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Motor myoneural junctions on frog intrafusal muscle fibers
J. ULTRASTRUCTURERESEARCH14, 191-211 (1966)
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M o t o r M y o n e u r a l Junctions on Frog Intrafusal Muscle Fibers 1 U L F KARLSSON 2 AND EBBA ANDERSSON-CEDERGREN 2
Department of Zoology, University of California at Los Angeles, Los Angeles, California Received June 8, 1964 Isolated frog muscle spindles were serially sectioned for electron microscopy, and motor innervation areas were studied for localization, mode of termination, and fine structure. Motor myoneural junctions are located in the polar regions of the muscle spindles, but in this investigation they were found still encapsulated in the spindle sheath and, to some degree, overlapping on sensory regions. The significance of this is discussed. Some differences were found with respect to the mode of termination of the nerve, and the question of dual-type motor endings is discussed. A tight nervemuscle plasma membrane relationship without interposed basement membrane was found. When end plate regions were prefrxed in formaldehyde, an increase in opaque material on the cytoplasmic side of the nmscle cell membrane was observed in the junctional fold area. The ultrastructure of different parts of the motor junction was evaluated, indicating a similarity to extrafusal motor junctions. Vesicles containing small opaque granules were observed among the common 400-600 ~ vesicles. Mitochondria in muscle and Schwann cells appeared to be larger than those in the nerve endings.
E l e c t r o p h y s i o l o g i c a l d a t a have i n d i c a t e d the existence of two different types of afferent responses f r o m a m p h i b i a n muscle spindles. W h e n spindles are subjected to a c t i v a t i o n b y nerve axons of large or small diameters (6, 7, 12), the responses corr e s p o n d to fast (twitch, phasic) or sl0w (tonic) m u s c u l a r c o n t r a c t i o n s , respectively. A n a t o m i c a l investigations on the light m i c r o s c o p i c a l level have described extrac a p s u l a r l o c a l i z a t i o n of m o t o r nerve endings on intrafusal muscle fibers (4) a n d have revealed i n d i c a t i o n s of two different types, designated as E n d b u s c h e l a n d g r a p e - t y p e endings (8). On the electron m i c r o s c o p i c level o f r e s o l u t i o n one r e p o r t has i n d i c a t e d a similarity in m o r p h o l o g y between extrafusal a n d i n t r a f u s a l m o t o r m y o n e u r a l j u n c t i o n s (13), b u t no detailed investigation has as yet been done r e g a r d i n g 1 This investigation was supported by Public Health Service Research Grant No. AM-04466 from the National Institute of Arthritis and Metabolic Diseases. "- Present address: Department of Anatomy, University of Umegt, Umegt, Sweden. 13
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comparative morphology and exact localization of motor myoneural junctions within the spindle system. The purpose of the present investigation was to study, on the electron microscopical level, the localization, the mode of termination, and the fine structure of the motor innervation areas of the intrafusal muscle fibers of the frog and to interpret the findings according to current physiological and anatomical views on the function of the muscle spindle. MATERIAL AND METHODS Single intrafusal muscle fiber bundles were isolated from Mm. extensores digitorum longi IV obtained from Rana temporaria and Rana pipiens. Whole muscles were fixed at different lengths for one-half to one hour in 1% osmium tetroxide in acetate-Veronal buffer. A few specimens were fixed in 4% formaldehyde in Ringer solution before the osmium fixation. After the fixation, further dissection was performed in Ringer solution. After conventional alcohol or acetone dehydration and embedding in Araldite or Vestopal, 0.2-1 mm by 0.2 mm sections were longitudinally or transversely serially cut on an LKB Ultrotome 4800. One transverse series was cut in 144 groups. The average thickness of the sections was estimated at 700 ~, and each group represented about 5/~ depth of tissue (11). The section staining and microscopical data were the same as those published elsewhere (11). RESULTS Iri the present material both transverse and longitudinal sections of frog muscle spindles have revealed the presence of morphologically distinct motor myoneural junctions. Criteria used for differentiation between these and sensory nerve endings, or other cell processes in contact with the muscle cell, were as follows: t. Nerve and muscle cell membranes are usually separated by a basement membrane at motor junctions (for exceptions, see below), but never at sensory junctions (10, 13), or intrafusal satellite cell contacts (10, 11). 2. Motor nerve endings are generally covered by expansions of Schwann cells where/is sensory nerve endings usually are not (2). 3. In longitudinal sections motor innervation areas exhibit some form of muscular specialization such as junctional folds and mitochondrial accumulations, whereas the sensory innervation areas and intrafusal satellite cell contact areas do not (2, 10, 11, 13). Further, single contact regions are considerably larger for motor than for sensory junctions in longitudinal sections (2). 4. The motor nerve endings are most often characterized by large amounts of vesicles 400-600 • in diameter. 5. Finally, in the encapsulated part of the spindle, the locality of motor innervation areas always is more polar than the equatorial sensory innervation areas (11).
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Sensory and m o t o r junctions on intrafusal muscle fibers were observed to be in very close proximity as studied in several specimens sectioned longitudinally or transversely. It definitely can be stated that m o t o r junctions do occur within the capsule of the muscle spindle and that the nerve endings reach and, even to some degree, overlap the sensory regions. Two examples of the intracapsular localization are shown in Figs. 1-3. In the specimen shown in Fig. 3, typical sensory nerve endings were identified about 40 # from where the m o t o r nerve ending disappeared out of the section, and in the same fiber a reticular zone (13) was observed only 150 # distant in the same direction. In one specimen transversely and serially cut it was possible to observe the m o t o r nerve endings overlapping the sensory innervation area on the same fiber (Fig. 1 b). With respect to neighboring bundles of intrafusal fibers, the overlap was found to be substantially more extensive. In the specimen transversely sectioned and partially illustrated in Fig. l a, the overlap between two partly joined intrafusal bundles was estimated to at least 100 #. It was not possible in this investigation to analyze the m o t o r innervation throughout the whole length of the muscle or at both sides of any particular sensory region. However, from the transversely sectioned series mentioned under Materials and Methods, a distance covering at least 150 # was estimated for the length of one m o t o r innervation area for one group of three intrafusal muscle fibers (Figs. 2a and 2d). The three fibers concerned were measured at 10 # (I), 7 # (//), and 5 # ( I I I ) in average diameter throughout the m o t o r innervation area. The nerve fiber illustrated in Fig. 2b was measured about 2 # in diameter in its immediate terminal course as far as it could be followed in the serial sections. After the myelin was no longer visible on the nerve that occurred in sections close to the one illustrated in Fig. 2b, the axon, now approximately 1 # in diameter and covered by a Schwann cell process, immediately exhibited morphological m o t o r ending characteristics and was traced to contacts with all three muscle fibers (Figs. 2a, 2c, and 2d). The nerve and the muscle fibers just mentioned were traced in cross sections at intervals of less than 5 # in order to analyze the contacts in detail. The muscle fibers were found to have relatively short contact areas with the m o t o r nerve endings at any portions along their course. The longest contact observed, as judged through consecutive section groups, was estimated at less than 25 # in length, and sometimes contact regions less than one section group long, representing less than 5/J, could be observed. It was considered likely that the different contact regions on different intrafusal muscle fibers were linked togethe r and that they had arisen from the same myelinated nerve fiber. Observations supporting such a view were gathered in the consecutive section groups where small axon branches were traced between the nerve endings at the contact areas of the different fibers (Fig. 2b). One of the fibers (III,
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i n Fig. 2 c ) h a d f e w e r j u n c t i o n s t h a n t h e o t h e r t w o f i b e r s w i t h i n t h e a n a l y z e d d i s t a n c e . N o o n e of t h e t h r e e m u s c l e f i b e r s s h o w e d a m o t o r i n n e r v a t i o n a r e a l o n g e r t h a n 80/~, a n d a t o n e e n d of t h e i n n e r v a t i o n a r e a t h e m u s c l e f i b e r b u n d l e e x h i b i t e d c h a r a c t e r i s t i c s of t h e s e n s o r y r e g i o n , e.g., m o r p h o l o g i c a l l y d i s t i n c t s e n s o r y n e r v e e n d i n g s (2, 11, 13) ( c o m p a r e w i t h s e n s o r y n e r v e e n d i n g s , SE, i n F i g . 1). T h e m o t o r n e r v e e n d i n g s measured mostly 1-3 # in thickness (perpendicular to the contacting surface), and less t h a n 4 # i n w i d t h ( p a r a l l e l t o t h e c o n t a c t i n g s u r f a c e ) . I n s o m e i n s t a n c e s a f e w d i s t i n c t j u n c t i o n a l f o l d s of t h e c o n t a c t i n g m u s c u l a r cell m e m b r a n e c o u l d b e o b s e r v e d .
