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The local effects of tetanus toxin on the electron microscopic structure of skeletal muscle fibres of the mouse
Journal of the neurological Sciences, 1973, 19:169-177
169
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The Local Effects of Tetanus Toxin on the Electron Microscopic Structure of Skeletal Muscle Fibres of the Mouse L. W. D U C H E N Department of Neuropathology, Institute of Psychiatry and The Maudsley Hospital, De Crespigny Park, London SE5 8AF (Great Britain) (Received 14 December, 1972)
INTRODUCTION
Recent studies have shown that although tetanus toxin causes intense and prolonged muscle spasm, slow muscle fibres are paralysed by the effect of the toxin on motor nerve terminals. In the preceding paper the electron-microscopic changes induced by tetanus toxin in motor end-plates of slow muscle fibres of the mouse were described (Duchen 1973). It was found that end-plates of fast muscle fibres of gastrocnemius were normal. In this paper the changes found with electron microscopy in the muscle fibres themselves are presented. Marked differences in the changes in slow and fast muscle were observed and although fast fibres were not paralysed by the toxin their internal structure became considerably abnormal. MATERIALS AND METHODS
A sublethal dose of tetanus toxin (Wellcome Research Laboratories), sufficient to cause prolonged local tetanism was injected into the muscle of one leg of male albino mice. After survival for periods of time ranging from 3 days to 6 months the animals were killed and tissues prepared for electron microscopy according to the methods described in the preceding paper. More than 40 animals were used in this investigation. The muscles examined by electron microscopy were mainly soleus (slow) and the outer 1-2 mm of gastrocnemius (fast) as well as, in a few cases, extensor digitorum longus. In some cases the muscles were prepared for electron microscopy after being studied in vitro by electrophysiological methods. For light microscopy (in which 140 mice were used) the tissues were prepared according to methods described previously (Duchen 1970). Many normal and control animals given antitoxin were also studied, no abnormalities being found in their muscles. This research was supported by grants from the National Fund for Research into Crippling Diseases, the Muscular Dystrophy Group of Great Britain and the Muscular Dystrophy Associations of America, Inc.
170
L . W . I)UCHEN RESULTS
Clinically there was intense spasm of the muscles of the leg from about 48 hr after the injection of toxin. The local tetanism persisted for up to 4 weeks in some animals. During the first few days there was, in some cases, a slight interstitial inflammatory reaction and a few muscle fibres appeared abnormally basophilic and contained central nuclei. This was interpreted as damage due to the needle and the injection. It was usually in gastrocnemius and always a very minor lesion and probably was of little significance in the development of the changes which were found later. Apart from the local damage, muscle fibres did not look abnormal with either light or electron microscopy during the first week.
Soleus By the end of the first week and then progressively for the next 2 weeks light microscopy showed that the muscle fibres of soleus became severely atrophied. Electron microscopy after the first week showed, in transverse sections, that the outline of the fibres of soleus was irregular instead of having the normal smooth contour. In the earlier stages of atrophy the basement membrane was in close contact with the sarcolemmal membrane but later when atrophy was more severe there was dissociation of the two membranes. There were then numerous small irregular folds of redundant basement membrane at various places around the muscle fibre similar to those described by Birks, Katz and Miledi (1959) in denervated muscle in the frog. From the end of the 1st week onwards there were changes in the appearances of the Z-lines in most of soteus fibres. The mildest abnormality consisted of the Z-line
Fig. 1. Longitudinal section of soleus muscle fibre at 12 days after the injection of tetanus toxin. The Z-line material (Z) appears rarefied and broken up into shorter than normal segments, x 30,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
171
Fig. 2. In the same soleus as Fig. 1 there were more severe lesions of the Z-line (Z) as shown here, where the Z-line material is spread out in an ill-defined smudge (between arrows). The transverse orientation of the Z-line was often broken at such places, x 30,000.
