneous activity in the EMG when the intoxicated animal was not too deeply anaesthetized. Occasionally a clonus (8-12 Hz) was clearly observed . The continuous activity became greater when the muscle was stretched slowly and maintained . The exaggerated stretch reflex was caused by increased gamma activity (TAxANO and Kntvo, 1973 ; TAxANO and HErlnTSCx, 1973; Hucx et al., 1981) and/or also due to the hypersensitivity of the alpha motoneurone. Such high activity of the muscle at maintained extension can neither be obtained in a deeply anaesthetized and intoxicated animal nor in the non-anaesthetized and non-intoxicated animal . When the muscle was stretched more strongly, the EMG activity was depressed. This inhibition may be caused by autogenic inhibition through group Ib afferent discharges from the Golgi tendon organs in the stretched muscle . The muscle activity could also be depressed by stretching the antagonistic flexor (e.g. tibialis anterior) muscles. This inhibition is caused by group Ia discharges from the muscle spindle and also group II and III discharges from other receptors of the antagonistic flexor muscles (for the EMG recordings see Fig. 3 in TAKANO and KIRCHNER, 1987). This rather simple experiment shows that autogenic as well as antagonistic inhibition remained intact in general tetanus. Inhibition of the stretch reflex induced by a sudden muscle stretch through contraction of the antagonistic muscle In this series of experiments the Type 2 preparation (see methods) was used for testing the antagonistic inhibition of the stretch reflex in response to sudden stretching . The peroneal nerve of the ipsilateral side was stimulated electrically by a single pulse. This stimulation served as an indirect stimulation of the flexor muscles and a concomitant stretching of the extensor triceps surae muscle . At the same time the afferent impulses elicited by the stimulation of the peroneal nerve conditioned (inhibited) the alphamotoneurone innervating the triceps surae muscle . At a stimulus strength two times the threshold of the muscle contraction (2T) almost all peroneal alpha motor nerve fibres were stimulated and, therefore, the maximal stretching of the triceps surae was achieved through maximal twitch of the flexor . The stretch reflex was induced and the electrical muscle response was recorded (Fig. 1, 2T). At 2T
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100
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20
50 ms 100
lntonal betrun condltioninp and tort atialulation :
FIG. 4. INHIBEI7ON CURVE OF THE LtON08YNAPTIC REFLEX DURING GENERAL TEEANUS .
The monosynaptic reflex was recorded from the cut end of the bundle from L, and S, ventral roots . The conditioning stimulus (4T) was given on the peroneal nerve at different times (abscissa in the logarithmic scale) before the test stimulation of the tibial nerve (2'I') to the gastrocnemius muscle . Each point is the averaged amplitude of reflex responses of 10 trials. The amplitude of reflex responses without inhibition is 100% . Cat, 100hr after i .v . injection of tetanus toxin 2 x 103 mMLD/kg .
stimulation the antagonistic Ia discharge inhibited the extensor motoneurones and the reflex response was depressed (smaller than in `4T without conditioning' in Fig. 1, see later) . At stronger stimulation, three or four times of threshold (3T or 4T), the smaller sensory fibres with higher threshold were also recruited and inhibited the extensor motoneurones more strongly . At the stimulus strength of 3T, the degree of muscle stretching cannot be smaller than the stimulation at 2T, (this is also visually controlled) but the reflex response was reduced strongly in amplitude (Fig. 1, 3T). The stretch reflex was totally depressed at the stimulus strength of 4T (Fig. 1, 4T). At the end of the experiment the peroneal nerve was cut proximal to the stimulated site, (as indicated by the arrow) . Now, the stretch reflex could no longer be inhibited (Fig. 1, 4T without conditioning). These experiments show that peroneal nerve stimulation causes a strong inhibition of the stretch reflex of the triceps surae muscle during a heavy tetanus intoxication . Relation between amplitude of the stretch reflex and interval between conditioning and test stimulations
In this series of experiments the Type 3 preparation was used (see methods) and the conditioning inhibitory stimulus on the peroneal nerve at 4T was given at different times before the short and rapid stretching of the triceps surae muscle. This was designed to obtain an approximate idea of the inhibition through which the stretch reflex was depressed (Fig. 2). The inhibition was strongest at the interval from 16 (C 16T) to 20 msec (C20T) resulting in almost total depression of the reflex . Notice that the group Ia impulse from the muscle spindle of the triceps surae has a handicap in reaching the motoneurone due to the time lag of the action potential generation in the muscle spindle. The inhibitory curve of Fig. 3 was obtained from the second animal of Type 3 preparation. The inhibition with maxima at ca 8 msec may be due to a complex postsynaptic inhibition by group I, II and III fibres . The delayed inhibition with its maximum at ca 80 msec may reflect the presynaptic inhibition . The delayed inhibition was not longer than that of the non-intoxicated animal, in contrast to local tetanus, where the presynaptic inhibition lasts longer (Tnlcnxo et al., 1989b) . This fact suggests that in generalized tetanus the spinal cord is scarcely affected by