Key to abbreviations ME motor nerve ending Mi mitochondria Nu nucleus OC outer spindle capsule
A axon branches, myelinated or unmyelinated C collagenous material E evaginations ef extracellular filamentous material if neurofilaments or intracellular filamentous material G Golgi vesicle complex IC inner spindle capsule IS intrafusal satellite ceils J junctional folds
P S
protrusions membrane-bound structures (Schwann cell processes?) Sch Schwann cell SE sensory nerve endings v vesicles
Fro. 1. (a) Transverse section of parts of two intrafusal muscle bundles to the left and right in the figure are separated by inner (IC) and outer (OC) spindle capsules. The picturedemonstrates the phase difference between sensory and motor innervation areas in neighboring intrafusal muscle fiber bundles. A myelinated axon branch (A) can be observed in the upper center region. Both muscle fibers illustrated in the group to the left are associated with numerous sensory nerve endings (SE) and intrafusal satellite cells (IS) (11). In the intrafusal muscle fiber bundle to the right, no nerve endings are seen associated with the upper muscle fiber. The lower muscle fiber, however, receives a motor nerve ending (ME). The opposing muscle cell cytoplasm is occupied partly by a peripheral nucleus (Nu). x approx. 5500. (b) Transverse section through part of a serially sectioned, compact sensory region (11) of a muscle spindle from a frog. The picture demonstrates the overlap of motor innervation upon sensory innervation areas on one single intrafusal muscle fiber. The muscle fibers are surrounded by an inner capsule (/C), an outer part of which (OC) is shown at the upper left. Associated with the muscle fibers are sensory nerve endings (SE) and intrafusal satellite cells (IS): At the upper center a motor nerve ending (ME) is seen making contact with one of the muscle fibers. This ending (ME) was traced in series of sections and found to be continuous with typical motor nerve endings. x approx. 5500. Fit. 2a-d. A transversely and serially sectioned motor innervation area is shown. The pictures illustrate sections at levels approximately 80 #, 110 ~, 165 #, and 200/~, respectively, from the start of the sectioning, as seen in the upper right corners. (a), (b), and (d) show a bundle of three intrafusal muscle fibers (/, II, and III) that are encapsulated by inner (IC) and outer (OC) spindle capsules. A capsule cell nucleus (Nu) is seen in the lower part of (a). Intrafusal muscle f i b e r / / i n (d) is associated with an intrafusal satellite cell, the nucleus of which is labeled IS. The myelinated nerve fiber seen to the left in (b) is surrounded by a Schwaun cell whose nucleus is labeled Nu. All three muscle fibers are contacted by branches of this nerve fiber. As displayed in (a), (c), and (d), the motor nerve endings (ME) are surrounded by Schwann cell cytoplasm, except at the side facing the muscle. Small unmyelinated axon branches (A), seen in the lower part of (b) and another in (d), were traced in cross sections and observed to link together the different contact areas on different fibers. The axon branch (A) to the left in (d) is associated with a Schwann cell nucleus (Nu). × approx. 5000.
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With respect to other structural features, the muscle at the contact regions had all the morphological characteristics previously reported, such as nuclei and accumulated mitochondria in the sarcoplasm. In another longitudinally sectioned specimen, a motor innervation area could be observed extending for at least 150 # on one single intrafusal muscle fiber (Fig. 3). Contact areas between the nerve ending and the muscle fiber could be followed in one section for 30 # with interdistances (arrows in Fig. 3) of less than 5/z. In the intervening spaces throughout the innervation area, collagen bundles, filamentous material, and extensions of Schwann cells were observed associated with the nerve ending. This indicates a possible continuum of the nerve ending from one end of the observed contact area to the other, In this specimen the thickness of the axoplasm perpendicular to the contacting surface was measured at about 3-4 # in most places of contact. The nerve endings previously mentioned all had a basement membrane interposed between the axon and muscle plasma membranes. However, in one specimen intimate contacts were detected at some locations along the 200 #-long motor innervation area. 1 The axoplasm was here found evaginated into sac- or ridge-like structures amounting to less than 2 ~u in diameter in the direction parallel to the muscle fiber (Fig. 4). A somewhat constricted part was found to join the sac-like evagination to the axoplasm (Fig. 4b). The evaginations were located in dilated junctional folds, and the plasma membranes of the evaginated axon and the muscle fiber were directly apposed without intervening basement membrane (Fig. 4). The intervening space between the two single-layered plasma membranes was measured at 100-150 A. None of the contact surfaces exceeded 3 # in length in any section. The estimated approximate interval between any two evaginations was at least 1 #. In a section less than 5 ,u apart from those illustrated in Fig. 4, only one evagination could be found. This would possibly indicate a restricted localization of these evaginations within a junctional region. Few membrane-bound structures were found in the evaginations. Scattered granular material and some 400-600 A vesicles were observed at the constriction toward the axoplasm proper (Fig. 4b). The axon plasma membrane often showed increased electron opacity (at P in Fig. 4a-c) at places opposite the entrances of the junctional folds, and a tendency for accumulation of 400-600 ~ vesicles was observed in the axoplasm at these places as has been reported for frog extrafusal motor nerve endings (3). These features were also found in specimens without evaginations (Figs. 5, 6, 7a, 7c, and 8b). The specimen with the peculiar evaginated structures showed shortened sarcomeres, 1.8 # long. Motor nerve ending axoplasm structures observed in all specimens of this investigation were found to conform to that reported for frog extrafusal motor junctions Presented by U. Karlsson at the Fifth International Congress for Electron Microscopy in Philadelphia in 1962 and appearing in the Proceedings (9).
