Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 375--386 (1973) 9 by Springer-Verlag 1973

Local Tetanus in Cats: Neuropharmaeokineties of 125I-Tetanus Toxin H. H. WellhSner, B. Hansel a n d U. C. Seib * Pharmakologisches Institut der Justus-Liebig-Universit~t GieBen Received June 22, 1972

Summary. 1. On unilateral injection of sublethal amounts of ~25I-tetanus toxin into one gastrocnemius muscle of cats, we found after 24 h: a) A steep gradient of radioactivity (teA) had developed in the sciatic nerve of the injected side only. b) The ventral roots of the spinal cord half segments supplying the injected muscle had a higher RA than the contralateral roots. In some of the cats the same was true to a varying degree for the dorsal roots too. c) The spinal cord half segments supplying the injected muscle had a RA much higher than the eontralateral half segments. Within the time of experiments, this surplus RA did not pass to the contralateral side. The rise in RA could be prevented with tetanus antitoxin injected intravenously immediately after the application of toxin or by ligature of the ventral roots. 2. On unilateral injection of 12~I-tetanus antitoxin or Na12SI no difference in RA was found between the spinal cord half segments. 3. On unilateral injection of a sublethal dose of 125Idetanus toxin into the skin of cats at the insertion area of a lateral intercostal nerve we found after 24--48 h: On the injected side in the intercostal nerves the RA is much higher than on the non-injected side. The difference is most prominent at the costal interspace under the toxin depot. The same is true for the spinal cord half segments. 4. On bilateral intracutaneous injection of t2aI-tetanus toxin and unilateral ligature of the lateral intercostal nerves, the RA in the nerves and spinal cord half segments was lower on the ligated side. Key words. Tetanus Toxin -- Pharmacokinetics -- Neural movement -- Tetanus Antitoxin. To d e m o n s t r a t e a n d to analyze the action of t e t a n u s t o x i n i n the spinal cord several kinds of neurophysiological experiments have been set u p the designs of which have two features i n c o m m o n : Firstly, t h e y have been m a d e in cats, a n d secondly, it was assumed t h a t d u r i n g the e x p e r i m e n t the t o x i n did n o t move to a significant e x t e n t i n the spinal cord from the side ipsilateral to the t o x i n depot to the eontralateral side (Sherrington, 1905; Acheson et al., 1942; Brooks et al., 1957; Wilson et al., 1960; K r y z h a n o v s k i i a n d D ' y a k o n o v a , 1964; Swerdlov a n d Alekseeva, * This work was supported by grant No. We 497/2 from the Deutsche Forschungsgemeinschaft. 25

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376

H . H . WellhSner et al. :

1966; K a n o a n d T a k a n o , 1969; T a k a n o a n d H e n a t s c h , 1973; P a a r a n d WellhSner, 1973). However, no q u a n t i t a t i v e d a t a have ever been presented to justify the l a t t e r assumption. The work of K r y z h a n o v s k i i et al. (1961) was confined to the peripheral nerves, spinal roots a n d ganglia. Doerr et al. (1936) stressed the difficulties to bioassay t e t a n u s t o x i n b y i n o c u l a t i n g susceptible a n i m a l s with tissue probes p r e s u m e d to be toxic. P a r t i c u l a r l y with m a t e r i a l from the spinal cord the m e t h o d does n o t work, as a n u n k n o w n p o r t i o n of the total t o x i n is firmly b o u n d to the tissue a n d a p p a r e n t l y is released to a n u n k n o w n degree into the test animals. A n e w m e t h o d became available when H a b e r m a n n (1970, 1972) succeeded to label t e t a n u s t o x i n with 125I. This approach has been used i n the work presented below to measure i n cats the localized a c c u m u l a t i o n of t e t a n u s t o x i n i n the spinal cord, i n the dorsal a n d v e n t r a l roots, a n d i n the peripheral nerves on i n t r a m u s c u l a r injection of labelled toxin. I n a d d i t i o n i t was i n v e s t i g a t e d whether i n t r a e u t a n e o u s i n j e c t i o n of labelled t o x i n results i n a spinal d i s t r i b u t i o n of r a d i o a c t i v i t y comparable with the d i s t r i b u t i o n after i n t r a m u s c u l a r application.