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the toxin and therefore the local toxin concentration in the spinal cord is far lower than in local tetanus (see discussion). The inhibitory curve of the monosynaptic reflex
In the previous experiments we had gathered the necessary experience to treat the generalized tetanus animal carefully, allowing us to additionally perform a laminectomy in order to get an inhibition curve of the monosynaptic reflex (Fig . 4). We observed the early antagonistic Ia inhibition with its maximum at ca 1 .5 msec. The second delayed inhibition with maximum of ca 20 msec may be due to a combination of group II, III postsynaptic inhibitions and the presynaptic inhibition, resulting in complete inhibition of the extensor motoneurone. Taken together these experiments clearly show that several kinds of inhibition of the motoneurone of the extensor muscle remained intact in heavy generalized tetanus. DISCUSSION
Most authors working on local tetanus in the second half of this century used tetanus toxin at very high doses (e.g. Bxooxs et al., 1957). At such high doses tetanus toxin blocks at first the inhibitory synapses and thereafter the animal dies before tetanus toxin invades the excitatory synapses. In our foregoing studies on local tetanus, we used mainly sublethal doses. Two mouse MLD/kg (10 -' cat MLD/kg) is the critical dose for eliciting local tetanus and blocking excitatory transmission of the motoneurone. In clinical tetanus the toxin at an approximately lethal dose spreads over the whole body. Suppose that the death rate without any care is 60%; meaning that 60% of the patients have more than one lethal dose and 40% of them a sublethal dose. Therefore the doses in this study, 1-5 times of the minimal lethal dose, are clinically relevant doses or slightly higher . Yet the spinal inhibition remained intact . We did not observe any sign of a local tetanus. How about the local concentration in the spinal cord segment in our preparations? HABERMANN and DIMPFEL (1973) reported that about 0.5% of the toxin injected i.v. could be found in the spinal cord and brain stem (not in the forebrain and cerebellum). They assumed a toxin concentration of about 10- ' S M in the spinal cord when they injected the toxin i.v. at the LDSO dose. HABHRMANN (1972) calculated for local tetanus a higher effective toxin concentration (i.e. 2~ x 10- "M) in the lumbar spinal cord when he injected tetanus toxin into the gastrocnemius muscle . The local concentration in the spinal cord in general tetanus at doses in the same range as in our study cannot reach that of local tetanus. When tetanus toxin at a very high dose is injected i.v., the animal dies without showing any hyperactivity of the motor system (`botulinum-like intoxication' after MATSUDA et al ., 1982). Experimental tetanus showing such flaccid paralysis might not be a conventional `general tetanus'. In our laboratory it was observed that several kinds of spinal inhibition were left intaçt in the rabbit injected i .v. at 2x lOSmMLD (ca 100 MLD). From this point of view general tetanus has a different pathogenesis from that in local tetanus. HABERMANN and Dnlpr->rr. (1973) injected the toxin at less than an LDSO dose i.v. and `some animals (rats) died between the 6th and 15th day, and all other animals also developed moderate to. severe tetanus. These symptoms had their maximum at the 10th day, and disappeared completely, by day 19'. As HABERMANN (1972) wrote, the symptoms of local tetanus persist for a long time, in agreement with our observations that the
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shortening of the muscle (at early period hyperactivity and later contracture without activity, KlxcxxER et al., 1980) lasts for many weeks. Fully developed clinical tetanus is characterized by generalized hyperactivity of muscles of the whole body . This general tetanus has been regarded as the summation of multiple local tetanus (e.g. KRYZHANOVSKY, 1967; HABI?RMANN, 1978). This hypothesis is based on the disinhibition hypothesis of the hyperactivity of the motor system without changing the excitatory transmission . As already stated in the introduction the spinal disinhibition is only evident in the early period of local tetanus. Tetanus toxin also blocks thereafter excitatory transmission even at low doses that just cause local tetanus. As demonstrated in this study, many kinds of inhibition of the spinal motoneurone appear to be left intact in general tetanus. Thus in general tetanus the functional mechanisms of the spinal motor system are operative, whereas during a local tetanus both inhibitory and excitatory transmission are disturbed. General tetanus and local tetanus are independent, although there might be some intervention of the former with the latter. The generation of the hyperactivity in tetanus resides in the higher central nervous system (HucK et al ., 1981), and not in the spinal cord as once suggested by e.g. KRYZHANOVSKY (1967) . We propose that general tetanus is not the summation of local tetanus. Local tetanus .appears to be produced in the spinal cord, whereas general tetanus is caused by supraspinal hyperactivity . Acknowledgement.~The authors thank Dr R. PetvNea for reading the manuscript and Mrs G. FEUERKEIL for technical assistance . Tetanus toxin was kindly supplied by Behringwerke AG (Marburg) .