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(3), e.g., c o m p a r t m e n t s of 400-600 A vesicles, neurofilaments, a n d small m i t o c h o n d r i a (Fig. 5). H o w e v e r , with the present technique no b e a d e d structures (3) of the n e u r o filaments c o u l d be detected which m e a s u r e d a b o u t 100 A (96 ___16 A for 26 m e a s u r e ments) in d i a m e t e r (Fig. 6). T h e m i t o c h o n d r i a a v e r a g e d 0.15 # (0.15_+0.04 # for 56 m e a s u r e m e n t s ) in d i a m e t e r in five different specimens. M i t o c h o n d r i a m e a s u r e m e n t s were at all times p e r f o r m e d o n the smallest d i a m e t e r of transversely sectioned organelles. T h e vesiculous p o r t i o n of the t e r m i n a l a x o p l a s m was f o u n d to c o n t a i n at least two types of vesicles. A g r a n u l e - c o n t a i n i n g vesicle was o b s e r v e d w i t h l o w frequency m e a s u r i n g a b o u t 900 A (940 ! 100/~ for 26 m e a s u r e m e n t s f r o m 2 specimens) in d i a m e t e r which always c o n t a i n e d a smaller electron o p a q u e granule v a r y i n g in size f r o m 200 to 800 A in d i a m e t e r (26 m e a s u r e m e n t s ) (Figs. 4, 5, 6, 7c, a n d 8a). The relative occurrence of this t y p e of vesicle was r o u g h l y e s t i m a t e d to a b o u t one in 25 of the m o r e c o m m o n 400-600 A vesicles. The S c h w a n n cell cover of the nerve endings was f o u n d c o m p l e t e o n the side a w a y f r o m the muscle (see Fig. 3). O n the side of the nerve ending facing the muscle, small m e m b r a n e - b o u n d profiles were o b s e r v e d in i n t i m a t e c o n t a c t w i t h the nerve e n d i n g p l a s m a m e m b r a n e a n d p r e d o m i n a n t l y occurring between two consecutive entrances of the j u n c t i o n a l folds (Fig. 6). These cell processes were c o n s i d e r e d likely to b e l o n g to the S c h w a n n cell. Often the S c h w a n n cell l a m e l l a covering the nerve ending was m e a s u r e d as thin as 2 0 0 0 / ~ , b u t closer to nuclei the a m o u n t of c y t o p l a s m was increased a n d thus a l l o w e d o b s e r v a t i o n s of its structures. T h e S c h w a n n cell c y t o p l a s m was c h a r a c t e r i z e d b y a n a b u n d a n c e of vesicular a n d t u b u l a r structures, a variety of r i b o s o m e - s i z e d particles, few m i t o c h o n d r i a ( a b o u t 0.2 # in diameter), a n d scattered larger granules. I n m a n y places large, r o u n d e d , extracellular spaces were f o u n d w i t h the smaller d i a m e t e r m e a s u r i n g a b o u t 1 # (Figs. 3 a n d 5). These spaces were p a r t l y o r c o m p l e t e l y filled w i t h a filamentous material, the unit filament
FIG. 3 a, b. Longitudinal section from a polar region of an intrafusal muscle fiber. The lower end of the picture (a) is continuous with the upper end of picture (b). To the right in both pictures sarcomeres with distinct cross bandings can be observed. On the outside of the muscle plasma membrane a motor nerve ending (ME) is seen coursing along the surface of the muscle fiber. The nerve ending can be seen to contact the muscle fiber extensively and with relatively long contact regions. The nerve ending could be followed for 150/~ along the muscle fiber. The short intervals between the contact regions (horizontal arrows) are seen to be occupied by Schwann cell cytoplasm and collagenous material (C). The contact regions of the nerve ending and the muscle fiber exhibit characteristics typical for motor myoneural junctions, such as peripheral muscle nuclei (Nu, in lower part of Fig. 3 a, and upper part of Fig. 3 b), relatively numerous mitochondria and junctional folds (J) in the muscle, compartments of vesicles (v), neurofilaments (if) and mitochondria (Mi) in the nerve ending. Sehwann cell (SC) is observed to cover the nerve ending on the side away from the muscle. Schwann cell nuclei (Nu) are seen in the upper part of (a) and in the lower part of (b). The cytoplasm of the Schwann cell in some places exhibits fenestrations filled with moderately dense filamentous material (el). Discontinuous lamellae (a) of the inner spindle capsule (IC) are seen surrounded by collagen bundles traversing in different directions, x approx. 6000.
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of w h i c h m e a s u r e d a b o u t 50 A in d i a m e t e r (Fig. 5). T h i s f i l a m e n t o u s m a t e r i a l c o u l d , at places, be o b s e r v e d to be c o n t i n u o u s w i t h b a s e m e n t m e m b r a n e m a t e r i a l (Fig. 5} a n d w i t h i d e n t i c a l m a t e r i a l f o u n d m i x e d w i t h c o l l a g e n b u n d l e s (see b e l o w ) . Intracapsular motor innervation areas were found within an inner discontinuous leaflet of t h e s p i n d l e c a p s u l e (Figs. 1-3) (10, 11). B e t w e e n this i n n e r leaflet a n d t h e S c h w a n n cell c o v e r i n g t h e m o t o r n e r v e e n d i n g , c o l l a g e n b u n d l e s of v a r y i n g sizes o c c u r r e d (Fig. 3). T h e s e c o l l a g e n b u n d l e s w e r e o r i e n t e d r o u g h l y p e r p e n d i c u l a r l y t o t h e l o n g i t u d i n a l axis of t h e m u s c l e fiber a n d m i x e d w i t h t h e f i l a m e n t o u s m a t e r i a l f o u n d in t h e e x t r a c e l l u l a r spaces m e n t i o n e d a b o v e . T h e m u s c u l a r c y t o p l a s m b e n e a t h t h e n e r v e e n d i n g was n o t f o u n d different f r o m t h a t p r e v i o u s l y r e p o r t e d f o r e x t r a f u s a l m o t o r m y o n e u r a l j u n c t i o n s (3). A s o b s e r v e d
FIa. 4a-c. Series of longitudinal sections of an intracapsular motor nerve ending in the frog muscle spindle. The consecutive pictures represent every second section. The muscle plasma membrane exhibits typical junctional folds. The sarcomeres in this specimen were found to be about 1.8 # long throughout the motor innervation area. The axoplasm in the upper parts of the micrographs contains 400-600 A vesicles, 900 A granulated vesicles (horizontal arrows), and small mitochondria (Mi). Toward the muscle surface, the axoplasm protrudes into smaller (P) or larger (E1-4) evaginations. The larger evaginations, either ridge- or sac-like, are seen to traverse the extracellular space down into the junctional folds where they make contact with the muscle without interposition of a basement membrane (double arrows). The evagination E3 connects with the axoplasm proper by a stalk that is visible only in the middle section (b} (sac-like evagination). The evagination El, presumably ridge-like, is seen to the left in all sections (a-c). A few 400-600 A vesicles, a few opaque granules and scattered amorphous material are seen within each evagination. Cytoplasmic processes (Schwann cell processes) are found on both sides of the evagination (Ea) (vertical arrows). Often the axon plasma membrane opposite the entrance to the junctional folds show increased density, x 27,500. FIG. 5. Higher magnification of part of the intracapsular motor myoneural junction. The muscle plasma membrane shows greater density at the contact region than in the region at the lower center. Junctional folds (J) are seen at a few places along the contact. A peripheral muscle nucleus (Nu), a Golgi vesicle complex (G), and an accumulation of relatively large mitochondria (Mi) in a matrix of smaller opaque particles and vesicles represent the muscular specialization at the motor myoneural junction. In the axoplasm to the right, mitochondria (Mi) and thin neurofilaments (if) are seen. Closer to the contact area, 400-600 A vesicles (v) are located. Interspersed among these vesicles and elsewhere, larger vesicles are found