Z. Zellforsch. 102, 466--482 (1969)

Effect of Oculomotor and Trigeminal Nerve Section on the Uhrastructure of Different Myoneural Junctions in the Rat Extraocular Muscles H . T]~RXVXINEN a n d K . I-IuIKURI$ Department of Anatomy, University of Helsinki, Helsinki 17, Finland Received September 8, 1969

Summary. The intramuscular nerves and myoneural junctions in the rat rectus superior, medialis and inferior muscles from 10 hours to about 10 days after section of the trigeminal and oculomotor nerves were studied with the electron microscope. Two different kinds of myoneural junctions are to be observed; one type derives from myelinated nerves and is similar to the ordinary myoneural junctions (motor end plates) of other striated skeletal muscles, while the other type derives from unmyelinated nerves, is smaller in size and has many myoneural synapses distributed along a single extrafusal muscle fibre. Section of the trigeminal nerve caused no changes in the myoneural synapses. After section of the oculomotor nerve degenerative changes occur in both the myelinated and unmyelinated nerves and in both types of myoneural junctions. In the axon terminals of both the myelinated and unmyelinated nerves the earliest changes are to be observed 10 to 15 hours after section of the nerve. First, swelling of the axoplasm, fragmentation of microtubules and mierofilaments and swelling of mitochondria takes place, somewhat later agglutination of the axonal vesicles and mitoehondria. The axon terminals are separated from the postsynaptic muscle membrane by hypertrophied teloglial cells about 24 hours after section of the nerve. The debris of the axon terminals is usually digested by the teloglial cells within 42 to 48 hours in both types of myoneural junction. Changes in the postsynaptic membrane arc observed in the myoneural junctions of the unmyelinated nerves as disappearance of the already earlier irregular infoldings, whereas no changes take place in the infoldings of the motor end plates. The postsynaptic sarcoplasm and its ribosomal content increase somewhat. The earliest changes occur along unmyelinated axons 10 to I5 hours and along myelinated axons 15 to 24 hours after nerve section. The unmyelinated axons are usually totally digested within 48 hours, whereas the myelinated axons took between 48 hours and 4 days to disappear. The degeneration, fragmentation and digestion of the myelin sheath begin between 24 and 42 hours and still continues l0 days after the operation. The results demonstrate that in the three muscles studied structures underlying the physiologically well known double innervation of the extraoccular muscles are all part of the oculomotor system. Key- Words: Extraoccular muscles - - Innervation - - Nerve Section - - Motor Endplates - Myoneural Junctions. Introduction I t has b e e n d e m o n s t r a t e d w i t h h i s t o l o g i c a l s t a i n i n g t e c h n i q u e s t h a t b o t h m y e l i n a t e d a n d u n m y e l i n a t e d n e r v e s t e r m i n a t e in t h e e x t r a o c u l a r m u s c l e s in all t h e m a m m a l s so far s t u d i e d a n d t h a t t h e u n m y e l i n a t e d n e r v e s h a v e m u l t i p l e e n d i n g s a l o n g one m u s c l e fibre (Boeke, 1926; H i n e s , 1931; W o o l l a r d , 1931; H i r a n o , 1941; K i r s c h e , 1951; F e i n d e l et al., 1952; A l p e r n a n d W o l t e r , 1956; K a d a n o f f a n d S p a s s o v a , 1963; Dziallas, 1965; K a d a n o f f , 1967). H i s t o c h e m i c a l * We are grateful to Professor Antti Telkk~, M.D. Head of the Electron Microscope Laboratory, University of Helsinki, for permission to use the facilities of the laboratory.