REFERENCES BBeoev, G. K., BIGALKE, H. and NELSON, P. G. (1987) Differential effects of tetanus toxin on inhibitory and excitatory synaptic transmission in mammalian spinal wrd neurons in culture: a presynaptic locus of action for tetanus toxin. J. Neurophysiol. 57, 121-I31. BROOKS, V. B., Cams, D. R. and ECCLES, J. C. (1957) The action of tetanus toxin on the inhibition of motoneurones. J. Physiol. 135, 655-672. C~1R173, D. R., FaLtx, D., G~t~, C. J. A. and MCCuLLOCx, R. M. (1973) Tetanus toxin and the synaptic release of GABA. Brain Res. 51, 358-362. H~ER~tvN, E. (1972) Distribution of'ZSI-tetanus toxin and'zs-toxoid in rats with local tetanus, as influenced by antitoxin . Naunyn-Schmiedebergs Arch. Pharmacol. 272, 75-88. HABERMANN, E. (1978) Tetanus. In Handbook ojClinical Neurology, Vol. 33, pp. 491-547 (VINKEN, P. J. and BRUVx, G. W., Eds) . Amsterdam: North Holland. H~eeR~rtx, E. and Dt~~eL, W. (1973) Distribution of'uI-tetanus toxin and'z3I-toxoid in rats with generalized tetanus, as influenced by antitoxin. Naunyn-Schmiedebergs Arch . Pharmacol. 276, 327-340. HuCK, S., KtRCfttvER, F. and Trtuxo, K. (1981) Rhythmic activity in the cerebellum and spinal cord of rabbits receiving tetanus toxin intravenously. Naunyn-Schmiedebergs Arch . Pharmacol. 317, 51-53. KANDA, K. and TAKANO, K. (1983) . Effect of tetanus toxin on the excitatory and inhibitory post-synaptic potentials in the cat motoneurone. J. Physiol., f ond. 335, 319-333. K~RCxxeR, F., KREfZSCHMAR, H. and Trtuxo, K. (1980) Study about the origin of contracturc of muscles in response to intramuscular injection of tetanus toxin. Proc . lUPS 14, 513. KRErzsc,~tastaR, H., KtRCt~txrae, F. and TAKANO, K. (1980) Relations between the effect of tetanus toxin on the neuromuscular transmission and histological functional properties of various muscles of the rat. Exp. Brain Res. 38, 181-187. KRYZHANOVSKY, G. N. (1967) The neural pathway of toxin: its transport to the central nervous system and the state of the spinal reflex apparatus in tetanus intoxication. In: Principles on Tetanus, pp . 155-168 (ECKMANN, L., Ed .) . Bern and Stuttgart : Hens Huber Publishers . MAISUDA, M., SUGIMOTO, N. and Ozu~rsuett, K. (1982) Acute botulinum like intoxication by tetanus toxin in mice and localization of the acute toxicity in the N-terminal-pepsin-fragment of the toxin. In : 6th /nternational Conjerence on Tetanus, pp . 2-37 . Lyon : Foundation Marcel Merieux.
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Oztnsustt, K., Dux-LuNß, L., Sußr~oro, N. and M~zsttnn, M. (1989) Isolation and purification by high performance liquid chromatography of a tetanus toxin fragment (Fragment [A-B]) derived from mildly pepsin-treated toxin. Toxicon 27, 1055-1057 . STRUPPLER, A. (1987) Das Nervensystem . In: Pathophysiologie~Pathobiochemie, pp. 193-258 (Lt.Nß, F., Ed .) . Stuttgart: Enke . T~NO, K. (1976x) Local tetanism a tool for understanding the stretch reflex . In Progress in Brain Res. 44, "Understanding the stretch reflex", pp . 491-502 (Hot~uu, S., Ed .). Amsterdam: Elsevier . T~tcwNO, K. (1976b) The effect of tetanus toxin on the extensor and flexor muscles on the hind leg of the cat. In : Animal, Plant and Microbial Toxins, Vol. 2, pp. 363-378 (OFÛAICA, A ., HAYA9FII, K. and $AWAI, Y., Eds) . London : Plenum Press. Tetuxo, K. and Hsrre~cH, H. D. (1973) Tension-extension diagram of the tetanus intoxicated muscle of the cat. Naunyn-Schmiedebergs Arch . Pharmacol. 276, 421 36. TAKANO, K. and KENO, M. (1973) . Gamma-bias of the muscle poisoned by tetanus toxin. Narxryn-Schmiedebergs Arch . Pharmaeol. 276, 413 20. TAKANO, K. and KtxCttxex, F. (1987) Pathogenesis of tetanus: clinically relevant new concepts . A mini review . In : Progress in venom and toxin research, pp . 68091 (GOPALAKRISHNAKONE, P. and TAN, C. K., Eds) . Singapore : National University . Tnruxo, K., KtxctrNm, F., Tr~x, P. and TtEar:ar, B. (1983) Effect of tetanus toxin on the monosynaptic reflex . Naunyn-Schmiedebergs Arch. Pharmacol. 323, 217-220. T~tceNO, K., KtxcHNm, F., GxzatstFa .r, A., MA~rsuDA, M., Ozu~tsun~t, N. and SußtMO~ro, N. (1989x) Blocking effects of tetanus toxin and its Fragment [A-B] on the excitatory and inhibitory synapses on the spinal motoneurone of the cat. Toxicon 27, 385-392 . TAICANO, K., KutcttN~e, F., T~tT, B. and TERIiAAR, P. (1989b) Presynaptic inhibition of the monosynaptic reflex during local tetanus in the cat. Toxicon 27, 431-438.
Toztoon Vol . 29, No . 2, pp . 2(11-209, 1991 . Printed in Great Britain .