Material and Methods Experiments were made in cats of either sex weighing between 1.8 and 3.5 kg. In the experiments with intramuscular injections all radioactive solutions were injected into the gastrocnemius muscles with the cats in general anesthesia (ether in the animals moving freely later on and 65 mg/kg chloralose i.p. in the other cats). In the experiments with intracutaneous injection the cats were anesthetized with ether and the skin was longitudinally cut over the sternal manubrium and on the dorsal side from C7--Thl2; the cuts were sutured some minutes later. This surgery was done to prevent distribution of toxin by lmyphatic flow from one side to the other. The skin was then opened with a cut parallel to the rib X and the cutaneous insertion of the lateral intercostal nerve IX (nomenclature: Sears, 1964) was located. With a 22 gauge needle an intracutaneous depot of toxin (106 cpm/kg body weight; 106 cpm dissolved in 0.05 ml saline with 0.1 ~ bovine serum albumin) was injected there from the hairy side (control by inspection from the other side). The wound was sutured therafter and the ether was removed. Tetanus toxin was obtained from Drs. Bizzini and Turpin, Institut Pasteur, and was labelled in this department (Habermann, 1970, 1972). Batches with slightly different specific radioactivities [cpm per g protein/toxicity (MLDmouse per g protein)] were obtained. A representative batch had the following characteristics MLDmou~e ~ 7.5 • 10-9 g toxin/kg (value prior to labelling 2.5--3.3 • 10-9), radioactivity = 875 • 109 cpm/g protein corresponding to 1.1 mCi/mg protein (windows of the Packard Autogamma spectrometer set at 48 and 100 keV). According to Fildes and Ledingham (1929), 1 MLD/kg eat is equivalent to 600 MLD/kg mouse. With this factor and the values given for the labelled toxin one obtains: 1 MLD/kg cat = 4.5 • 10-9 g 12~I-tetanus toxin corresponding to 3.8 • l0 s cpm. 60 d later this value would be still 1.9 • l0 s cpm. The cats received a standard dose of 109 cpm/kg. The batches were never used longer than 60 d. Therefore the dose was in the range of 1 MLD or below.

Local Tetanus with 1251J-Tetanus Toxin in Cats

377

Some cats underwent long-time general anesthesia and laminectomy to enable ligature of the ventral roots. The surgery was conventional. The provisions of intensive care are described elsewhere (Wellh6ner et a l . , 1973). The animals were sacrificed by exsanguination 24--36 h after the injection of the radioactive solutions and dissected. Probes from neural tissues were taken according to the following scheme: The tibial, peroneal and sciatic nerves and, when intracutaneous injections had been made, the intercostal nerves were removed on both sides in their full length and divided into pieces of 2 cm or 3 cm. The ventral and dorsal roots L5-$2 (intradural parts) were taken. The spinal cord was removed and divided with a razor blade below the entry of the ventral roots into segments; the segments were then subdivided into a right and left half. In addition, samples were taken from the brain, the chorioid plexus, the dura mater, the muscles, the skin, the parenchymatous organs, the liquor, the urine, and the blood. The probes were transferred to Eppendorf vessels immediately. The vessels were weighed and counted in a Packard Autogamma spectrometer for 50 min with the windows set at 48 keV and 100 keV. The radioactivity R was calculated as R --

N 1 - - NO

t (gl - g2)

(1)

t counting time (50 min), iV0 counts of an e m p t y vessel in t, N 1 counts of a filled vessel in t, gl weight of the e m p t y vessel, g2 weight of the filled vessel. Calculation of the standard deviation: Generally, if F is a function of the variables x 1 . . . X n and s ( x l ) . . . s ( x n ) are the standard deviations of the x j . . . Xn, t h e n the standard deviation of F is calculated from

~(r) =

[~-

(x~)