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cholinesterase techniques (Kupfer, 1960; Hess, 1961, 1962; Hess and Pilar, 1963; Cheng, 1963 ; Wolter and O'Keefe, 1963 ; Wolter, 1964; Zenker and Anzenbacher, 1964; Dietert, 1965; N a m b a et al., I968a) have likewise shown two differently innervated muscle fibres; the one type possessing one large myoneural junction structurally similar to t h a t of other striated skeletal muscles (,,motor end plate"), the other type being unique among extrafusal muscle fibres of mammalian striated muscles in containing multiple small myoneural junctions in a single muscle fibre. Thus both singly and multiply innervated extrafusal muscle fibres are present, and are somewhat different in ultrastructural appearance (c/. Ter~vi~inen, 1968) possibly secondary to the type and demands of their innervation seen physiologically as the well known fast and slow contractions of the extraocular muscles. Electrophysiological studies suggest that these two movement systems are totally independent of each other (Rashbass, 1961, Robinson, 1965; Dement, 1964) and behave as if they were regulated by separate neuronal systems (Robinson, 1964) which, in addition, can be separately paralysed in diseases of the central nervous system (Davson, 1962; Starr, 1967). I t is extremely important to evaluate the anatomical basis of this dual innervation, from myelinated nerves on the one hand and from unmyelinated nerves in the other, to a greater degree than has been done at the present. The few denervation studies that have as yet been carried out have mainly been made with out-of-date techniques, leaving uncertain the origin of the unmyelinated nerves ending as multiple small myoneural junctions in the extraocular muscles. Fibre analysis of the nerves leading to the extraocular muscles of various mammals has shown two fibre sizes, for example in the oculomotor nerve about 4 - - 6 ~ and 9--11 ~ (Bj6rkman and Wohlfart, 1936 ; Donaldson, 1960), which would explain the origin of both the unmyelinated and myelinated nerves. However, section of the rabbit oculomotor nerve is reported only to cause degeneration of the fine structure of the myelinated nerves and their myoneural junctions in the rectus superior muscle, while leaving the unmyelinated nerves intact (Cheng et al., 1967; Cheng-Minoda et al., 1968). Workers using older histological techniques, on the other hand, have reported that the unmyelinated nerves and their endings degenerated partially (Tozer and Sherington, 1910) or totally (Hines, 1931) after section of the oculomotor nerve. The aim of the present experiments, in which the trigeminal and oculomotor nerves were cut, was both to evaluate the anatomical pathway of the unmyelinated nerves ending as small multiple myoneural junctions and to study the so far unknown changes in their fine structure after nerve section. The changes in the myoneural junctions derived from myelinated nerves will not be comprehensively reported in the present work because they have been carefully disclosed earlier both in the extraocular muscles (Cheng-Minoda et al., 1968) and in other striated muscles (eg. Nickel and Waser, 1968). Material and Methods

The material consisted of 16 adult Sprague-Dawley rats. The denervation was performed under ether anaesthesia under a dissecting microscope. The left trigeminal and oculomotor nerves were approached by an extracranial route after resection of a small piece of the zygomatic arch and removal of the lateral portion of the Harderian gland. In 5 of the rats the

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trigeminal nerve was severed immediately after its arrival in the orbital cavity by resecting a small piece of the nerve. From eleven of the rats a small piece was removed from the oculomotor nerve immediately after the nerve had entered the orbit about I mm centrally of the ganglion ciliarc. The success of the latter operation was confirmed by maximal dilatation of the ipsilateral pupil, external strabismus, absence of the light reflex and slight ptosis of the upper lid. The contralateral unoperated side served as a control. The animals were killed by decapitation under light ether anaesthesia 10, 15, 24, 42, 48 hours and 4, 7, and 10 days after partial resection of the oculomotor nerve, and the superior, medial and inferior recti were removed. The same muscles were removed identically 10, 15, 24, 42 hours and 7 days after surgical interference with the trigeminal nerve. Immediately after the animals were killed the muscles were cut transversely into three small pieces and fixed for electron microscopy for two hours in 2.5 % glutaraldehyde buffered at pH 7.4 with phosphate. The specimens were washed in 0.25 M sucrose for 12 hours to 6 days, postfixed at pit 7.4 with 1% osmium tetroxide phosphate buffer, dehydrated in a graded ethyl alcohol series, and embedded in Epon 812 (Luft, 1961). The sections were afterstained with the lead citrate (Reynolds, 1963), and examined with a Philips EM-200 electron microscope. Results