appearing rarefied, not straight and sometimes broken into shorter than usual segments as if some myofibrils were thinner than normal (Fig. 1). At various places the Z-line material was spread into a smudge which sometimes was as wide as the two adjacent 1-bands together (Fig. 2). In these fibres the usual regular transverse striations were broken or angulated at the points where the smudges appeared. In more severe focal lesions (Fig. 3) there was a total disruption of the sarcomeric arrangements of filaments and Z-line dense material was interspersed with filaments, granules and tubules. Disruptions such as these were usually limited in extent to up to about 6 sarcomeres in length and usually did not involve the full width of the muscle fibre. All gradations of severity of damage to Z-lines were found in the same muscle. Lesions were most severe between the 10th and 21st days but were seen occasionally even at 4 months. They were not found in gastrocnemius fibres. Other abnormalities in fibres of soleus included many dense bodies, probably lysosomes, either singly or within membrane-bound sacs as was described in the preceding paper (Duchen 1973). Lipid and multivesicular bodies were numerous in occasional fibres and a fairly common finding was of irregularly-shaped membranebound spaces containing amorphous material (Fig. 4). Some of these spaces seemed to communicate with T-tubules. In a few fibres of 1 animal killed at 12 days the central region of the fibre was devoid of myofilaments but contained many small and irregularly-shaped mitochondria and contorted bands of tubules. These bands were composed of up to 4 tubules, which were like T-tubules, in width. Changes in soleplate nuclei seen in soleus were described in the preceding paper.
172
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7,
r
Fig. 3. In this soleus muscle fibre at 12 days there is a region of total disruption of the sarcomeric arrange, ment of filaments. The Z-lines are broken up and small patches of dense material derived from them lic intermingled with myofilaments and glycogen granules, ~ 30,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
173
Fig. 4. Transverse section of soleus muscle fibres at 2 weeks after the injection of toxin. Membrane-bound spaces (*) are filled with amorphous material. At several points these spaces appear to communicate with T-tubules (arrows), × 30,000.
174
L.W. DUCHEN
By the 6th 7th week the internal structure of the fibres of soleus was usually normal in appearance. Occasional centrally-placed nuclei were present.
Gastrocnemius The fast muscle fibres in the periphery of gastrocnemius showed a much lesser degree of atrophy than did fibres of soleus. The atrophy was most apparent by the 2nd-3rd week when small folds of redundant basement m e m b r a n e were fairly common. In occasional fibres dense bodies or m e m b r a n e - b o u n d multivesicular bodies were seen but this was an u n c o m m o n finding. N o Z-line abnormalities, very striking changes in slow muscle, were seen in the fast fibres of any of the animals studied. Tubular abnormalities were, however, very common. After the 2nd week transverse sections showed that many fibres contained bands of tubules, which looked like T-tubules, closely applied to each other in pairs or in groups of up to 6 tubules (Fig. 5). These tubules seemed to be transversely orientated. They were not apparently associated with tubules of the sarcoplasmic reticulum. Other abnormalities consisted of aggregates composed of tubules which resembled the lateral cisternae of the triads. These aggregates were first seen in fast muscle fibres of gastrocnemius at 2 weeks. At this stage they were found in only a few of the fibres and usually consisted of 10-20 tubules lying in the long axis of the fibre. At later stages the aggregates usually contained m a n y more tubules and they lay both longitudinally and transversely within the
Fig. 5. Transverse section of gastrocnemius muscle fibre at 3 weeks. Groups of tubules are seen lying between myofilaments.Their appearance and plane of orientation suggests that they are, or are derived from, T-tubules, x 90,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
175
Fig. 6. In this longitudinal section of a muscle fibre of gastrocnemius 4 m o n t h s after the injection of tetanus toxin m a n y tubules lie in aggregations between the myofibrils. Tubules are cut in both transverse and longitudinal planes and some appear to open into dilated sacs, x 30,000.