(2)

i=1 Specially, in form (1) t is assumed to have an s ( t ) = O, g l and g2 are assumed to have the same s ( g ) and 2/1 and N 0 are assumed to have s (_hT1) = ]/hV1 and s(N0) = ]/~T0, respectively. The latter assumption requires t h a t the Poisson character of the radioactive disintegration process is preserved for the counts, i.e. t h a t no serious distortion is introduced by the counter. This assumption has been tested for our counter and window settings according to Weise (1971, form 8.23) and was found to be justified. Application of (2) to (1) t h e n leads to N~ + N o

s (R) = R .

s 2 (g)

( ~ z ~00)+ + 2 (z~ - g~)~'

(3)

In most instances the second term under the square root was much smaller t h a n the first one. s (R) could t h e n be assumed to be the standard error in a Poisson distribution passing into a normal distribution. With this approximation two radioaetivities R 1 and R 2 can be tested for a significant difference with R1 - - R2

= V~)

+ ~(R~)

(see Weise, 1971, form 7.24 and 7.25) where z is the standard normal variable. 25*

(4)

H. It. Wellh5ner et al.:

378

Results Injection oI Toxin into the Gastrocnemius Muscles After 24 h, a major part of the toxin was still found in the gastrocnemius muscles. This has been quantified in six cats, where the muscles have been excised in toto and the residual radioactivities were measured ( T a b l e 1). A m o n g t h e tissues in t h e v i c i n i t y of t h e g a s t r o c n e m i n s m u s c l e s Table 1. gesidual radioactivity (cpm) in gastrocnemius muscles 24 h after injection of labelled tetanus toxin. Cat 28 moving freely, other cats anesthetized Cat No. M.gastrocn.

28

left right left right left right left right left right left right

29 30 31 34 41

cpm injected

residual

0 2.5 X 106 2.2 X l0 G 2.2 X 106 2.5 X 106 2.5 X 106 3.0 X t0 ~ 3.0 X 106 3,0 X 106 3.0 X 106 2.4 X 106 2.4 X 106

338 0.65 X 106 1.11 X 106 1.18 X 106 1.02 X 106 1.01 X 106 1.72 X 10~ 1.38 X 106 1.08 X 106 0.97 X 106 1.t3 X 106 1.31 X 106

cpm/g 104 ~

lO3

102

o-..-o

left

~

ripht

._2 0

2 9

4 prox.

6

8

10

12

14

cm 16

dist.

Fig. 1. Radioactivity in the sciatic nerves 29 h after injection of 0.6X 106 epm 125I-tetanus toxin (equiv. to ca. 0,7 X 10-6g protein) per kg cat into the right gastrocnemius muscle. Cat moving freely. Proximal to the spinal cord (left side of the figure) the differences between the radioaetivities of corresponding pieces from the right and left nerve are still significant

Local Tetanus with ~e~IiTetanus Toxin in Cats

379

the f a t in t h e fossa p o p l i t e a was p a r t i c u l a r l y rich in a c t i v i t y . T h e r e was no i n c r e a s e d a c t i v i t y in p r o b e s t a k e n f r o m t h e a d d u c t o r muscles in t h e v i c i n i t y of t h e sciatic nerve. The r a d i o a c t i v i t y i n t h e sciatic n e r v e was m e a s u r e d in 44 specimens. I n 31 of t h e m a g r a d i e n t like in Fig. 1 was found. A c o n t i n u o u s l y increasing steepness of t h e g r a d i e n t was the e x c e p t i o n r a t h e r t h a n t h e rule. I n t h e v e n t r a l r o o t s a n e n r i c h m e n t of r a d i o a c t i v i t y was f o u n d which was v e r y high in some animals b u t a l w a y s confined s t r i c t l y to t h e segments where t h e g a s t r o c n e m i u s muscle is i n n e r v a t e d f r o m (consistent results from 19 spinal cord sides). A n e x a m p l e is given in Fig.2a_ I n t h e s a m e figure, t h e a c t i v i t y in t h e dorsal r o o t s is v e r y small. W i t h r e g a r d to t h e classical findings (for review see K r y z h a n o v s k i , 1967) one m i g h t be ............. dorsal I L6