Normal Structure My o n eu ral j u n c ti o n s which were d e r i v e d f r o m either m y e l i n a t e d or unm y e l i n a t e d nerves are observed in all th e th r e e e x t r a o c u l a r muscles studied. T h e m y e l i n a t e d n er v e form a m y o n e u r a l j u n c t i o n in a p p r o x i m a t e l y t h e middle t h i r d of t h e muscle fibres, which possess n u m e r o u s large m i t o c h o n d r i a an d a well d e v e l o p e d sarcoplasmic r e t i c u l u m w i t h T tubules. Th e large s y n a p t i c a x o n t e r m i n a l is filled with t h e c o m m o n " e m p t y " 500 A s y n a p t i c vesicles a n d m i t o ch o n d r i a, an d is apposed to t h e t h i c k e n e d a n d folded p o s t s y n a p t i c m e m b r a n e of t h e muscle fibre (Fig. 1). On t h e other hand, t h e u n m y e l i n a t e d nerves f o r m n u m e r o u s m y o n e u r a l j u n ct io n s along a considerable length of t h e muscle fibre. My o n eu r al j u n ct i o n s of this t y p e are observed m a i n l y in t h e distal t h i r d of t h e extraocular muscles, a n d h e n c e f o r w a r d will be called " m u l t i p l e e n d i n g s " in t h e present work to clearly distinguish t h e m f r o m th e o r d i n a r y m y o n e u r a l j u n ct i o n s d e r i v e d f r o m m y e l i n a t e d nerves. T h e fine s t r u c t u r e of t h e multiple endings also differs f r o m t h a t of the m y o n e u r a l j u n c t io n s in th e f u r t h e r respect t h a t the a x o n term i n al s are small in size a n d a m o n g n u m e r o u s e m p t y 500 A vesicles also

Fig. 1. Myelinated nerve (M) forming a myoneural junction in the extraocular muscles by apposing to a regularly folded and thickened postsynaptic muscle plasma membrane (PM). Note the large size of the terminal axon filled with vesicles 500 A in diameter, the zone of mitochondria between the vesicles of the axon terminal and the neurotubules and neurofibrils of the motor axon. TC Teloglial cell. • 20,000 Fig. 2. Unmyelinated axon (A) enveloped by a Schwann cell (S) making a myoneural junction with a slow-type muscle fibre. Myoneural junctions of this type are called multiple ending in the present work. Note that the axon terminals (AX) are rather small compared with the myoneural junction from myelinated nerve in Fig. 1, and that the postsynaptic muscle membrane is irregularly folded in this particular case. In the axon terminal empty vesicles about 500 A in diameter dispersed rather homogeneously throughout the axoplasmic matrix, and some mitochondria and neurotubules are seen. In the axon (A), one larger vesicle of about 1000 A with a dense core (arrow) is seen close to the myelinated figures. Note also the rather dark cytoplasm of the Schwann cells in the teloglial region (T). F fast-type muscle fibre. • 13,600

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contain some larger dense-cored vesicles in diameter about 1000 A. The postsynaptic m e m b r a n e is thickened as in the myoneural junction b u t the a m o u n t of postsynaptic infolding varies considerably, sometimes even being absent, and is never so regular as in the myoneural junction. The small terminal axons are

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either g a t h e r e d close t o each o t h e r to form a few small groups (Type I) or widely s e p a r a t e d from each o t h e r (Type I I , see Teriiv~inen, 1968). I n a d d i t i o n , these a x o n t e r m i n a l s are o b s e r v e d to s y n a p s e to a muscle fibre in which t h e mitochondrial c o n t e n t a n d t h e a m o u n t of sarcoplasmic r e t i c u l u m are less t h a n in t h e muscle fibres i n n e r v a t e d b y the m y e l i n a t e d nerves (Fig. 2).

Structure alter Section oI the Trigeminal Nerve T r i g e m i n a l nerve section d i d n o t cause d e g e n e r a t i v e changes in t h e m y o n e u r a l j u n c t i o n s or m u l t i p l e endings during t h e 10-hour to 7 - d a y follow-up period. I n two of t h e three muscles r e m o v e d 15 hours after t h e operation, p a r t i a l degeneration of b o t h m y o n e u r a l j u n c t i o n s a n d m u l t i p l e endings is o b s e r v e d in one of t h e animals. Since none of t h e o t h e r specimens showes a n y signs of d e g e n e r a t i v e m y o n e u r a l synapses, this o b s e r v a t i o n is i n t e r p r e t e d as p r o b a b l y resulting from p a r t i a l lesion of t h e o c u l o m o t o r nerve (see below) during t h e t r i g e m i n a l operation.