displaying a denser inner core (vertical arrows). At the contact area the axoplasm can be seen to protrude (horizontal arrows), especially opposite the junctional folds (J). Here 400-600 A vesicles and an increased density of the axon plasma membrane may be seen. Extracellular filamentous material (el)in the lower center is seen to be continuous with the basement membrane material. In the space between the plasma membranes of the muscle and the nerve cell, membrane-bound structures (S) (Schwann cell processes?) are associated with the axon plasma membrane. × 33,000. FIG. 6. High magnification of part of the motor myoneural junction in the frog muscle spindle. To the right of the muscle cell nucleus (Nu) the muscle plasma membrane exhibits several junctional folds (J). Opposite the entrances of these folds the axoplasm may be seen protruding (horizontal arrows), and in some places the axon plasma membrane displays higher electron density than elsewhere. In between each of these protrusions, membrane-bound cell processes (S) (Schwann cell processes?) can be located close to the axon plasma membrane. The oblique arrow in the lower part of the picture points to a part of the axon plasma membrane that appears triple-layered. The oblique arrow in the upper center points to a larger vesicle with a dense core. To the lower right, thin neurofilaments (if) and two mitochondria (Mi) are seen. × 70,000.
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at mammalian junctions (I), accumulations of tightly packed mitochondria located juxtanuclearly (Fig. 8 a) were often found. The individual mitochondria were seldom estimated to be less than 0.25 # in diameter. In specimens prefixed in formaldehyde, an electron opaque thick coating was observed associated with the sarcoplasmic side of the muscle plasma membrane in the region of the junctional folds (Fig. 7a, b, c). This material appeared more prominent at the plasma membrane between the junctional folds and at their sides than at the bottom of them. As many muscles had been fixed at different lengths, it was possible to observe the nerve-muscle relationship at different lengths of the sarcomeres. In the frog a frequency of 3-4 junctional folds per sarcomere has been estimated (3), which appears as a less complicated pattern than that found in mammalia (i). When analyzing the appearance of the motor myoneura] junction at different sarcomere lengths, it was observed that at rest length, or with stretched sarcomeres (for example, the specimens illustrated in Figs. 3 and 7), the typical frequency was 3-4 junctional folds per sarcomere. At shortened sarcomeres the differences observed were grossly a wavy appearance of the whole junctional area (Fig. 8a) and the number of the junctional folds per sarcomere changed (Figs. 8a and 8b). The sarcoplasmic characteristics of all muscle fibers were analyzed. In the transversely sectioned series it was found that all muscle fibers changed their relative content along their course, such as myofilaments, sarcotubular systems, and amount of mitochondria. Even their overall diameter changed (compare fiber I in Figs. 2a and 2 d). No apparent differencescould be found between different intrafusal muscle fibers which would characterize them as twitch or slow (15), although no high resolution analysis was made. DISCUSSION The present investigation has uncovered certain morphological features typical for motor myoneural junctions in muscle spindles of the frog. The intrafusal motor nerve ending differs from the extrafusal in that it is enclosed in the same capsule as are the sensory nerve endings. Thus it is clear that the criterion stated by Cajal (4) FIG. 7 a--c. Three micrographs showing different examples of the appearance of the motor myoneural junction areas when prefixed in 4% formaldehyde before postfixation in osmium tetroxide. All three, and especially the center one (b), display a thick coat of electron opaque material located at and associated with the inside of the muscle plasma membrane. This material is predominantly located in between and on the side walls of the junctional folds (arrows). Otherwise the morphology of the junctional area is similar to that illustrated in Figs. 5 and 6, except that very few 400-600 ,~ vesicles are observed in the axoplasm in the ending illustrated in (b). In Fig. 7a indications of crowding of the 400-600 /~ vesicles are seen in association with the axon plasma membrane opposite the entrances to the junctional folds. A frequency of 3-4 junctional folds per sarcomere is exhibited in (b). The sarcomere length in (b) was measured to about 2.7 #. x 40,000.
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and accepted by Katz (13) concerning the exclusive extracapsular existence of m o t o r m y o n e u r a l junctions o n intrafusal muscle fibers must be reconsidered. The close proximity of the m o t o r nerve endings to the sensory nerve endings therefore can be t h o u g h t of as shortening the distance for the contraction wave influencing the sensory nerve endings and thus facilitating the subsequent afferent impulse. The mere fact of encapsulation also brings up the question of pharmacological effect on the m o t o r nerve endings. It has been stated that curare is less effective for intrafusal than for extrafusal nerve endings (6, 12). It m a y be justifiable to assume that the spindle capsule would act as a general diffusion barrier and as a specific one if it is considered to contribute to maintaining a special environment for the sensory nerve endings (10). Electrophysiological data (6, 7, 12) have disclosed the presence of two different types of afferent responses related to twitch and slow activation of the muscle spindle. The anatomical correlate for this p h e n o m e n o n has been assigned partly to the existence of Endbuschel- and grape-type endings occurring on the intrafusal muscle fibers (8). I n the present investigation no definite qualitative differences could be f o u n d between several m o t o r junctions studied. With respect to the m o r p h o l o g y of the muscle fibers, no apparent differences could be f o u n d which would characterize them as twitch or slow (15). The differences observed were related quantitatively to the length of individual m o t o r m y o n e u r a l contacts, the spread of the innervation areas along the muscle fibers, the dimensions of the nerve endings and extent of ramification of one single nerve fiber. A l t h o u g h the size of the nerve supplying the innervation areas analyzed was n o t observed, the nerve fiber constituting endings in the transversely sectioned specimen (Fig. 2) was quite thin. It also had extensive ramifications and f o r m e d relatively short contact regions with all the intrafusal muscle fibers concerned. Only in exceptional cases did the thickness of the nerve endings exceed 3/~. However, in two longitudinally sectioned specimens where lengthy contacts were observed (Figs: 3 and 4), a thickness of 3-4 ,u was measured. With respect to the spread over one intrafusal muscle fiber, the former specimen exhibited lengths maximally estimated to 80 #, while the latter
FIG. 8. This micrograph demonstrates the morphology of the motor myoneural junction in a s p e c i where the sarcomere length was measured to an average of 1.8 #. The appearance should be compared with the one exhibited in Fig. 7b where the sarcomere length was measured to 2.7 #. (a) The whole motor nerve ending (ME) can be observed folded and making an impression into the muscle surface. The Schwann cell cover (SC) is seen to follow the folding of the nerve ending. In the center of the axoplasm a 900 A granulated vesicle can be seen (vertical arrow). To the lower right, a small part of a 15 #-long and 3/z-wide accumulation of mitochondria (Mi) can be seen. Very few junctional folds are observed in the muscle plasma membrane in contact with the motor nerve ending, x 27,500. (b) Another part of the same junctional area as illustrated above. Here the sarcomeres can be observed to be about 1.8 /z long. The frequency of junctional folds is about 6 per sarcomere (vertical arrows), x 43,500. men