~

ventral

i

L7

~S2

cpm/9

cpm/g 500

0 left side

0

500

1000

1500

right side

Fig. 2 a. Radioactivity in the dorsal and ventral roots 24 h after injection of 10~ cpm ~z~I-tetanus toxin (equiv. to ca. 1.2 X 10-~ g protein) per kg cat into the right gastrocnemius muscle. Cat anesthetized :~z~:~:~:~:~dorsal I ventral

........ i

L7 " lfffrfllf . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

cpm/g 1400 1000

600

200 left side

C) 6

260 right side

cpm/g 600 1000

Fig. 2 b. Radioactivity in the dorsal and ventral roots 24 h after injection of l0 Gcpm lzsI-tetanus toxin per kg cat into either gastrocnemius muscle. Cat anesthetized. Note the considerable radioactivity in the dorsal roots

380

H.H. Wellh6ner et

al. :

inclined to expect this. However, the amount of radioactivity in the dorsal roots varied considerably and could become as high as in the ventral roots. Our most extreme finding is presented in Fig. 2 b. Knowing about the distribution of radioactivity in the ventral roots, one expects an elevation of radioactivity also in the respective spinal cord segments. This proved to be justified in 22 out of 22 cats, where tetanus toxin has been injected unilaterally or bilaterally into the gastroenemius muscles. An elevation was always found in L7 and S 1 and eight times also in L 6. An example is given in Fig. 3. I n 12 experiments with injection of toxin into either gastroenemius muscle, the maximum of activity was found in the half segments of the same cord level in nine eats. A second important result is apparent from Fig.3: Within the first 24 h after intramuseular injection of l~sI-tetanus toxin, the radio-

600

cpm/g

:~i.~~ left

500

I

right

400 300 200 100

t =::~:~ L4

L5

.....~=.i

~-d~

L6

L7

$1

Fig. 3. Radioactivity in the spinal cord half segments 24 h after injection of 106 epm 125I-tetanus toxin (equiv. to ca. 1.2 • 10-6 g protein) into the right gastroenemius muscle. Cat moving freely

500

left I right

cprn/g

400 300 200 100 L5

L6

ilii L7

St

$2

Fig. 4. Radioactivity in the spinal cord half segments 24 h after injection of 10Gepm 125I-tetanus toxin (equiv. to ca. 1.2 • 10-G g protein) per kg cat into the right gastrocnemius muscles and 50000 IU tetanus antitoxin per kg eat into the jugular vein. C~t moving freely

Local Tetanus with 125I-Tetanus Toxin in Cats

381

activity in the spinal cord does not pass to the contralateral side. This result has been found in 6 out of 6 animals. According to the classical theory tetanus toxin reaches the spinal cord via the ventral roots only. This assumption was tested in 4 cats. The ventral roots L 5 - - $ 2 were cut or ligated and labelled toxin was injected intramuscularly thereafter, lkTo enrichment of radioactivity in the spinal cord could be detected one day later. The question might be raised whether the radioactivity in the spinal cord represents tetanus toxin or a split product lacking immune reactivity. A test has been made therefore with tetanus antitoxin, a high dose of which was administered intravenously immediately after intramuscular injection of labelled toxin. No significant elevation of spinal cord radioactivity was found (Fig. 4), and the same was true for the ventral roots. However, in the sciatic nerve of the injected side there was a gradient of radioactivity. Injection o/125I-Tetanus Antitoxin or Na125I into the Gastrocnemius Muscle To get further evidence whether the enrichment of radioactivity in the ventral roots and the spinal cord is specific for 125I-tetanus toxin, control experiments have been made with intramuscular injection of Nal~sI (two cats) and l~sI-tetanus antitoxin (one cat). 125I-tetanus antitoxin (25 ~Ci/ml) was prepared by H a b e r m a n from Fermoserum (Behringwerke). On unilateral injection of the material into one gastrocnemius muscle, no side difference in radioactivity was found in the spinal cord (Table 2), in the ventral roots and in the sciatic nerves with the exception of the 4 cm nearest to the gastrocnemius muscle. Moreover, the absolute activity in the spinal cord was very low. On unilateral injection of Na125I a similar result as with labelled antitoxin was obtained (Table 3). Table 2 Radioactivity (cpm/g wet weight) in spinal cord half segments one day after injection of 3 • 106 cpm (equivalent to 150 ~l) 125I-tetanus-antitoxin (Fermoserum) into the right gastrocnemius muscle. Cat, 3.0 kg, moving freely Segment

left

right

L4 L5 L6 L7 $1

214-4 184-5 214-5 204-6 24 4- 14

154-4 19• 18~:5 184-6 94-14

H . H . WellhSner et al. :