Structure alter Section o/the Oculomotor Nerve A f t e r section of t h e o c u l o m o t o r nerve s t r u c t u r a l changes were o b s e r v e d in t h e axon, in t h e s y n a p t i c a x o n t e r m i n a l , in t h e S c h w a n n cells a r o u n d t h e axons a n d t h e s y n a p t i c a x o n t e r m i n a l ( " t e l o g l i a cell"), a n d in t h e muscle fibre. Section of t h e o c u l o m o t o r nerve causes d e g e n e r a t i o n of t h e m y o n e u r a l j u n c t i o n s a n d m u l t i p l e endings in all the t h r e e muscles studied. The t i m e sequence of d e g e n e r a t i o n of t h e a x o n a n d of the s y n a p t i c a x o n t e r m i n a l is different in t h e m y e l i n a t e d nerves, b u t r a t h e r similar in t h e u n m y e l i n a t e d nerves. On t h e o t h e r h a n d , because similarities are o b s e r v e d in t h e sequence a n d velocity of degener a t i o n of t h e a x o n t e r m i n a l s of b o t h t h e m y o n e u r a l j u n c t i o n s a n d t h e m u l t i p l e endings, the changes o b s e r v e d along the two t y p e s of nerves are also briefly r e p o r t e d here. Changes in t h e Multiple E n d i n g s a n d U n m y e l i n a t e d Nerves The m a t r i x of t h e a x o n t e r m i n a l of t h e m u l t i p l e endings is less osmiophilic t h a n in t h e u n o p e r a t e d controls 10 hours after nerve section, h a v i n g a s o m e w h a t pale a n d swollen a p p e a r a n c e (Fig. 3). Some fine filaments are seen in t h e m a t r i x Fig. 3. In this multiple ending two axon terminals (AX) are seen 10 hours after section of the oculomotor nerve. The axonal vesicles are unevenly distributed and tend to form small aggregations, and the axoplasmic matrix appears somewhat pale and swollen. The mitochondria and microtubules look normal. The teloglial cell (TC) appears pale and hypertrophied, and contains fine cytoplasmic filaments and a few microtubules. The muscle fibre appears to be unaltered. • 13,600 Fig. 4. Part of a multiple ending at high magnification 15 hours after section of the oculomotor nerve, demonstrating absence of neurotubules and fragmentation of neurofilaments. The intra-axonal vesicles appear structurally normal, although their tendency to aggregate close to each other is evident. The mitochondria are slightly hypertrophied. • 34,000 Fig. 5. Remnants of what were probably two axon terminals are seen in this multiple ending 24 hours after section of the oculomotor nerve. One of the axon terminals has lost the axonal plasma membrane (AX), but in the other terminal the teloglial cell membrane and the plasma membrane of the axon are still distinguishable, although the membrane of the terminal axon appears to be distorted (arrow). Note also the myelin-like figures in the teloglial cell. TC teloglial cell. MF muscle fibre. • 20,000