INTACT INHIBITION OF THE STRETCH REFLEX DURING GENERAL TETANUS K. TAxnxo,l F. KmcxNERI and M. MIZOTE 2 rAbteilung Pathoneurophysiologie, Universität Gôttingen, Hltmboldtallee 23, D-3400 Gôttingen, Germany, and ~Teikyo University of Technology, 2289 Uruido, Ichihara, Chiba 29U-O1, Japan (Received 25 April 1990; Accepted 18 July 1990)
K. TAxANO, F. Kmcl-n~rEtt and M. MizoTE. Intact inhibition of the stretch reflex during general tetanus . Toxicon 29, 201-209, 1991 .-Tetanus toxin at lethal doses (2-10 x 10 3 mouse minimal lethal doses per kg body weight, mMLD/kg) was injected i.v. into 10 cats under pentobarbital anaesthesia . After the appearance of the first sign of generalized tetanus the animal was anaesthetized by a mixture of urethane and chloralose . Experiments were performed several hr thereafter when the toxin action was anticipated to be optimal. The stretch reflexes were elicited manually, by the contraction of the antagonistic muscles or by a stretch device . In toxin treated animals the spontaneous electromyographic activity was inhibited by strong stretching of the tested muscle or by that of the antagonistic muscle. The stretch reflex of the extensor muscle elicited by a contraction of the flexor muscle was inhibited by electrical stimulation of the flexor afferent fibres . The stretch reflex elicited by a stretch device as well as the electrically elicited monosynaptic reflex were inhibited by conditioning stimulation of the antagonistic nerve. The inhibition curves were almost the same as those of healthy animals. It is concluded that the spinal inhibitions, such as antagonistic group Ia, autogenic group Ib, groups II and III and the presynaptic inhibitions, were kept intact in severe general tetanus. INTRODUCTION THERE are many studies on the pathogenesis of tetanus. In most of these investigations
tetanus toxin was injected into the muscle, the peripheral nerve or into the spinal cord . Consequently the animal showed typical signs of a local tetanus. In such preparations Bxooxs et al. (1957) found a selective block of inhibition of the motoneurone without an alteration of the monosynaptic reflex . Further, CuxTrs et al. (1973) could also demonstrate the reduction of presynaptic inhibition of the spinal cord . In review articles (e .g. HABERMANN, 1978) and text books of neurology (e.g . STRUPPLER, 1987) it has been stated that the hyperactivity of the motor system of tetanus is caused by the general disinhibition of the nervous system or at least by the disinhibition of the motoneurone in the spinal cord . However, this may not be the case in clinical tetanus. Studies on local tetanus from our laboratory (TAKANO, 1976x; TAI{ANO et al., 1983) showed that there was no selective blocking of inhibitory synapses when toxin, at not very high doses, was injected into the muscle of the cat. We injected tetanus toxin into the gastrocnemius muscle of the cat at doses of 0.0005 to 5 cat minimal lethal doses. Even at 201
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0.001 minimal lethal dose we always observed local tetanus of the injected muscle and the inhibition of the monosynaptic reflex was blocked at the earlier period and the monosynaptic reflex itself was blocked at a later stage. This finding was supported by intracellular recording observing the change of the IPSP and the EPSP by KANDA and TAKANO (1983) until five days after the injection of tetanus toxin at the dose of 0.05 of a minimal lethal dose. B11tGEY et al. (1987) found similar effects of tetanus toxin on cultured cells. Recently we reported that even a fragment of tetanus toxin (Fragment [A-B]) blocked both types of synapses of the spinal motoneurone (TAKANO et al., 1989a) . OztrrsuMl et al. (1989) reported a `weakness' produced by injection of Fragment [A-B]. They differentiated `weakness' from flaccid paralysis which is caused by blocking of the endplate . The presynaptic, delayed and prolonged inhibition of the monosynaptic reflex in the cat with local tetanus was observed until the reflex faded and the underlying dorsal root potential was observed even after the monosynaptic reflex was blocked (TAKANO et al., 19896) . Thus the presynaptic inhibition in the spinal cord may be blocked only when the local toxin concentration is abnormally high, unlike the case of the natural disease (for further discussion see TAKAIVO et al., 19896) . A local tetanus also occurs more or less in clinical tetanus, but the most remarkable manifestation of the disease results in the generalized hyperactivity of muscles in the whole body, that is general tetanus. In most cases the first clinical sign of tetanus is lockjaw, regardless of the location of the infected wound. This fact indicates that the toxin produced in the wound by Clostridium tetani is transported by blood circulation into the muscles of the whole body, and is then transported via the nerves into the spinal cord or motor nuclei of the brain. The first symptom appears in the muscles like the masseter, which lie nearest to the central nervous system and/or have many muscle spindles (Tnxnxo and KANO, 1973 ; TAKANO and HENATSCH, 1973). When tetanus toxin is injected i.v. the animal shows a generalized hyperactivity of the muscles in the whole body. Hence, for simulation of clinical tetanus, the animal injected i.v. with the toxin is more relevant . To test the spinal inhibition in general tetanus, tetanus toxin at lethal doses was injected i.v. and the acute electrophysiological experiments were performed during periods of the maximal manifestation of the convulsion and tremor shortly before the death of animals. MATERIALS AND METHODS Cats under light sodium pentobarbital anaesthesia (25-30mg/kg i .p .) received 2000 (0 10,000 mouse minimal lethal doses per kg body weight (mMLD/kg) of tetanus toxin (T~, Hehringwerke, Marburg) by i.v. injection . One cat minimal lethal dose is approximately 2000 mMLD/kg . Extreme care was taken in the treatment of the animâl to avoid any kind of stimulations which may cause the death of the animal . When animals injected with tetanus toxin showed any small tetanus symptoms which are mostly some disquiet in their behaviour, and the slight extension of the forelimbs, a mixture of chloralose (40 mgng) and urethane (400 mgng) was injected i .p. Additional anaesthesia was given if necessary . Electrophysiological experiments began several hr after the beginning of the anaesthesia in expectation of the further spread of the toxin and the development of the disease. All animals diod between 68 and 120 hr after toxin injection . Four types of preparation were used. Type 1 preparation
For this there was no treatment other than insertion of the needle electrodes into the triceps surge muscle (extensor) for recording of the electromyogram (EMG) in the first part of the experiment. In the second part of the experiment the ankle tendons of the extensor (triceps surge) and the flexor muscles (tibialis anterior etc .) were cut for slow manual stretching. Two cats received 10'mMLD/kg of tetanus toxin . (see the scheme in Fig . 1) After a small skin incision a pair of stimulating electrodes were laid on the deep peroneal nerve (to the flexor muscles) . The animal was fixed at the hip and knee joints without surgical invasion . The foot could move freely Type 1 preparation
Intact Inhibition in General Tetanus
203 fMi
v
2T
4T
3T
4T Without conditioning
10 ms
FIG. 1 . ARRANGEMENT OF THE EXPERIMENT (TYPE 2) AND THE STRETCH REFLEX RESPONSE THE CAT IN GENERAL TETANUS .