176
t.. w. I)U('HEN
fibre (Fig. 6). These tubular aggregates were seen in some fibres of all the animals studied including those surviving for 4 months. DISCt~SSION The pathological changes induced by tetanus toxin in the structure of the muscle fibres are probably secondary to its effects on motor nerve terminals at which the release of transmitter is inhibited. For reasons unknown the toxin exerts a more severe effect on nerve terminals of slow muscle fibres than of fast and there is thus a greater degree of impairment of neuromuscular transmission in slow muscle than in fast (Duchen and Tonge 1973). The changes in slow muscle fibres of soleus can, therefore, be interpreted as being secondary to the "functional denervation" caused by the presynaptic block of transmission and these changes would include the atrophy, the activation of lysosomes and the distintegration of Z-lines. The Z-line changes are similar to those described in human cases of tetanism by Eyrich, Agostini, Schulz. Mtiller, Reichenmiller and Wiemers (1967) which suggests that this toxin exerts a similar block of transmission in some types of muscle in man as well as in experimental animals. The changes in Z-line structure are similar to those found in "target" and "target-like" fibres in human muscle with neurogenic atrophy (Engel 1961 ; Shafiq, Milhorat and Gorycki 1967; Schotland 1969). Z-line alterations were also found in denervated muscle in the rat (Pellegrino and Franzini 1963) and in muscle fibres of the mouse paralysed by botulinum toxin (Duchen 1971) in which "functional denervation" is similar to that caused by tetanus toxin. The absence of severe pathological changes in the fibres of gastrocnemius is in keeping with the absence of any severe disturbance of synaptic transmission though there was a temporary mild degree of atrophy. The tubular abnormalities in the fast fibres are similar to those seen after botulinum toxin (Duchen 1971) where atrophy was severe and to those found in fast fibres which were denervated by nerve section and allowed to become reinnervated (author's unpublished observations). It seems possible that in the mouse the formation of tubular aggregates is a non-specific reaction occurring only in fast muscle fibres which have become atrophied and have then recovered their size again and that once these aggregates are formed they remain within the muscle fibre permanently. Groups of tubules have been reported in biopsies of human muscle from patients with a wide variety of clinical conditions (see Mair and Tom6 1972) most of which are thought to be "myopathic" in origin. These tubules are similar in some respects to those seen in the mouse muscles but at present it is not possible to say whether their pathogenesis is similar. The fact that, in the mouse, the tubules are found after recovery from atrophy of neurogenic origin suggests that in some myopathic processes there is also a disturbance of the relationship between nerve and muscle. ACKNOWI.EI)GEMENTS
I should like to thank Dr. R. O. Thompson, Wellcome Research Laboratories, for the gift of the tetanus toxin and Mr. A. J. Davey, Mrs. Betty Smith and Miss Lesley Warwick for the microscopic preparations and photographs.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
177
SUMMARY
Electron microscopic studies were made of slow (soleus) and fast (superficial gastrocnemius) skeletal muscle fibres after the local injection of a sublethal dose of tetanus toxin into the leg of the mouse. Slow muscle fibres, which are paralysed by the toxin, showed severe atrophy, disintegration of Z-lines, abnormalities of tubules and the appearance of lysosomes and multivesicular bodies. Pathological changes in slow fibres persisted for several weeks but within 6 weeks of the administration of toxin their structure was back to normal. Fast muscle fibres, which are not paralysed by sublethal doses of toxin, showed only a mild degree of atrophy. Few abnormalities were found in their internal structure but they did include the appearance of aggregations of tubules. REFERENCES BmKS, R., B. KATZ AND R. MILEDI (1959) Dissociation of the "surface membrane complex" in atrophic muscle fibres, Nature (Lond.), 184: 150%1508. DUCHEN,L. W. (1970 ) Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles, J. Neurol. Neurosurg. Psyehiat., 33: 40--54. DUCI-IEN,L. W. (1971) Changes in the electron microscopic structure of slow and fast skeletal muscle fibres of the mouse after the local injection of botulinum toxin, J. neurol. Sci., 14:61-74. DUCHEN, L. W. (1973) The effects of tetanus toxin on the motor end-plates of the mouse: an electron microscopie study, J. neurol Sci., 19:153-167. DUCI-IEN, L. W. AND D. A. TONGE (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse, J. PhysioL (Lond.), 228 : 15%172. ENGEL,W. K. (1961) Muscle target fibres: a newly recognized sign of denervation, Nature (Lond.), 191 : 389-390. EYRICH, K., B. AGOSTINI, A. SCHULZ,E. MLrLLER, H. NOETZEL, H. E. REICHENMILLERAND g . WIEMERS (1967) Klinische und morphologische Beobachtungen yon Skelettmuskelver/inderungen beim Tetanus, Dtsch. reed. Wschr., 92: 530-540. MAre, W. G. P. AND F. M. S. Tor~ (1972) Atlas of the Ultrastructure of Diseased Human Muscle, ChurchillLivingstone, London, pp. 54-55. PELLEGRINO,A. AND C. FRANZINI (1963) An electron microscope study of denervation atrophy in red and white skeletal muscle fibers, J. Cell Biol., 17: 32%349. SCnOrLAND, D. L. (1969) An electron microscopic study of target fibers, target-like fibers and related abnormalities in human muscle, J. Neuropath. exp. Neurol., 28 : 214-228. SHAFIQ, S. A., A. T. MILI-IORATAND M. A. GORYCKI (1967) Fine structure of human muscle in neurogenic atrophy, Neurology (Minneap.), 17 : 934-948.