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specimens amounted to a minimal spread of 150-200/J. Therefore, the present authors are inclined to conclude that there is a differentiation occurring in the motor myoneural junctions of the frog muscle spindles with one type of ending displaying long contacts over great lengths of the fiber with relatively thick nerve endings (long type) and the other type having shorter contact areas, thinner nerve endings and an extensive arborescent pattern on a relatively short length of the muscle fiber (short type). The relative absence of junctional folds observed in the short type specimen might be of significance (5) but must be interpreted with caution as it is possible to section through fold-free areas at the amphibian motor myoneural junction (13, 18). The interpretation mentioned with respect to different types of nerve endings conforms with light microscopical data (8), thus classifying the long type as Endbuschel and the short type as grape-type ending. Functional interpretations will necessarily be speculation even if morphological considerations may be applied. In that case, however, a similar neuromuscular transmission of the two types of junctions can be assumed and the differences considered quantitative. On purely physical grounds the probability of efficient transmission might be higher at a longer contact area supplied by a thicker nerve ending than that of a shorter contact area supplied by a thinner nerve ending. In one of the motor junctions of the long type a peculiar membrane relationship was found, essentially showing evaginations of the axoplasm into dilated junctional folds which contacted the muscle fiber without interposition of a basement membrane (Fig. 4). The meaning of this is obscured by the fact that it was observed in only one specimen and that it may represent a possible aberration in normal morphology. The axon plasma membrane opposite the entrances of the junctional folds was found to have increased electron opacity and a tendency for 400-600 A vesicles to accumulate in the axoplasm at these condensations. This is in agreement with previous observations (3). The cell processes interposed between the axon plasma membrane and the basement membrane (3, 17, 18) in this investigation were found to predominantly occupy locations between the entrances of the junctional folds (Fig. 6). The topographical localization of the axon protrusions, the axon plasma membrane opacities, the small vesicles, and the above-mentioned cell processes may indicate a functional relationship with respect to synaptic transmission. The content of the nerve endings described in this investigation was found not to deviate from previous investigations of extrafusal muscle fibers (3). To our knowledge the granular vesicle found has not been described previously in motor nerve endings but can be found in published pictures of endings on extrafusal muscle fibers (3). However, identical vesicles have been described as present elsewhere in the frog peripheral nervous system (2, 16, 19). The prevailing interpretation of the contents of these granular, 900 A vesicles has been to identify them with similar vesicles in the
INTRAFUSAL MOTOR MYONEURAL JUNCTIONS
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central nervous system referred to as "neurosecretory granules" (14). However, conclusions with respect to its functional relationship to the m o t o r myoneural junction can presently not be made since the nature of the contents of '~neurosecretory granules" has not yet been established. When subjecting the muscle to formaldehyde as a prefixative, as compared to conventional primary osmium tetroxide fixation, an apparent difference in amount of electron dense material was observed associated with the muscle plasma membrane at the sarcoplasmic side in the junctional area (Figs. 4-6 compared to Fig. 7). This would suggest the existence of proteinaceous material at this location. It could be that formaldehyde is a good preservative for proteins such as acetylcholinesterase, which would be anticipated to occur at this location. The results presented regarding differences in morphology of junctional areas at different sarcomere lengths of the muscle fiber indicate that mechanical forces in the process of contraction exert influence at this location. The authors are deeply indebted to Professor F. S. Sj6strand for support and valuable criticism and for the privilege of using his laboratory facilities. Thanks are also due to Mr. Herman Kabe for skillful photographic assistance, and to Dr. R. L. Schultz and Mrs. Kay McQuaid for correcting the English in the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 1 1.
12. 13. 14. 15. 16. 17. 18. 19.
ANDERSSON-CEDERGREN,E., Y. Ultrastruet. Res. Suppl. 1 (1959). ANDERSSON-CEDERGREN,E. and KARLSSON, U. (in preparation). BIRKS,R., HUXLEY, H. E. and KATZ, B., J. Physiol. (London) 150, 134 (1960). CAJAL, S. R. Y, Rev. Trim. Histol. Norm. y Patol., No. 1, !888. Quoted from Cajal, S. R. y, Histologie du Syst6me Nerveux, Vol. 1, pp. 485 seq. Madrid, 1952. COTEUX,R., Exptl. Cell Res. Suppl. 5, 294 (1958). EYZAGUIRRE,C., J. Neurophysiol. 20, 523 (1957). -J. Neurophysiol. 21, 465 (1958). GRAY, E. G., Proc. Roy. Soc. B 146, 416 (1957). KARLSSON,U., Proc. Intern. Congr. Electron Microscopy, 5th, Philadelphia, 1962, Vol. 2, U-4. Academic Press, New York, 1962. KARLSSON,U. and ANDERSSON-CEDERGREN,E. (in preparation). KARLSSON, U., ANDERSSON-CEDERGREN,E. and OTTOSON, O., J. Ultrastruct. Res. in press. KATZ, B., J. Exptl. Biol. 26, 201 (1949). - - - - Phil. Trans. Roy. Soc. London, B 243, 221 (1960). PALAY, S. L., in Progress in Neurobiology, Vol. 2, p. 31. Harper (Hoeber), New York, 1957. PEACHEY,L. D. and HUXLEY, A. F., Y. Ceil Biol. 13, 177 (1962). PICK, J., Anat. Record 144, 295 (1962). REGER, J. F., Anat. Record133, 327 (1959). ROBERTSON,J. D., Am. J. Phys. Med. 39, 1 (1960). TAxi, J., Compt. Rend. Acad. 252, 174 (1961).
191
M o t o r M y o n e u r a l Junctions on Frog Intrafusal Muscle Fibers 1 U L F KARLSSON 2 AND EBBA ANDERSSON-CEDERGREN 2
Department of Zoology, University of California at Los Angeles, Los Angeles, California Received June 8, 1964 Isolated frog muscle spindles were serially sectioned for electron microscopy, and motor innervation areas were studied for localization, mode of termination, and fine structure. Motor myoneural junctions are located in the polar regions of the muscle spindles, but in this investigation they were found still encapsulated in the spindle sheath and, to some degree, overlapping on sensory regions. The significance of this is discussed. Some differences were found with respect to the mode of termination of the nerve, and the question of dual-type motor endings is discussed. A tight nervemuscle plasma membrane relationship without interposed basement membrane was found. When end plate regions were prefrxed in formaldehyde, an increase in opaque material on the cytoplasmic side of the nmscle cell membrane was observed in the junctional fold area. The ultrastructure of different parts of the motor junction was evaluated, indicating a similarity to extrafusal motor junctions. Vesicles containing small opaque granules were observed among the common 400-600 ~ vesicles. Mitochondria in muscle and Schwann cells appeared to be larger than those in the nerve endings.