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of the axon terminal, the mitochondria and neurotubules appear intact but the intra-axonal vesicles tend to aggregate into clumps. The teloglial cells are hypertrophied but no changes in the postsynaptic membrane or sarcoplasm are observed. Between 10 and 15 hours after section of the oeulomotor nerve the neurotubules and neurofilaments are fragmented and decomposing, the mitochondria appear swollen and the aggregation of the axonal vesicles has proceeded further (Fig. 4). The changes oberved varies from one junction to another 15 and especially 24 hours after the operation. The axolemma beging to disappear (Fig. 5) and the swollen axoplasm, possibly fluid in vivo, is mostly seen without the axonal membrane in side the teloglial cells (Fig. 6). The membrane-containing material, such as mitochondria and vesicles, form dark intensely osmiophilie, aggregations inside the teloglial cell, there being usually no synaptic contact between the axon and the postsynaptie membrane (Fig. 6). Some dense structureless dark aggregations of partially digested axon contents are sometimes still seen inside the hypertrophied and pale teloglial cells containing increased amounts of cytoplasmic microtubules 42 and also rarely 48 hours after section of the oculomotor nerve, although the axonal debris has almost always totally disappeared and been digested by the tcloglial cells by that time. The previous site of the junction between the unmyelinated nerves and the muscle fibre is very difficult to detect after the disintegration and disappearance of the axon remnants, because of its small size and variable appearance. In places, however, painstaking search reveals Schwann cells quite close to the slow-type muscle fibre, which show regionally increased thickening of the muscle plasma membrane (Fig. 7). At such places, probably representSng the previous site of a multiple ending, the postsynaptic membrane is flat without infoldings but. no other changes are to be seen even up to 10 days after nerve degeneration. The amount of the postsynaptic sarcoplasm and its content of free ribosomes as well as rough endoplasmic reticulum appears to be slightly increased. The teloglial cells are no longer hypertrophied. In the extraocular muscles of normal unoperated animals, both myelinated and unmyelinated nerves are seen inside the endoneural tubes (Fig. 8). Mitochondria, neurofilaments and neurotubules, as well as some dense-cored vesicles, occur in the axoplasm of the unmyelinated nerves, which are invested by the Schwann cells. The cytoplasm of the Schwann cells is darker than that of the axons, and in the cytoplasm mitochondria, filaments, microtuhules, some glycogen, free ribosomes and granular endoplasmic reticulum are present. Along the umnyelinated nerves 10 hours after section of the oculomotor nerve degenerative changes are .seen, such as dissolution of neurofilaments and neurotubules with concomitant increase of the investing Schwann cell cytoplasm. The Schwann cells are hypertrophied, the amount of intracellutar nficrotubules and filaments has increased, and the aggregation of axoplasmic debris and mitochondria inside the Schwann cells is always marked 15 to 24 hours after the operation (Fig. 9). Between 42 and 48 hours after the operation digestion of the axon remnants is usually totally accomplished and unmyelinated axons are no longer visible. The size of the Schwann cells diminishes after the 4 t h postoperative day and thereafter only Schwann cell processes containing increased amounts of intracellular microtubules are seen inside the endoneural tube (Fig. 10).

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Fig. 6. Probable site of multiple ending 24 hours after section of the oculomotor nerve. Note the dark, intensely osmiophilie aggregations composed of partially decomposed mitochondria and axonal vesicles (arrow) inside the teloglial cell rich in intracellular microtubules and filaments (TC). There is no membrane between the aggregate and the teloglial cell and the synaptic cleft is formed by the teloglial membrane and postsynaptic muscle (MF) plasma membrane. The pale areas inside the teloglial cells resemble the pale swollen axoplasmic matrix, being possibly fluid in vivo. • 20,000 Fig. 7. Probable site of multiple ending 10 days after section of the oculomotor nerve. The Schwann cell chain is seen in close contact with the muscle fibre, the plasma membrane of which shows regionally increased electron density (arrows) and is devoid of postsynaptic infoldings. There is some collagen between the Schwann cells and the muscle membrane. The Schwann cell processes are no longer hypertrophied and show the same electron density as before the operation. • 13,600 Changes in the M y o n e u r a l J u n c t i o n s and M y e l i n a t e d N e r v e s T h e m a t r i x of t h e a x o n t e r m i n a l a n d the m i t o c h o n d r i a a p p e a r s o m e w h a t swollen, a n d the i n t r a - a x o n a l vesicles a n d lnitochondria t e n d to a p p r o a c h each other, b u t no o t h e r changes are usually e v i d e n t 10 to 15 hours after t h e

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Fig. 8. Myelinated (M) and unmyelinated nerves (UN) both invested by Schwann cells (SC) x inside the endoneural tube (E). The axonal matrix appears paler than that of the Schwann cells, and in both the myelinated and unmyeIinated nerves neurofilaments, neurotubules, and some mitochondria can be seen. In the unmyclinated nerves occasional dense-cored vesicles are also visible (arrow). The Schwann cells, where they invest, the unmyelinated axons, form typical mesaxons. In contrast to the axoplasm, free ribosomes and granular endoplasmie reticulum are seen in the cytoplasm of the Schwann cells (ER). C Collagen. • 13,600 Fig. 9. Intramuscular myelinated and unmyelinated nerves 24 hours after section of the oculomotor nerves, showing the difference in degeneration rate. In the myelinated nerves, detachment of the axon from the myelin can be seen, the mitoehondria appear somewhat swollen, and the Schwann cell slightly hypertrophied, but the myelin sheath is normal (compare with the normal structure in Fig. 8). On the other hand, the unmyelinated axon (A) is visible only as a dark aggregation consisting mainly of mitochondria inside a greatly hypertrophied and pale Sehwann cell (SC). In the Schwa.nn cell numerous intracellular tubules and fine filaments can be seen. Some membranous material suggestive of decomposing axonal membrane is seen close to the aggregation. • 34,600 Fig. 10. Mixed myelinated and umnyelinated nerves l0 days after section of the oculomotor nerve. At the previous site of unmyelinated nerves (compare with the normal structure in Fig. 8) only Schwann cell processes (SC) are seen, containing ribosomes, mitochondria, endoplasmie reticulum and intracellular microtubules and microfilaments. There are still remnants of degenerating myelin (M) in abundance inside hypertrophied Schwann cells. • 20,000 Fig, 11. The myoneural junction 4 days after section of the oculomotor nerve, At the previous site of the terminal axon only processes of the teloglial cells (TC) are present. The infoldings of the postsynaptie muscle membrane appear normal. In the postsynaptie sarcopla.sm the mitochondria are mainly round, the amount of the sureoplasm is large, and in the sarcoplasm numerous free ribosomes and folds of granular endoplasmic retieulum can be seen among the typical agr0mutar ones. • 13,600