382

Table 3. Radioactivity (cpm/g wet weight) in spinal cord half segments 24 h after injection of 2 • 10~ cpm Na~2~I/kg cat into the right gastroenemius muscle. Cat moving freely Segment

left

right

L4 L5 L6 L7 $1

206:~ 9 275~ 9 314• 498::j:19 831•

282-t:12 284~10 334~10 474i22 649•

Table 4. Radioactivity (cpm/g wet weight) in spinal cord half segments from a cat 36 h after injection of 106 cpm 12~I-tetanus toxin (equiv. to ca. 1.2 • 10 -8 g protein) into the skin at the insertion of the left lateral intercostal nerve IX. Same animal as in Fig. 5 Segment

left

right

Th6 Th 7 Th 8 Th 9 T h 10 Thll Th12

157~: 8 274 :j: i1 256 :[: 11 309 J : 11 303 ::[: 12 148• 7 94J: 7

72~8 101 ::]: 7 115 • 7 66 • 7 68 ::I: 6 69~6 61i6

Intracutaneous Injection o~ 125I- Tetanus T o x i n Experiments with unilateral or bilateral injections of labelled toxin into the thoracic skin have been made in 15 animals. One cat obtained Na12~I, another one labelled toxin into the skin of the hindlimb a t the lateral side of toe V. The essential results can be outlined b y Figs.5a, 5b and Table 4 which refer to the same cat unilaterally injected with labelled toxin. The radioactivity is found predominantly in the intercostal nerve under the toxin depot b u t also, to a smaller extent in the neighbouring nerves. We made a gross autoradiography b y pressing the thoracic skin on an X-ray fihn a n d found t h a t the radioactivity h a d spread towards the axilla (see the l y m p h drainage of this area in Ratzlaff, 1970). This is in correspondancc with the distribution of radioactivity (Fig.5a a n d Table 4). Occasionally, in some cats a high radioactivity was found in the spinal cord at Th3, although the toxin was injected a t Th9. I t should be mentioned in this connection t h a t the sternal l y m p h nodes in the cat v a r y greatly as to their position and m a y be sometimes found a t the articulations of the second a n d t h i r d ribs with the sternum (Ottaviani and Cavalli, 1933). The part of the external oblique muscle underlying the toxin depot was found to be moderately radioactive as compared with other musculature in spite of the fact t h a t the depot was definitely intracutaneous. I n 6 cats labelled toxin was injected on b o t h sides a n d the lateral intercostal nerves to the skin were ligated on one side. On the ligated side the radioactivity was lower in the spinal

Local Tetanus with 125I-Tetanus Toxin in Cats 15

383

9103cpm/g ~r~; left right

10

II

IV

Ill

V

VII

Vl

VIII

IX

X

XI

XII

Nil

a: Lateral intercostal nerves 2000

~i~i~,i~ileft

cprn/g

right 1500 1000 500

I1

II1

IV

V

Vl

VII

VIII

IX

X

XI

X[[

XIII

b: Proximal intercostal nerves Fig. 5 a and b. I~adioactivity in the intercostal nerves 36 h after injection of 106 cpm ~zsI-tetanus toxin (equiv. to ac. 1.2 X 10-6 g protein) per kg ear into the skin at the right intercostal space I X

cord as compared with the contralateral side, but the radioactivity was still higher than in a non-injected side.--With injections of Na125I no difference in radioactivity was found in the spinal eord.--One experiment has been made where tabelled toxin was injected on either side into the skin of the hind limb at the toeV. The peroneal nerve was ligated thereafter on one side. The radioactivity did rise in the spinal cord segments L6-$2, buth the side-differences were insignificant. Other .Findings. High radioaetivities were found in the regional lymph nodes, in the thyroid glands, and in the wall of the stomach. Extremely low radioactivities were found in the liquor recovered after laminectomy by puncture of the dura mater.