(EMG) OF
The triceps surge muscle was stretched by the contraction of the flexor muscle which was elicited by the electrical stimulation of the peroneal nerve at the popliteal fossa. The afferent impulses conditioned the motoneurones of the triceps surge muscle . 2T, 3T, 4T are stimuli strength two, three and four times the threshold of the motor nerve. At 2T and 3T the stretch reflex was depressed and at 4T the reflex was totally blocked in general tetanus. At the end of the experiment, the peroneal nerve was cut at the central side of the stimulation as indicated by the arrow in the scheme (4T without conditioning). Because of the slow rise time of the muscle contraction the group Ia input of the stretch reflex arrives at the motoneurone not in the effective period of the antagonistic Ia inhibition but only later. Therefore, the slight inhibition in `2T' does not mean that the inhibition was depressed. Cat, 40 hr after i.v. injection of tetanus toxin at lO~mMLD/kg . when the nerve to the muscle was stimulated . When the peroneal nerve was electrically stimulated (rectangular pulse with a duration of 0.2 rllsec and a stimulus strength two to four times of threshold of the motor nerve, 2T4T) the flexor muscles contracted and the extensor muscles were stretched by the flexor (ascent time ca 25 cosec according to the contraction time of the flexor muscles). The reflex response was recorded by needle electrodes inserted into the triceps surge muscle as in the Type 1 preparation (the same in Types 3 and 4) . Two cats received l0` or 5 x 10 3 mMLD/kg of toxin. Type 3 preparation (see the scheme in Fig. 2) In addition to the operation of the Type 2 preparation the tendon to the triceps surge muscle was cut. The muscle was freed from the surrounding tissue and connected with an electromagnetic stretch device . The zerolength of the triceps surge muscle was adjusted by extending it to a point, where the tension was 1 N. The muscle was stretched several mm from the zero-length with a triangular pulse (ascent time 2.5 cosec) . The peroneal nerve was cut and the central end was stimulated for the conditioning . The stretch apparatus was triggered by a delay circuit to stretch the muscle with variable delay time after the conditioning electrical stimulation of the peroneal nerve (rectangular pulse with a duration of 0.2 cosec at a stimulus strength 4T). Three cats received 10~, 5 x 10 3 or 2 x l03 mMLD/kg of toxin. Type 4 preparation With a deep anaesthesia (brief ether anaesthesia was used additionally if necessary) a laminectomy was performed. The monosynaptic reflex was recorded from a part of the central cut end of the ventral root of ~ and/or S, . The conditioning stimulus was given to the peroneal nerve and the testing stimulus for the
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et a!.
EMi
D
,
C
i C100T
COT [2T C6 T
C15oT
CBT C12T Cl6T
T
[2oT
C2oT
C150T
- C30T [00T C50T
" C300T
ron ~s
~- [10T
FIG .
i.
ARRANGEANT OF THE aYVme~AtFrrr (TYFE 3) AND THE STRETCH REFLEX RESPONSE THE CAT IN GENERAL TETANUS .
(EMG) OF
The triceps some muscle was stretched by a stretch device (rise time, 2.5 msec) triggered by the test pulse fT) at different times after electrical stimulation of the antagonistic peroneal nerve (conditioning stimulus, C). Responses of the stretch reflex (EMG) with and without conditioning stimulus were shown. The number between C and T shows the interval between conditioning stimulus and test pulse in cosec. Note the different time scales in a, b, c, d, and that the motoneurone is already inhibited by the electrical conditioning stimulus when the Ia reflex impulse elicited by the muscle stretch without delay (COT) arrives at the motoneurone. Cat, 70hr after i.v . injection of tetanus toxin at 2 x lO;mMLD/kg. monosynaptic reflex was applied to the tibial nerve (for further information about this type of preparation, see TAKANO et al., 1983) . Two cats received 5 x 10' or 2 x 10' mMLD/kg of toxin. For further details of the stimulation in each type of the experiments, see results . RESULTS
Inhibition of the continuous activity of the muscle
The Type 1 preparation was used for this experiment (see methods) . Acute experiments were performed two days after the injection of tetanus toxin at 10°mMLD/kg. The
Intact Inhibition in General Tetanus
20 5
-100 C Y O Y
"
" O.
ee 0
1
"
" "
"
" "
2 5 10 10 50 100 200 interrel between conditioning :tinulu: and test stretch ( ms ~
500
FIG. 3 . INHIBITION CURVE OF 3TRETCH REFLIX DURING GENERAL TETANUS.