169
© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
The Local Effects of Tetanus Toxin on the Electron Microscopic Structure of Skeletal Muscle Fibres of the Mouse L. W. D U C H E N Department of Neuropathology, Institute of Psychiatry and The Maudsley Hospital, De Crespigny Park, London SE5 8AF (Great Britain) (Received 14 December, 1972)
INTRODUCTION
Recent studies have shown that although tetanus toxin causes intense and prolonged muscle spasm, slow muscle fibres are paralysed by the effect of the toxin on motor nerve terminals. In the preceding paper the electron-microscopic changes induced by tetanus toxin in motor end-plates of slow muscle fibres of the mouse were described (Duchen 1973). It was found that end-plates of fast muscle fibres of gastrocnemius were normal. In this paper the changes found with electron microscopy in the muscle fibres themselves are presented. Marked differences in the changes in slow and fast muscle were observed and although fast fibres were not paralysed by the toxin their internal structure became considerably abnormal. MATERIALS AND METHODS
A sublethal dose of tetanus toxin (Wellcome Research Laboratories), sufficient to cause prolonged local tetanism was injected into the muscle of one leg of male albino mice. After survival for periods of time ranging from 3 days to 6 months the animals were killed and tissues prepared for electron microscopy according to the methods described in the preceding paper. More than 40 animals were used in this investigation. The muscles examined by electron microscopy were mainly soleus (slow) and the outer 1-2 mm of gastrocnemius (fast) as well as, in a few cases, extensor digitorum longus. In some cases the muscles were prepared for electron microscopy after being studied in vitro by electrophysiological methods. For light microscopy (in which 140 mice were used) the tissues were prepared according to methods described previously (Duchen 1970). Many normal and control animals given antitoxin were also studied, no abnormalities being found in their muscles. This research was supported by grants from the National Fund for Research into Crippling Diseases, the Muscular Dystrophy Group of Great Britain and the Muscular Dystrophy Associations of America, Inc.
170
L . W . I)UCHEN RESULTS
Clinically there was intense spasm of the muscles of the leg from about 48 hr after the injection of toxin. The local tetanism persisted for up to 4 weeks in some animals. During the first few days there was, in some cases, a slight interstitial inflammatory reaction and a few muscle fibres appeared abnormally basophilic and contained central nuclei. This was interpreted as damage due to the needle and the injection. It was usually in gastrocnemius and always a very minor lesion and probably was of little significance in the development of the changes which were found later. Apart from the local damage, muscle fibres did not look abnormal with either light or electron microscopy during the first week.
Soleus By the end of the first week and then progressively for the next 2 weeks light microscopy showed that the muscle fibres of soleus became severely atrophied. Electron microscopy after the first week showed, in transverse sections, that the outline of the fibres of soleus was irregular instead of having the normal smooth contour. In the earlier stages of atrophy the basement membrane was in close contact with the sarcolemmal membrane but later when atrophy was more severe there was dissociation of the two membranes. There were then numerous small irregular folds of redundant basement membrane at various places around the muscle fibre similar to those described by Birks, Katz and Miledi (1959) in denervated muscle in the frog. From the end of the 1st week onwards there were changes in the appearances of the Z-lines in most of soteus fibres. The mildest abnormality consisted of the Z-line
Fig. 1. Longitudinal section of soleus muscle fibre at 12 days after the injection of tetanus toxin. The Z-line material (Z) appears rarefied and broken up into shorter than normal segments, x 30,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
171
Fig. 2. In the same soleus as Fig. 1 there were more severe lesions of the Z-line (Z) as shown here, where the Z-line material is spread out in an ill-defined smudge (between arrows). The transverse orientation of the Z-line was often broken at such places, x 30,000.
appearing rarefied, not straight and sometimes broken into shorter than usual segments as if some myofibrils were thinner than normal (Fig. 1). At various places the Z-line material was spread into a smudge which sometimes was as wide as the two adjacent 1-bands together (Fig. 2). In these fibres the usual regular transverse striations were broken or angulated at the points where the smudges appeared. In more severe focal lesions (Fig. 3) there was a total disruption of the sarcomeric arrangements of filaments and Z-line dense material was interspersed with filaments, granules and tubules. Disruptions such as these were usually limited in extent to up to about 6 sarcomeres in length and usually did not involve the full width of the muscle fibre. All gradations of severity of damage to Z-lines were found in the same muscle. Lesions were most severe between the 10th and 21st days but were seen occasionally even at 4 months. They were not found in gastrocnemius fibres. Other abnormalities in fibres of soleus included many dense bodies, probably lysosomes, either singly or within membrane-bound sacs as was described in the preceding paper (Duchen 1973). Lipid and multivesicular bodies were numerous in occasional fibres and a fairly common finding was of irregularly-shaped membranebound spaces containing amorphous material (Fig. 4). Some of these spaces seemed to communicate with T-tubules. In a few fibres of 1 animal killed at 12 days the central region of the fibre was devoid of myofilaments but contained many small and irregularly-shaped mitochondria and contorted bands of tubules. These bands were composed of up to 4 tubules, which were like T-tubules, in width. Changes in soleplate nuclei seen in soleus were described in the preceding paper.