Discussion I t has b e e n s h o w n t h a t t h e r a d i o a c t i v i t y does n o t pass f r o m one t o t h e o t h e r side o f t h e spinal c o r d d u r i n g t h e first 24 h a f t e r t o x i n i n j e c t i o n . T h i s justifies s u b s e q u e n t l y t h e a s s u m p t i o n s o f t h e n e u r o p h y s i o l o g i s t s

384

H. It. WellhSner et al. :

(see introduction). Moreover it has been shown that the accumulation of radioactivity one day after intramuscular injection is strictly confined to the spinal cord segments innervating the muscle and an ascent or descent of radioactivity within the spinal cord is not yet apparent. I t seems to be of some additional value for neurophysiological experiments in the spinal cord to know that the maximum of radioactivity is in the majority of eases, but not always, found at the same level. This would eventually imply that in some cats the gastroencmius muscle of one side is mainly innervated from S 1 and on the other side from L7. -- The results reported above with intramuscular injection of tetanus toxin are in agreement with the findings of Habermann (1970, 1972) in rats. The same is true for the findings with intravenous and intramuscular injection of antitoxin. The results from the experiments with antitoxin and ~Tal~sI are in favour of the assumption that the radioactivity in the spinal cord represents toxin and that the ability to enrich in the spinal cord is a property which is in some way specific for tetanus toxin and some related substances, as for instance tetanus toxoid (Habermann, 1972). I t is known that a neural ascent might be observed with a greater variety of substances (for review see Kristenson, 1970). The results we obtained in the ventral and dorsal roots confirm Kryzhanovskii et al. (1961) ; apparently due to our method, we frequently found an elevated radioactivity also in the dorsal roots. However, it has been shown by ligature of the ventral roots that the toxin in the dorsal roots could not enter the spinal cord (see also WellhSner et al., 1973). -- The gradient of radioactivity in the sciatic nerve is high. No attempt is made from the present work to speculate about the compartment of the nerve eventually involved in the ascent of toxin. Autoradiographic studies have been made by Fedinec (1967). Other histological work has been performed by Zaeks and Sheff (1970). The results from the experiments with intraeutaneous injection indicate that in fact local tetanus might develop ff only the skin has been hurt. They show in addition that mechanical destruction or irritation of a muscle as for instance with a needle is not a prerequisite to create local tetanus, i.e. local tetanus is not a highly artificial situation. On the other hand it is apparent that the results give only weak evidence to the conclusion that tetanus toxin ascends in nerves free of motor axons, since even with strictly intracutaneons injection one cannot avoid the contamination of neighbouring muscles. An a t t e m p t with a less equivocal method has been made by Hensel et al. (1973). Acknowledgement. Drs. Bizzini and Dr. Turpin provided the toxin, Prof. Habermann and Dr. g~iker labelled it, the Behring-Werke provided counting facilities in the beginning of the work, and Miss Liehau gave valuable technical assistance.