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operation. The plasma membrane of the axon terminal is usually invisible, the mitochondria and intra-axonal vesicles form a dense mass of debris inside the teloglial cell which surround the axon terminal and separate it from the postsynaptic membrane 24 hours after the operation. The debris of the former a x o a terminals is almost totally digested by the teloglial cells in most of the myoneural junctions examined 42 to 48 hours after the operation. On the 4 t h day after the operation, the axon remnants have always disappeared and the postsynaptic sarcoplasm and its content of free ribosomes is increased (Fig. 11). Some rough endoplasmic reticulum appeares in the postsynaptic sarcoplasm, the mitochondria are round, and the tubules of the sarcoplasmic reticulum are somewhat wider than in the control muscles. There are no changes in the infoldings of the postsynaptic membrane such as were observed in the multiple endings. The total degeneration of the myclinated part of the axon is slower and causes some increase of the Schwann cell cytoplasm and of its intracellular organelles 15 hours after the operation, while at places the axon become detached from the myelin, although the myelin and the axon usually have a normal fine structure. The axoplasm is sometimes slightly changed at 15 hours but usually only after 24 hours its osmiophilicity increases, and axoplasmic structures such as mitochondria are swollen and partially agglutinated inside the still apparently intact myelin sheath. The axon membrane is usually still visible, although detached from the myelin sheath. Concomitantly with the further increase of the cytoplasm, the microtubules and endoplasmic reticulum of the Schwann cell between 24 and 48 hours after the operation, agglutination of the intra-axonal membranous material is always marked and resultes in intensely osmiophilic aggregates inside a pale matrix. At this time some disintegration and fragmentation of the myelin sheath is also observed sometimes 24 hours but usually 42 hours after the operation. As a rule, the intra-axonal material is no longer visible on the 4 t h postoperative day (Fig. 10), whereas the disintegration of the myelin sheath proceedes further and myelin remnants inside the greatly hypertrophied Schwann cells are still observed in abundance on the 10th postoperative day when the present experiment ended. Discussion

The present study demonstrates that section of the oculomotor nerve causes degenerative changes in the fine structure of the axon terminals of the myoneural junctions and multiple endings, of the myelinated and unmyclinated axons, and in the Schwann cells along both types of axons, as well as in the postsynaptic muscle fibre. The structural changes observed, and likewise the time sequence of their disappearance, are essentially identical in the axons and in their terminals in both types of myoneural junctions, although their occurrence is slightly slower in the larger terminals of the myelinated axons and particularly inside the myelin sheath. The changes observed include swelling of the axoplasm and intra-axonal mitochondria, agglutination of the vesicles of the axon terminals, dissolution of the axon membrane and interruption of the axonal continuity, resulting in intensely osmiophilic, dark aggregations of axonal remnants inside the Schwann cells along the course of the former nerves up to their terminals.