The stretch reflex was conditioned at different times before the test stimulation (abscissa in the logarithmic scale). The same ezperimcnt as in Fig. 2, but from another animal . Each point is the averaged amplitude of reflex responses of 10 trials . The amplitude of reflex responses without inhibition is 100% . Cat, SOhr after i.v . injection of tetanus toxin at 5 x 10'mMLD/kg .
extensor muscles but not the flexor in the hind leg (as in local tetanus; TAKANO, 1976b; Kx>rrrzscxMAx et al., 1980) showed a high amount of spontaneous activity in the EMG when the intoxicated animal was not too deeply anaesthetized. Occasionally a clonus (8-12 Hz) was clearly observed . The continuous activity became greater when the muscle was stretched slowly and maintained . The exaggerated stretch reflex was caused by increased gamma activity (TAxANO and Kntvo, 1973 ; TAxANO and HErlnTSCx, 1973; Hucx et al., 1981) and/or also due to the hypersensitivity of the alpha motoneurone. Such high activity of the muscle at maintained extension can neither be obtained in a deeply anaesthetized and intoxicated animal nor in the non-anaesthetized and non-intoxicated animal . When the muscle was stretched more strongly, the EMG activity was depressed. This inhibition may be caused by autogenic inhibition through group Ib afferent discharges from the Golgi tendon organs in the stretched muscle . The muscle activity could also be depressed by stretching the antagonistic flexor (e.g. tibialis anterior) muscles. This inhibition is caused by group Ia discharges from the muscle spindle and also group II and III discharges from other receptors of the antagonistic flexor muscles (for the EMG recordings see Fig. 3 in TAKANO and KIRCHNER, 1987). This rather simple experiment shows that autogenic as well as antagonistic inhibition remained intact in general tetanus. Inhibition of the stretch reflex induced by a sudden muscle stretch through contraction of the antagonistic muscle In this series of experiments the Type 2 preparation (see methods) was used for testing the antagonistic inhibition of the stretch reflex in response to sudden stretching . The peroneal nerve of the ipsilateral side was stimulated electrically by a single pulse. This stimulation served as an indirect stimulation of the flexor muscles and a concomitant stretching of the extensor triceps surae muscle . At the same time the afferent impulses elicited by the stimulation of the peroneal nerve conditioned (inhibited) the alphamotoneurone innervating the triceps surae muscle . At a stimulus strength two times the threshold of the muscle contraction (2T) almost all peroneal alpha motor nerve fibres were stimulated and, therefore, the maximal stretching of the triceps surae was achieved through maximal twitch of the flexor . The stretch reflex was induced and the electrical muscle response was recorded (Fig. 1, 2T). At 2T
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100
O C O
E
10
20
50 ms 100
lntonal betrun condltioninp and tort atialulation :
FIG. 4. INHIBEI7ON CURVE OF THE LtON08YNAPTIC REFLEX DURING GENERAL TEEANUS .
The monosynaptic reflex was recorded from the cut end of the bundle from L, and S, ventral roots . The conditioning stimulus (4T) was given on the peroneal nerve at different times (abscissa in the logarithmic scale) before the test stimulation of the tibial nerve (2'I') to the gastrocnemius muscle . Each point is the averaged amplitude of reflex responses of 10 trials. The amplitude of reflex responses without inhibition is 100% . Cat, 100hr after i .v . injection of tetanus toxin 2 x 103 mMLD/kg .
stimulation the antagonistic Ia discharge inhibited the extensor motoneurones and the reflex response was depressed (smaller than in `4T without conditioning' in Fig. 1, see later) . At stronger stimulation, three or four times of threshold (3T or 4T), the smaller sensory fibres with higher threshold were also recruited and inhibited the extensor motoneurones more strongly . At the stimulus strength of 3T, the degree of muscle stretching cannot be smaller than the stimulation at 2T, (this is also visually controlled) but the reflex response was reduced strongly in amplitude (Fig. 1, 3T). The stretch reflex was totally depressed at the stimulus strength of 4T (Fig. 1, 4T). At the end of the experiment the peroneal nerve was cut proximal to the stimulated site, (as indicated by the arrow) . Now, the stretch reflex could no longer be inhibited (Fig. 1, 4T without conditioning). These experiments show that peroneal nerve stimulation causes a strong inhibition of the stretch reflex of the triceps surae muscle during a heavy tetanus intoxication . Relation between amplitude of the stretch reflex and interval between conditioning and test stimulations
In this series of experiments the Type 3 preparation was used (see methods) and the conditioning inhibitory stimulus on the peroneal nerve at 4T was given at different times before the short and rapid stretching of the triceps surae muscle. This was designed to obtain an approximate idea of the inhibition through which the stretch reflex was depressed (Fig. 2). The inhibition was strongest at the interval from 16 (C 16T) to 20 msec (C20T) resulting in almost total depression of the reflex . Notice that the group Ia impulse from the muscle spindle of the triceps surae has a handicap in reaching the motoneurone due to the time lag of the action potential generation in the muscle spindle. The inhibitory curve of Fig. 3 was obtained from the second animal of Type 3 preparation. The inhibition with maxima at ca 8 msec may be due to a complex postsynaptic inhibition by group I, II and III fibres . The delayed inhibition with its maximum at ca 80 msec may reflect the presynaptic inhibition . The delayed inhibition was not longer than that of the non-intoxicated animal, in contrast to local tetanus, where the presynaptic inhibition lasts longer (Tnlcnxo et al., 1989b) . This fact suggests that in generalized tetanus the spinal cord is scarcely affected by