172
1~. \V. I)[J('HI!N
7,
r
Fig. 3. In this soleus muscle fibre at 12 days there is a region of total disruption of the sarcomeric arrange, ment of filaments. The Z-lines are broken up and small patches of dense material derived from them lic intermingled with myofilaments and glycogen granules, ~ 30,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
173
Fig. 4. Transverse section of soleus muscle fibres at 2 weeks after the injection of toxin. Membrane-bound spaces (*) are filled with amorphous material. At several points these spaces appear to communicate with T-tubules (arrows), × 30,000.
174
L.W. DUCHEN
By the 6th 7th week the internal structure of the fibres of soleus was usually normal in appearance. Occasional centrally-placed nuclei were present.
Gastrocnemius The fast muscle fibres in the periphery of gastrocnemius showed a much lesser degree of atrophy than did fibres of soleus. The atrophy was most apparent by the 2nd-3rd week when small folds of redundant basement m e m b r a n e were fairly common. In occasional fibres dense bodies or m e m b r a n e - b o u n d multivesicular bodies were seen but this was an u n c o m m o n finding. N o Z-line abnormalities, very striking changes in slow muscle, were seen in the fast fibres of any of the animals studied. Tubular abnormalities were, however, very common. After the 2nd week transverse sections showed that many fibres contained bands of tubules, which looked like T-tubules, closely applied to each other in pairs or in groups of up to 6 tubules (Fig. 5). These tubules seemed to be transversely orientated. They were not apparently associated with tubules of the sarcoplasmic reticulum. Other abnormalities consisted of aggregates composed of tubules which resembled the lateral cisternae of the triads. These aggregates were first seen in fast muscle fibres of gastrocnemius at 2 weeks. At this stage they were found in only a few of the fibres and usually consisted of 10-20 tubules lying in the long axis of the fibre. At later stages the aggregates usually contained m a n y more tubules and they lay both longitudinally and transversely within the
Fig. 5. Transverse section of gastrocnemius muscle fibre at 3 weeks. Groups of tubules are seen lying between myofilaments.Their appearance and plane of orientation suggests that they are, or are derived from, T-tubules, x 90,000.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
175
Fig. 6. In this longitudinal section of a muscle fibre of gastrocnemius 4 m o n t h s after the injection of tetanus toxin m a n y tubules lie in aggregations between the myofibrils. Tubules are cut in both transverse and longitudinal planes and some appear to open into dilated sacs, x 30,000.
176
t.. w. I)U('HEN
fibre (Fig. 6). These tubular aggregates were seen in some fibres of all the animals studied including those surviving for 4 months. DISCt~SSION The pathological changes induced by tetanus toxin in the structure of the muscle fibres are probably secondary to its effects on motor nerve terminals at which the release of transmitter is inhibited. For reasons unknown the toxin exerts a more severe effect on nerve terminals of slow muscle fibres than of fast and there is thus a greater degree of impairment of neuromuscular transmission in slow muscle than in fast (Duchen and Tonge 1973). The changes in slow muscle fibres of soleus can, therefore, be interpreted as being secondary to the "functional denervation" caused by the presynaptic block of transmission and these changes would include the atrophy, the activation of lysosomes and the distintegration of Z-lines. The Z-line changes are similar to those described in human cases of tetanism by Eyrich, Agostini, Schulz. Mtiller, Reichenmiller and Wiemers (1967) which suggests that this toxin exerts a similar block of transmission in some types of muscle in man as well as in experimental animals. The changes in Z-line structure are similar to those found in "target" and "target-like" fibres in human muscle with neurogenic atrophy (Engel 1961 ; Shafiq, Milhorat and Gorycki 1967; Schotland 1969). Z-line alterations were also found in denervated muscle in the rat (Pellegrino and Franzini 1963) and in muscle fibres of the mouse paralysed by botulinum toxin (Duchen 1971) in which "functional denervation" is similar to that caused by tetanus