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These structural changes are similar to those that have been reported to occur after nerve section in various degenerating synapses (de Robertis, 1956; c/. also Alksne et al., 1966) including the myoneural junction (Bauer et al., 1962; Reger, 1959 ; Birks et al., 1960 ; Nickel and Waser, 1968, 1969 ; Song, 1968 ; Lentz, 1969), and along degenerating nerves (Vial, 1958; Ohmi, 1959; Glimstedt and Wohlfart, 1960; Lampert, 1967; c]. also Alksne et al., 1966). Excluding the absence of of postsynaptic infoldings in the multiple endings, the structural changes observed in the postsynaptie region as slight regional hypertrophy of the postsynaptie sareoplasm and increase of its content of free and membrane-bound ribosomes are similar in the two types of myoneural junctions. The follow-up period was too short in the present work to demonstrate more profound changes (Nickel and Waser, 1968) in the sareoplasm of the postsynaptie muscle fibre. The disappearance of the postsynaptie infoldings from the multiple endings during degeneration might indicate that the infoldings, in contrast to the myoneural junction, are not a characteristic feature of these endings. This is also seen in the intact multiple endings, which possess infoldings that are very irregular and variable in both structure and amount. The present anatomical observations, showing that agglutination of synaptie vesicles occurs within 24 hours, indicate that in the both types of myoneural junctions in the extraoeular muscles functional capacity is lost within 24 hours of nerve section (Usherwood et al.., 1968). In regard to the velocity of degeneration, which was observed in the present study to be rather high (total disappearance of axon terminals often took only about 2 days), figures ranging from 2 to 7 days are reported in the literature. These discrepancies are probably partly due to the fact that different muscles have been studied in different animals, but it also remains to be shown what part is played by the length of the distal stump of the degenerating nerve on the degeneration velocity of the synaptic axon terminal. In addition, such factors as a change of temperature (Usherwood et al., 1968) and changes in the blood flow through the tissue (Garret, 1969) cause very great differences in the rate of degeneration of the axons. Most of the earlier light microscopic research done with classical histological techniques on a variety of animals (see p. 466)suggested that both myelinated and unmyelinated nerves form nerve endings in the extraocular muscle fibres, the latter type being multiple in the muscle fibre and usually referred to as grapelike endings (" terminaison en grappe ") or multiple endings (c]. Ter/~v/s 1968). Since then, the electron microscope has revealed the presence of an ordinary myoneural junction (motor end plate) in the extraocular muscles of a wide variety of mammals (Cheng and Breinin, 1965; Pilar and Hess, 1966; Cheng et al., 1967; v. Dfiring, 1967; Cheng-Minoda et al., 1968; Mukuno, 1968; Namba et al., 1968b; Ter~v~inen, 1968). Although structurally different myoneural junctions (Cheng and Breinin, 1965; Mayr et al., 1966; Pilar and Hess, 1966; Cheng-Minoda et al., 1968; Namba et al., 1968b) of as many as six types (Mukuno, 1968) have been observed in the extraocular muscles with the electron microscope, except in the rat (Ter~vKinen, 1968), it remains uncertain whether only structurally different myoneural junctions have been described or the observations have really been concerned with functionally different myoneural junctions classifiable in regard of the type of nerve from which these were derived.