Intact Inbibidon in General Tetanus
207
the toxin and therefore the local toxin concentration in the spinal cord is far lower than in local tetanus (see discussion). The inhibitory curve of the monosynaptic reflex
In the previous experiments we had gathered the necessary experience to treat the generalized tetanus animal carefully, allowing us to additionally perform a laminectomy in order to get an inhibition curve of the monosynaptic reflex (Fig . 4). We observed the early antagonistic Ia inhibition with its maximum at ca 1 .5 msec. The second delayed inhibition with maximum of ca 20 msec may be due to a combination of group II, III postsynaptic inhibitions and the presynaptic inhibition, resulting in complete inhibition of the extensor motoneurone. Taken together these experiments clearly show that several kinds of inhibition of the motoneurone of the extensor muscle remained intact in heavy generalized tetanus. DISCUSSION
Most authors working on local tetanus in the second half of this century used tetanus toxin at very high doses (e.g. Bxooxs et al., 1957). At such high doses tetanus toxin blocks at first the inhibitory synapses and thereafter the animal dies before tetanus toxin invades the excitatory synapses. In our foregoing studies on local tetanus, we used mainly sublethal doses. Two mouse MLD/kg (10 -' cat MLD/kg) is the critical dose for eliciting local tetanus and blocking excitatory transmission of the motoneurone. In clinical tetanus the toxin at an approximately lethal dose spreads over the whole body. Suppose that the death rate without any care is 60%; meaning that 60% of the patients have more than one lethal dose and 40% of them a sublethal dose. Therefore the doses in this study, 1-5 times of the minimal lethal dose, are clinically relevant doses or slightly higher . Yet the spinal inhibition remained intact . We did not observe any sign of a local tetanus. How about the local concentration in the spinal cord segment in our preparations? HABERMANN and DIMPFEL (1973) reported that about 0.5% of the toxin injected i.v. could be found in the spinal cord and brain stem (not in the forebrain and cerebellum). They assumed a toxin concentration of about 10- ' S M in the spinal cord when they injected the toxin i.v. at the LDSO dose. HABHRMANN (1972) calculated for local tetanus a higher effective toxin concentration (i.e. 2~ x 10- "M) in the lumbar spinal cord when he injected tetanus toxin into the gastrocnemius muscle . The local concentration in the spinal cord in general tetanus at doses in the same range as in our study cannot reach that of local tetanus. When tetanus toxin at a very high dose is injected i.v., the animal dies without showing any hyperactivity of the motor system (`botulinum-like intoxication' after MATSUDA et al ., 1982). Experimental tetanus showing such flaccid paralysis might not be a conventional `general tetanus'. In our laboratory it was observed that several kinds of spinal inhibition were left intaçt in the rabbit injected i .v. at 2x lOSmMLD (ca 100 MLD). From this point of view general tetanus has a different pathogenesis from that in local tetanus. HABERMANN and Dnlpr->rr. (1973) injected the toxin at less than an LDSO dose i.v. and `some animals (rats) died between the 6th and 15th day, and all other animals also developed moderate to. severe tetanus. These symptoms had their maximum at the 10th day, and disappeared completely, by day 19'. As HABERMANN (1972) wrote, the symptoms of local tetanus persist for a long time, in agreement with our observations that the
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shortening of the muscle (at early period hyperactivity and later contracture without activity, KlxcxxER et al., 1980) lasts for many weeks. Fully developed clinical tetanus is characterized by generalized hyperactivity of muscles of the whole body . This general tetanus has been regarded as the summation of multiple local tetanus (e.g. KRYZHANOVSKY, 1967; HABI?RMANN, 1978). This hypothesis is based on the disinhibition hypothesis of the hyperactivity of the motor system without changing the excitatory transmission . As already stated in the introduction the spinal disinhibition is only evident in the early period of local tetanus. Tetanus toxin also blocks thereafter excitatory transmission even at low doses that just cause local tetanus. As demonstrated in this study, many kinds of inhibition of the spinal motoneurone appear to be left intact in general tetanus. Thus in general tetanus the functional mechanisms of the spinal motor system are operative, whereas during a local tetanus both inhibitory and excitatory transmission are disturbed. General tetanus and local tetanus are independent, although there might be some intervention of the former with the latter. The generation of the hyperactivity in tetanus resides in the higher central nervous system (HucK et al ., 1981), and not in the spinal cord as once suggested by e.g. KRYZHANOVSKY (1967) . We propose that general tetanus is not the summation of local tetanus. Local tetanus .appears to be produced in the spinal cord, whereas general tetanus is caused by supraspinal hyperactivity . Acknowledgement.~The authors thank Dr R. PetvNea for reading the manuscript and Mrs G. FEUERKEIL for technical assistance . Tetanus toxin was kindly supplied by Behringwerke AG (Marburg) .