toxin. The absence of severe pathological changes in the fibres of gastrocnemius is in keeping with the absence of any severe disturbance of synaptic transmission though there was a temporary mild degree of atrophy. The tubular abnormalities in the fast fibres are similar to those seen after botulinum toxin (Duchen 1971) where atrophy was severe and to those found in fast fibres which were denervated by nerve section and allowed to become reinnervated (author's unpublished observations). It seems possible that in the mouse the formation of tubular aggregates is a non-specific reaction occurring only in fast muscle fibres which have become atrophied and have then recovered their size again and that once these aggregates are formed they remain within the muscle fibre permanently. Groups of tubules have been reported in biopsies of human muscle from patients with a wide variety of clinical conditions (see Mair and Tom6 1972) most of which are thought to be "myopathic" in origin. These tubules are similar in some respects to those seen in the mouse muscles but at present it is not possible to say whether their pathogenesis is similar. The fact that, in the mouse, the tubules are found after recovery from atrophy of neurogenic origin suggests that in some myopathic processes there is also a disturbance of the relationship between nerve and muscle. ACKNOWI.EI)GEMENTS
I should like to thank Dr. R. O. Thompson, Wellcome Research Laboratories, for the gift of the tetanus toxin and Mr. A. J. Davey, Mrs. Betty Smith and Miss Lesley Warwick for the microscopic preparations and photographs.
EFFECTS OF TETANUS TOXIN ON EM STRUCTURE OF SKELETAL MUSCLE FIBRES
177
SUMMARY
Electron microscopic studies were made of slow (soleus) and fast (superficial gastrocnemius) skeletal muscle fibres after the local injection of a sublethal dose of tetanus toxin into the leg of the mouse. Slow muscle fibres, which are paralysed by the toxin, showed severe atrophy, disintegration of Z-lines, abnormalities of tubules and the appearance of lysosomes and multivesicular bodies. Pathological changes in slow fibres persisted for several weeks but within 6 weeks of the administration of toxin their structure was back to normal. Fast muscle fibres, which are not paralysed by sublethal doses of toxin, showed only a mild degree of atrophy. Few abnormalities were found in their internal structure but they did include the appearance of aggregations of tubules. REFERENCES BmKS, R., B. KATZ AND R. MILEDI (1959) Dissociation of the "surface membrane complex" in atrophic muscle fibres, Nature (Lond.), 184: 150%1508. DUCHEN,L. W. (1970 ) Changes in motor innervation and cholinesterase localization induced by botulinum toxin in skeletal muscle of the mouse: differences between fast and slow muscles, J. Neurol. Neurosurg. Psyehiat., 33: 40--54. DUCI-IEN,L. W. (1971) Changes in the electron microscopic structure of slow and fast skeletal muscle fibres of the mouse after the local injection of botulinum toxin, J. neurol. Sci., 14:61-74. DUCHEN, L. W. (1973) The effects of tetanus toxin on the motor end-plates of the mouse: an electron microscopie study, J. neurol Sci., 19:153-167. DUCI-IEN, L. W. AND D. A. TONGE (1973) The effects of tetanus toxin on neuromuscular transmission and on the morphology of motor end-plates in slow and fast skeletal muscle of the mouse, J. PhysioL (Lond.), 228 : 15%172. ENGEL,W. K. (1961) Muscle target fibres: a newly recognized sign of denervation, Nature (Lond.), 191 : 389-390. EYRICH, K., B. AGOSTINI, A. SCHULZ,E. MLrLLER, H. NOETZEL, H. E. REICHENMILLERAND g . WIEMERS (1967) Klinische und morphologische Beobachtungen yon Skelettmuskelver/inderungen beim Tetanus, Dtsch. reed. Wschr., 92: 530-540. MAre, W. G. P. AND F. M. S. Tor~ (1972) Atlas of the Ultrastructure of Diseased Human Muscle, ChurchillLivingstone, London, pp. 54-55. PELLEGRINO,A. AND C. FRANZINI (1963) An electron microscope study of denervation atrophy in red and white skeletal muscle fibers, J. Cell Biol., 17: 32%349. SCnOrLAND, D. L. (1969) An electron microscopic study of target fibers, target-like fibers and related abnormalities in human muscle, J. Neuropath. exp. Neurol., 28 : 214-228. SHAFIQ, S. A., A. T. MILI-IORATAND M. A. GORYCKI (1967) Fine structure of human muscle in neurogenic atrophy, Neurology (Minneap.), 17 : 934-948.