Local Tetanus with 125I-Tetanus Toxin in Cats

385

References Acheson, G. H., Ratnoff, O. D., Schoenbach, E. B.: The localized action on the spinal cord of intramuscularly injected tetanus toxin. J. exp. IVied. 75, 465--480 (1942). Brooks, V. B., Curtis, R. D., Eccles, J. C. : The action of tetanus toxin on the inhibition of motoneurones. J. Physiol. (Loud.) 135, 655--672 (1957). Doerr, R., Seidenberg, S., Magrassi, Fl.: Kritische und experimentelIe Studien zur Frage des Nachweises yon Tetanustoxin im peripheren Nerven. Z. Hyg. Infekt.Kr. 118, 92--116 (1936). Fedinec, A. A. : Absorption and distribution of tetanus toxin in experimental animals. In: L. Eckmann (Edit.): Principles on tetanus, pp. 168--175. Bern: Huber 1967. Eildes, P., Ledingham, J. C. G. : A system of bacteriology in relation to medicine. Vol. III, pp. 298--372. London: His Majesty's Stationary Office 1929. IIabermann, E. : Pharmakokinetische Besonderheiten des Tetanustoxins und ihre Beziehungen zur Pathogenese des lokalen bzw. generalisierten Tetanus. NaunynSchmiedebergs Arch. Pharmak. 267, 1--19 (1970). Habermann, E. : Distribution of 125I-tetanus toxin and 125I-toxoid in rats with local tetanus as influenced by antitoxin, l~aunyn-Schmiedeberg's Arch. Pharmacol. 272, 75--88 (1972). Hensel, B., Seib, U. C., Wellh5ner, H. I-I. : Vagal ascent and distribution of 125Itetanus toxin after injection into the anterior wall of the stomach. NaunynSchmiedeberg's Arch. Pharmacol. 276, 395--402 (1973). Kano, M., Takano, K. : Gamma activity of rigid cat caused by tetanus toxin. Jap. J. Physiol. 19, 1--10 (1969). Kristenson, K. : Transport of fluorescent protein tracer in peripheral nerves. Aeta neuropath. (Berl.) 16, 293--300 (1970). Kryzhanovskii, G. N. : The neural pathway of toxin. In: L. Eckmann (Edit.): Principles on tetanus. Bern: Huber 1967. Kryzhanovskii, G. N., D'yakonova, M. V. : Changes in the efferent pathway of the spinal cord in tetanus poisoning. Bull. exp. Biol. Med. 58, 1021-1024 (1964). Kryzhanovskii, G. iN., Pevnitskii, L. A., Grafova, V. N., Polgar, A. A. : Routes of entrance of tetanus toxin into the central nervous system and some problems connected with the pathogenesis of experimental tetanus. Part I I : Experiments on mice, guinea pigs, rabbits and cats. Bull. exp. Biol. Mad. 52, 984--898 (1961). Ottaviani, G., Cavalli, M. : Contributi all' anatomia del sistema linfatico del gatto. Nuova Vet. 11, 169--191 (1933). Paar, G. H., WellhSner, H. H. : The action of tetanus toxin on preganglionic sympathetic reflex discharges. Naunyn-Schmiedeberg's Arch. Pharmacol. 276, 437--445 (1973). l~atzlaff, M. H.: The superficial lympathic system of the cat. Lymphology 4, 151--159 (1970). Sears, T. A. : The fibre calibre spectra of the sensory and motor fibres in the intercostal nerves of the cat. J. Physiol. (Lond.) 172, 150--161 (1964). Sherrington, C. S. : On reciprocal innervation of antagonistic muscles-8, note. Proc. roy. Soc. B 76, 269--297 (1905). Swerdlov, Yu, S., Alekseeva, V. I. : Effect of tetanus toxin on presynaptic inhibition in the spinal cord. Fed. Proc. (Trans. Suppl.) 25, T 931--T 936 (1966). Takano, K., Henatsch, It.-D. : Tension-extension diagram of the tetanus-intoxicated muscle of the cat. Naunyn-Sehmiedeberg's Arch. Pharmacol. 276, 421--436 (1973).

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Weise, L.: Statistische Auswertung yon Kernstrahlungsmessungen. iKfinchen: Oldenbourg 1971. WellhSner, H. H., Seib, U. C., Hensel, ]3. : Local tetanus in cats: The influence of neuromuscular activity on spinal distribution of 1~5I labelled tetanus toxin. Naunyn-Schmiedeberg's Arch. 13harmacol. 276, 387--394 (1973). Wilson, V. J., Diecke, F. 13. J., Talbot, W. H. : Action of tetanus toxin on conditioning of spinal motoneurons, g. Neurophysiol. 28, 659--656 (1960). Zacks, S. I., Sheff, M. F. : 13athobiological aspects of the action of tetanal toxin in the nervous system and skeletal muscle. I n : S. Ehrenpreis and O. C. Solnitzky (Edit.): Neuroscienees Res., Vol. III, pp. 210--287. New York: Academic Press 1970. Hans H. Wellh5ner 13harmakologisches Institut Universit~t Gieflen Schubertstr. 1/VII D-6300 Giel]en Federal Republic of Germany