REFERENCES BBeoev, G. K., BIGALKE, H. and NELSON, P. G. (1987) Differential effects of tetanus toxin on inhibitory and excitatory synaptic transmission in mammalian spinal wrd neurons in culture: a presynaptic locus of action for tetanus toxin. J. Neurophysiol. 57, 121-I31. BROOKS, V. B., Cams, D. R. and ECCLES, J. C. (1957) The action of tetanus toxin on the inhibition of motoneurones. J. Physiol. 135, 655-672. C~1R173, D. R., FaLtx, D., G~t~, C. J. A. and MCCuLLOCx, R. M. (1973) Tetanus toxin and the synaptic release of GABA. Brain Res. 51, 358-362. H~ER~tvN, E. (1972) Distribution of'ZSI-tetanus toxin and'zs-toxoid in rats with local tetanus, as influenced by antitoxin . Naunyn-Schmiedebergs Arch. Pharmacol. 272, 75-88. HABERMANN, E. (1978) Tetanus. In Handbook ojClinical Neurology, Vol. 33, pp. 491-547 (VINKEN, P. J. and BRUVx, G. W., Eds) . Amsterdam: North Holland. H~eeR~rtx, E. and Dt~~eL, W. (1973) Distribution of'uI-tetanus toxin and'z3I-toxoid in rats with generalized tetanus, as influenced by antitoxin. Naunyn-Schmiedebergs Arch . Pharmacol. 276, 327-340. HuCK, S., KtRCfttvER, F. and Trtuxo, K. (1981) Rhythmic activity in the cerebellum and spinal cord of rabbits receiving tetanus toxin intravenously. Naunyn-Schmiedebergs Arch . Pharmacol. 317, 51-53. KANDA, K. and TAKANO, K. (1983) . Effect of tetanus toxin on the excitatory and inhibitory post-synaptic potentials in the cat motoneurone. J. Physiol., f ond. 335, 319-333. K~RCxxeR, F., KREfZSCHMAR, H. and Trtuxo, K. (1980) Study about the origin of contracturc of muscles in response to intramuscular injection of tetanus toxin. Proc . lUPS 14, 513. KRErzsc,~tastaR, H., KtRCt~txrae, F. and TAKANO, K. (1980) Relations between the effect of tetanus toxin on the neuromuscular transmission and histological functional properties of various muscles of the rat. Exp. Brain Res. 38, 181-187. KRYZHANOVSKY, G. N. (1967) The neural pathway of toxin: its transport to the central nervous system and the state of the spinal reflex apparatus in tetanus intoxication. In: Principles on Tetanus, pp . 155-168 (ECKMANN, L., Ed .) . Bern and Stuttgart : Hens Huber Publishers . MAISUDA, M., SUGIMOTO, N. and Ozu~rsuett, K. (1982) Acute botulinum like intoxication by tetanus toxin in mice and localization of the acute toxicity in the N-terminal-pepsin-fragment of the toxin. In : 6th /nternational Conjerence on Tetanus, pp . 2-37 . Lyon : Foundation Marcel Merieux.
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Oztnsustt, K., Dux-LuNß, L., Sußr~oro, N. and M~zsttnn, M. (1989) Isolation and purification by high performance liquid chromatography of a tetanus toxin fragment (Fragment [A-B]) derived from mildly pepsin-treated toxin. Toxicon 27, 1055-1057 . STRUPPLER, A. (1987) Das Nervensystem . In: Pathophysiologie~Pathobiochemie, pp. 193-258 (Lt.Nß, F., Ed .) . Stuttgart: Enke . T~NO, K. (1976x) Local tetanism a tool for understanding the stretch reflex . In Progress in Brain Res. 44, "Understanding the stretch reflex", pp . 491-502 (Hot~uu, S., Ed .). Amsterdam: Elsevier . T~tcwNO, K. (1976b) The effect of tetanus toxin on the extensor and flexor muscles on the hind leg of the cat. In : Animal, Plant and Microbial Toxins, Vol. 2, pp. 363-378 (OFÛAICA, A ., HAYA9FII, K. and $AWAI, Y., Eds) . London : Plenum Press. Tetuxo, K. and Hsrre~cH, H. D. (1973) Tension-extension diagram of the tetanus intoxicated muscle of the cat. Naunyn-Schmiedebergs Arch . Pharmacol. 276, 421 36. TAKANO, K. and KENO, M. (1973) . Gamma-bias of the muscle poisoned by tetanus toxin. Narxryn-Schmiedebergs Arch . Pharmaeol. 276, 413 20. TAKANO, K. and KtxCttxex, F. (1987) Pathogenesis of tetanus: clinically relevant new concepts . A mini review . In : Progress in venom and toxin research, pp . 68091 (GOPALAKRISHNAKONE, P. and TAN, C. K., Eds) . Singapore : National University . Tnruxo, K., KtxctrNm, F., Tr~x, P. and TtEar:ar, B. (1983) Effect of tetanus toxin on the monosynaptic reflex . Naunyn-Schmiedebergs Arch. Pharmacol. 323, 217-220. T~tceNO, K., KtxcHNm, F., GxzatstFa .r, A., MA~rsuDA, M., Ozu~tsun~t, N. and SußtMO~ro, N. (1989x) Blocking effects of tetanus toxin and its Fragment [A-B] on the excitatory and inhibitory synapses on the spinal motoneurone of the cat. Toxicon 27, 385-392 . TAICANO, K., KutcttN~e, F., T~tT, B. and TERIiAAR, P. (1989b) Presynaptic inhibition of the monosynaptic reflex during local tetanus in the cat. Toxicon 27, 431-438.