E l e c t r o p h y s i o l o g i c a l d a t a have i n d i c a t e d the existence of two different types of afferent responses f r o m a m p h i b i a n muscle spindles. W h e n spindles are subjected to a c t i v a t i o n b y nerve axons of large or small diameters (6, 7, 12), the responses corr e s p o n d to fast (twitch, phasic) or sl0w (tonic) m u s c u l a r c o n t r a c t i o n s , respectively. A n a t o m i c a l investigations on the light m i c r o s c o p i c a l level have described extrac a p s u l a r l o c a l i z a t i o n of m o t o r nerve endings on intrafusal muscle fibers (4) a n d have revealed i n d i c a t i o n s of two different types, designated as E n d b u s c h e l a n d g r a p e - t y p e endings (8). On the electron m i c r o s c o p i c level o f r e s o l u t i o n one r e p o r t has i n d i c a t e d a similarity in m o r p h o l o g y between extrafusal a n d i n t r a f u s a l m o t o r m y o n e u r a l j u n c t i o n s (13), b u t no detailed investigation has as yet been done r e g a r d i n g 1 This investigation was supported by Public Health Service Research Grant No. AM-04466 from the National Institute of Arthritis and Metabolic Diseases. "- Present address: Department of Anatomy, University of Umegt, Umegt, Sweden. 13
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comparative morphology and exact localization of motor myoneural junctions within the spindle system. The purpose of the present investigation was to study, on the electron microscopical level, the localization, the mode of termination, and the fine structure of the motor innervation areas of the intrafusal muscle fibers of the frog and to interpret the findings according to current physiological and anatomical views on the function of the muscle spindle. MATERIAL AND METHODS Single intrafusal muscle fiber bundles were isolated from Mm. extensores digitorum longi IV obtained from Rana temporaria and Rana pipiens. Whole muscles were fixed at different lengths for one-half to one hour in 1% osmium tetroxide in acetate-Veronal buffer. A few specimens were fixed in 4% formaldehyde in Ringer solution before the osmium fixation. After the fixation, further dissection was performed in Ringer solution. After conventional alcohol or acetone dehydration and embedding in Araldite or Vestopal, 0.2-1 mm by 0.2 mm sections were longitudinally or transversely serially cut on an LKB Ultrotome 4800. One transverse series was cut in 144 groups. The average thickness of the sections was estimated at 700 ~, and each group represented about 5/~ depth of tissue (11). The section staining and microscopical data were the same as those published elsewhere (11). RESULTS Iri the present material both transverse and longitudinal sections of frog muscle spindles have revealed the presence of morphologically distinct motor myoneural junctions. Criteria used for differentiation between these and sensory nerve endings, or other cell processes in contact with the muscle cell, were as follows: t. Nerve and muscle cell membranes are usually separated by a basement membrane at motor junctions (for exceptions, see below), but never at sensory junctions (10, 13), or intrafusal satellite cell contacts (10, 11). 2. Motor nerve endings are generally covered by expansions of Schwann cells where/is sensory nerve endings usually are not (2). 3. In longitudinal sections motor innervation areas exhibit some form of muscular specialization such as junctional folds and mitochondrial accumulations, whereas the sensory innervation areas and intrafusal satellite cell contact areas do not (2, 10, 11, 13). Further, single contact regions are considerably larger for motor than for sensory junctions in longitudinal sections (2). 4. The motor nerve endings are most often characterized by large amounts of vesicles 400-600 • in diameter. 5. Finally, in the encapsulated part of the spindle, the locality of motor innervation areas always is more polar than the equatorial sensory innervation areas (11).
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Sensory and m o t o r junctions on intrafusal muscle fibers were observed to be in very close proximity as studied in several specimens sectioned longitudinally or transversely. It definitely can be stated that m o t o r junctions do occur within the capsule of the muscle spindle and that the nerve endings reach and, even to some degree, overlap the sensory regions. Two examples of the intracapsular localization are shown in Figs. 1-3. In the specimen shown in Fig. 3, typical sensory nerve endings were identified about 40 # from where the m o t o r nerve ending disappeared out of the section, and in the same fiber a reticular zone (13) was observed only 150 # distant in the same direction. In one specimen transversely and serially cut it was possible to observe the m o t o r nerve endings overlapping the sensory innervation area on the same fiber (Fig. 1 b). With respect to neighboring bundles of intrafusal fibers, the overlap was found to be substantially more extensive. In the specimen transversely sectioned and partially illustrated in Fig. l a, the overlap between two partly joined intrafusal bundles was estimated to at least 100 #. It was not possible in this investigation to analyze the m o t o r innervation throughout the whole length of the muscle or at both sides of any particular sensory region. However, from the transversely sectioned series mentioned under Materials and Methods, a distance covering at least 150 # was estimated for the length of one m o t o r innervation area for one group of three intrafusal muscle fibers (Figs. 2a and 2d). The three fibers concerned were measured at 10 # (I), 7 # (//), and 5 # ( I I I ) in average diameter throughout the m o t o r innervation area. The nerve fiber illustrated in Fig. 2b was measured about 2 # in diameter in its immediate terminal course as far as it could be followed in the serial sections. After the myelin was no longer visible on the nerve that occurred in sections close to the one illustrated in Fig. 2b, the axon, now approximately 1 # in diameter and covered by a Schwann cell process, immediately exhibited morphological m o t o r ending characteristics and was traced to contacts with all three muscle fibers (Figs. 2a, 2c, and 2d). The nerve and the muscle fibers just mentioned were traced in cross sections at intervals of less than 5 # in order to analyze the contacts in detail. The muscle fibers were found to have relatively short contact areas with the m o t o r nerve endings at any portions along their course. The longest contact observed, as judged through consecutive section groups, was estimated at less than 25 # in length, and sometimes contact regions less than one section group long, representing less than 5/J, could be observed. It was considered likely that the different contact regions on different intrafusal muscle fibers were linked togethe r and that they had arisen from the same myelinated nerve fiber. Observations supporting such a view were gathered in the consecutive section groups where small axon branches were traced between the nerve endings at the contact areas of the different fibers (Fig. 2b). One of the fibers (III,
194
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i n Fig. 2 c ) h a d f e w e r j u n c t i o n s t h a n t h e o t h e r t w o f i b e r s w i t h i n t h e a n a l y z e d d i s t a n c e . N o o n e of t h e t h r e e m u s c l e f i b e r s s h o w e d a m o t o r i n n e r v a t i o n a r e a l o n g e r t h a n 80/~, a n d a t o n e e n d of t h e i n n e r v a t i o n a r e a t h e m u s c l e f i b e r b u n d l e e x h i b i t e d c h a r a c t e r i s t i c s of t h e s e n s o r y r e g i o n , e.g., m o r p h o l o g i c a l l y d i s t i n c t s e n s o r y n e r v e e n d i n g s (2, 11, 13) ( c o m p a r e w i t h s e n s o r y n e r v e e n d i n g s , SE, i n F i g . 1). T h e m o t o r n e r v e e n d i n g s measured mostly 1-3 # in thickness (perpendicular to the contacting surface), and less t h a n 4 # i n w i d t h ( p a r a l l e l t o t h e c o n t a c t i n g s u r f a c e ) . I n s o m e i n s t a n c e s a f e w d i s t i n c t j u n c t i o n a l f o l d s of t h e c o n t a c t i n g m u s c u l a r cell m e m b r a n e c o u l d b e o b s e r v e d .