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Possibly because the structure of the different myoneural junctions in the extraocular muscles varies considerably (Zenker and Gruber, 1967; Tertiviiinen, 1968), the literature available on the anatomicM basis of the innervation of the extraocular muscle fibres is somewhat confusing. I n addition, the early histological gold and silver staining and methylene blue techniques (for earlier literature see e. g. Hines, 1931 ; Woollard, 1931 ; Kirsche, 1951) did not always clearly reveal that mixed trunks of myelinated and unmyelinated nerves really form completely independent innervation systems of the extraocular muscle fibres. Possibly this led m a n y of the earlier authors to report these as "accessory endings", "terminal discs", or endings arising from unmyelinated side branches of myelinated nerves. The myoneural junctions were not only classified at t h a t time as epilemmM or hypolemmal, but sensory, motor, parasympathetic (autonomic) as well as sympathetic innervation (Wolter, 1951, 1954; c[. also Davson, 1962) of the extraocular muscle fibres were considered possible. Later studies with the rat have clearly shown the difference between the myoneural junction and the multiple endings, and demonstrated t h a t the ultrastructure of the multiple endings deriving from umnyelinatcd nerves is t h a t of a cholinergic motor excitatory synapse (Tertiv~inen, 1968). Because multiple endings can also be seen to have acetylcholinesterase activity in the electron microscope (Ters 1969a), the evidence now available strongly suggests their cholinergic excitatory nature. Because section of the oculomotor nerve but not of the trigeminal nerve, causes degeneration of both the myoneural ]unctions and the multiple endings in the extrafusat muscle fibres in the three extraocular muscles studied, which are generally known to be supplied by the oculomotor nerve in the rat, the present. work establishes beyond doubt t h a t the anatomical pathway of the physiologically well known double cholinergic excitatory innervation of the extraocular muscles is based on the oculomotor system. I n addition, the possibility t h a t the unmyelinated nerves ending as small multiple endings in the extraocular muscles take their origin from the superior cervical ganglion (Armaly, 1959) or ganglion ciliate (Kure et al., 1927; Brecher and Mitchell, 1957) seems to be excluded, since exstirpation of the ganglion ciliate (Tert~v~inen and Huikuri, 1969) or bilateral removal of the superior cervical ganglion (Tergvt~inen and Rauanheimo, 1969) does not result in changes in their fine structure in the rat. In our opinion, an exception to the above rule, there being but these two synapse types in the rat extraocular muscles, is that a very few of the unmyelinated nerves form specialized myoneural contacts with closemembrane apposition (synaptic cleft of only 100 A or less, devoid of postsynaptie membrane thickening and of basement membrane between the synaptie membranes, see Tertivtiinen, 1969b). This kind of organization is considered by electrophysiologists to represent a site of low electrical resistance of the membrane (Ruch et at., 1966), and structurally, too, it resembles some (sensory-?) endings in intrafusal muscle fibres of the rat (Merrilles, 1960; Hennig, 1969), frog (Karlsson and Anderson-Cedergren, 1966) and cat (Adal, 1969) striated skeletal muscles. This special type of ending may thus be sensory, since the rat extraocular muscles are reported to be devoid of ordinary muscle spindles (Cilimbaris, 1910). The only previous studies using modern electron microscopic techniques known to us (Cheng et al., 1967; Cheng-Minoda et al., 1968) are at variance with the present results, since it, was reported t h a t in rabbit superior recti only the myelinated nerves and their myoneural junctions degenerate, while the

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u n m y e l i n a t e d fibres p r e s e r v e d t h e i r n o r m a l fine s t r u c t u r e after section of t h e o c u l o m o t o r nerve. H o w e v e r , in our opinion t h e p u b l i s h e d p h o t o m i c r o g r a p h s (see, Cheng-Minoda et al., 1968) of a m i x e d m y e l i n a t e d a n d u n m y e l i n a t e d nerve 4 d a y s (Fig. 4, page 603) a n d u n m y e l i n a t e d nerves 4 d a y s (Fig. 6, p. 605) a n d 14 d a y s (Fig. 7, p. 606) after section of t h e o c u l o m o t o r nerve show o n l y S c h w a n n cell processess. The nerve a n d t h e S c h w a n n cell processes, a l t h o u g h n o t e x a c t l y similar in t h e fine s t r u c t u r e of t h e i r c y t o p l a s m , are r a t h e r difficult to d i s c r i m i n a t e on t h e basis of t h e cell c y t o p l a s m , b u t are identified b y us as S c h w a n n cells, because t h e s t r u c t u r e s labelled as axons are d e v o i d of t h e i n v e s t i n g S c h w a n n cell processes a n d resulting m e s a x o n s t y p i c a l of u n m y e l i n a t e d nerves (Gasser, 1955; Gasser, 1958; Elfvin, 1958; Cavsey, 1960). According to t h e p r e s e n t observations, t h e u n m y e l i n a t e d nerves t o t a l l y d i s a p p e a r 4 d a y s after nerve section, leaving only S c h w a n n cell processes, as was also d e m o n s t r a t e d in t h e a b o v e - c i t e d photom i c r o g r a p h s in our opinion. I t t h u s seems a p p a r e n t t h a t t h e r e are no discrepancies b e t w e e n our results in t h e r a t a n d t h e results of Cheng-Minoda a n d her coworkers (op. cit.) in t h e r a b b i t , their results being similar to ours, b u t i n t e r p r e t e d in a different way. References

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-

Dr. H. Ter~iv~inen Dept. of Anatomy, University of Helsinki Siltavuorenpenger 20 Helsinki 17, Finland Present address:

Washington University School of Medicine Department of Physiology and Biophysics 660 South Euclid Avenue St. Louis, Missouri 63110, U.S.A.