Key to abbreviations ME motor nerve ending Mi mitochondria Nu nucleus OC outer spindle capsule
A axon branches, myelinated or unmyelinated C collagenous material E evaginations ef extracellular filamentous material if neurofilaments or intracellular filamentous material G Golgi vesicle complex IC inner spindle capsule IS intrafusal satellite ceils J junctional folds
P S
protrusions membrane-bound structures (Schwann cell processes?) Sch Schwann cell SE sensory nerve endings v vesicles
Fro. 1. (a) Transverse section of parts of two intrafusal muscle bundles to the left and right in the figure are separated by inner (IC) and outer (OC) spindle capsules. The picturedemonstrates the phase difference between sensory and motor innervation areas in neighboring intrafusal muscle fiber bundles. A myelinated axon branch (A) can be observed in the upper center region. Both muscle fibers illustrated in the group to the left are associated with numerous sensory nerve endings (SE) and intrafusal satellite cells (IS) (11). In the intrafusal muscle fiber bundle to the right, no nerve endings are seen associated with the upper muscle fiber. The lower muscle fiber, however, receives a motor nerve ending (ME). The opposing muscle cell cytoplasm is occupied partly by a peripheral nucleus (Nu). x approx. 5500. (b) Transverse section through part of a serially sectioned, compact sensory region (11) of a muscle spindle from a frog. The picture demonstrates the overlap of motor innervation upon sensory innervation areas on one single intrafusal muscle fiber. The muscle fibers are surrounded by an inner capsule (/C), an outer part of which (OC) is shown at the upper left. Associated with the muscle fibers are sensory nerve endings (SE) and intrafusal satellite cells (IS): At the upper center a motor nerve ending (ME) is seen making contact with one of the muscle fibers. This ending (ME) was traced in series of sections and found to be continuous with typical motor nerve endings. x approx. 5500. Fit. 2a-d. A transversely and serially sectioned motor innervation area is shown. The pictures illustrate sections at levels approximately 80 #, 110 ~, 165 #, and 200/~, respectively, from the start of the sectioning, as seen in the upper right corners. (a), (b), and (d) show a bundle of three intrafusal muscle fibers (/, II, and III) that are encapsulated by inner (IC) and outer (OC) spindle capsules. A capsule cell nucleus (Nu) is seen in the lower part of (a). Intrafusal muscle f i b e r / / i n (d) is associated with an intrafusal satellite cell, the nucleus of which is labeled IS. The myelinated nerve fiber seen to the left in (b) is surrounded by a Schwaun cell whose nucleus is labeled Nu. All three muscle fibers are contacted by branches of this nerve fiber. As displayed in (a), (c), and (d), the motor nerve endings (ME) are surrounded by Schwann cell cytoplasm, except at the side facing the muscle. Small unmyelinated axon branches (A), seen in the lower part of (b) and another in (d), were traced in cross sections and observed to link together the different contact areas on different fibers. The axon branch (A) to the left in (d) is associated with a Schwann cell nucleus (Nu). × approx. 5000.
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INTRAFUSAL MOTOR MYONEURAL JUNCTIONS
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With respect to other structural features, the muscle at the contact regions had all the morphological characteristics previously reported, such as nuclei and accumulated mitochondria in the sarcoplasm. In another longitudinally sectioned specimen, a motor innervation area could be observed extending for at least 150 # on one single intrafusal muscle fiber (Fig. 3). Contact areas between the nerve ending and the muscle fiber could be followed in one section for 30 # with interdistances (arrows in Fig. 3) of less than 5/z. In the intervening spaces throughout the innervation area, collagen bundles, filamentous material, and extensions of Schwann cells were observed associated with the nerve ending. This indicates a possible continuum of the nerve ending from one end of the observed contact area to the other, In this specimen the thickness of the axoplasm perpendicular to the contacting surface was measured at about 3-4 # in most places of contact. The nerve endings previously mentioned all had a basement membrane interposed between the axon and muscle plasma membranes. However, in one specimen intimate contacts were detected at some locations along the 200 #-long motor innervation area. 1 The axoplasm was here found evaginated into sac- or ridge-like structures amounting to less than 2 ~u in diameter in the direction parallel to the muscle fiber (Fig. 4). A somewhat constricted part was found to join the sac-like evagination to the axoplasm (Fig. 4b). The evaginations were located in dilated junctional folds, and the plasma membranes of the evaginated axon and the muscle fiber were directly apposed without intervening basement membrane (Fig. 4). The intervening space between the two single-layered plasma membranes was measured at 100-150 A. None of the contact surfaces exceeded 3 # in length in any section. The estimated approximate interval between any two evaginations was at least 1 #. In a section less than 5 ,u apart from those illustrated in Fig. 4, only one evagination could be found. This would possibly indicate a restricted localization of these evaginations within a junctional region. Few membrane-bound structures were found in the evaginations. Scattered granular material and some 400-600 A vesicles were observed at the constriction toward the axoplasm proper (Fig. 4b). The axon plasma membrane often showed increased electron opacity (at P in Fig. 4a-c) at places opposite the entrances of the junctional folds, and a tendency for accumulation of 400-600 ~ vesicles was observed in the axoplasm at these places as has been reported for frog extrafusal motor nerve endings (3). These features were also found in specimens without evaginations (Figs. 5, 6, 7a, 7c, and 8b). The specimen with the peculiar evaginated structures showed shortened sarcomeres, 1.8 # long. Motor nerve ending axoplasm structures observed in all specimens of this investigation were found to conform to that reported for frog extrafusal motor junctions Presented by U. Karlsson at the Fifth International Congress for Electron Microscopy in Philadelphia in 1962 and appearing in the Proceedings (9).
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(3), e.g., c o m p a r t m e n t s of 400-600 A vesicles, neurofilaments, a n d small m i t o c h o n d r i a (Fig. 5). H o w e v e r , with the present technique no b e a d e d structures (3) of the n e u r o filaments c o u l d be detected which m e a s u r e d a b o u t 100 A (96 ___16 A for 26 m e a s u r e ments) in d i a m e t e r (Fig. 6). T h e m i t o c h o n d r i a a v e r a g e d 0.15 # (0.15_+0.04 # for 56 m e a s u r e m e n t s ) in d i a m e t e r in five different specimens. M i t o c h o n d r i a m e a s u r e m e n t s were at all times p e r f o r m e d o n the smallest d i a m e t e r of transversely sectioned organelles. T h e vesiculous p o r t i o n of the t e r m i n a l a x o p l a s m was f o u n d to c o n t a i n at least two types of vesicles. A g r a n u l e - c o n t a i n i n g vesicle was o b s e r v e d w i t h l o w frequency m e a s u r i n g a b o u t 900 A (940 ! 100/~ for 26 m e a s u r e m e n t s f r o m 2 specimens) in d i a m e t e r which always c o n t a i n e d a smaller electron o p a q u e granule v a r y i n g in size f r o m 200 to 800 A in d i a m e t e r (26 m e a s u r e m e n t s ) (Figs. 4, 5, 6, 7c, a n d 8a). The relative occurrence of this t y p e of vesicle was r o u g h l y e s t i m a t e d to a b o u t one in 25 of the m o r e c o m m o n 400-600 A vesicles. The S c h w a n n cell cover of the nerve endings was f o u n d c o m p l e t e o n the side a w a y f r o m the muscle (see Fig. 3). O n the side of the nerve ending facing the muscle, small m e m b r a n e - b o u n d profiles were o b s e r v e d in i n t i m a t e c o n t a c t w i t h the nerve e n d i n g p l a s m a m e m b r a n e a n d p r e d o m i n a n t l y occurring between two consecutive entrances of the j u n c t i o n a l folds (Fig. 6). These cell processes were c o n s i d e r e d likely to b e l o n g to the S c h w a n n cell. Often the S c h w a n n cell l a m e l l a covering the nerve ending was m e a s u r e d as thin as 2 0 0 0 / ~ , b u t closer to nuclei the a m o u n t of c y t o p l a s m was increased a n d thus a l l o w e d o b s e r v a t i o n s of its structures. T h e S c h w a n n cell c y t o p l a s m was c h a r a c t e r i z e d b y a n a b u n d a n c e of vesicular a n d t u b u l a r structures, a variety of r i b o s o m e - s i z e d particles, few m i t o c h o n d r i a ( a b o u t 0.2 # in diameter), a n d scattered larger granules. I n m a n y places large, r o u n d e d , extracellular spaces were f o u n d w i t h the smaller d i a m e t e r m e a s u r i n g a b o u t 1 # (Figs. 3 a n d 5). These spaces were p a r t l y o r c o m p l e t e l y filled w i t h a filamentous material, the unit filament
FIG. 3 a, b. Longitudinal section from a polar region of an intrafusal muscle fiber. The lower end of the picture (a) is continuous with the upper end of picture (b). To the right in both pictures sarcomeres with distinct cross bandings can be observed. On the outside of the muscle plasma membrane a motor nerve ending (ME) is seen coursing along the surface of the muscle fiber. The nerve ending can be seen to contact the muscle fiber extensively and with relatively long contact regions. The nerve ending could be followed for 150/~ along the muscle fiber. The short intervals between the contact regions (horizontal arrows) are seen to be occupied by Schwann cell cytoplasm and collagenous material (C). The contact regions of the nerve ending and the muscle fiber exhibit characteristics typical for motor myoneural junctions, such as peripheral muscle nuclei (Nu, in lower part of Fig. 3 a, and upper part of Fig. 3 b), relatively numerous mitochondria and junctional folds (J) in the muscle, compartments of vesicles (v), neurofilaments (if) and mitochondria (Mi) in the nerve ending. Sehwann cell (SC) is observed to cover the nerve ending on the side away from the muscle. Schwann cell nuclei (Nu) are seen in the upper part of (a) and in the lower part of (b). The cytoplasm of the Schwann cell in some places exhibits fenestrations filled with moderately dense filamentous material (el). Discontinuous lamellae (a) of the inner spindle capsule (IC) are seen surrounded by collagen bundles traversing in different directions, x approx. 6000.
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of w h i c h m e a s u r e d a b o u t 50 A in d i a m e t e r (Fig. 5). T h i s f i l a m e n t o u s m a t e r i a l c o u l d , at places, be o b s e r v e d to be c o n t i n u o u s w i t h b a s e m e n t m e m b r a n e m a t e r i a l (Fig. 5} a n d w i t h i d e n t i c a l m a t e r i a l f o u n d m i x e d w i t h c o l l a g e n b u n d l e s (see b e l o w ) . Intracapsular motor innervation areas were found within an inner discontinuous leaflet of t h e s p i n d l e c a p s u l e (Figs. 1-3) (10, 11). B e t w e e n this i n n e r leaflet a n d t h e S c h w a n n cell c o v e r i n g t h e m o t o r n e r v e e n d i n g , c o l l a g e n b u n d l e s of v a r y i n g sizes o c c u r r e d (Fig. 3). T h e s e c o l l a g e n b u n d l e s w e r e o r i e n t e d r o u g h l y p e r p e n d i c u l a r l y t o t h e l o n g i t u d i n a l axis of t h e m u s c l e fiber a n d m i x e d w i t h t h e f i l a m e n t o u s m a t e r i a l f o u n d in t h e e x t r a c e l l u l a r spaces m e n t i o n e d a b o v e . T h e m u s c u l a r c y t o p l a s m b e n e a t h t h e n e r v e e n d i n g was n o t f o u n d different f r o m t h a t p r e v i o u s l y r e p o r t e d f o r e x t r a f u s a l m o t o r m y o n e u r a l j u n c t i o n s (3). A s o b s e r v e d
FIa. 4a-c. Series of longitudinal sections of an intracapsular motor nerve ending in the frog muscle spindle. The consecutive pictures represent every second section. The muscle plasma membrane exhibits typical junctional folds. The sarcomeres in this specimen were found to be about 1.8 # long throughout the motor innervation area. The axoplasm in the upper parts of the micrographs contains 400-600 A vesicles, 900 A granulated vesicles (horizontal arrows), and small mitochondria (Mi). Toward the muscle surface, the axoplasm protrudes into smaller (P) or larger (E1-4) evaginations. The larger evaginations, either ridge- or sac-like, are seen to traverse the extracellular space down into the junctional folds where they make contact with the muscle without interposition of a basement membrane (double arrows). The evagination E3 connects with the axoplasm proper by a stalk that is visible only in the middle section (b} (sac-like evagination). The evagination El, presumably ridge-like, is seen to the left in all sections (a-c). A few 400-600 A vesicles, a few opaque granules and scattered amorphous material are seen within each evagination. Cytoplasmic processes (Schwann cell processes) are found on both sides of the evagination (Ea) (vertical arrows). Often the axon plasma membrane opposite the entrance to the junctional folds show increased density, x 27,500. FIG. 5. Higher magnification of part of the intracapsular motor myoneural junction. The muscle plasma membrane shows greater density at the contact region than in the region at the lower center. Junctional folds (J) are seen at a few places along the contact. A peripheral muscle nucleus (Nu), a Golgi vesicle complex (G), and an accumulation of relatively large mitochondria (Mi) in a matrix of smaller opaque particles and vesicles represent the muscular specialization at the motor myoneural junction. In the axoplasm to the right, mitochondria (Mi) and thin neurofilaments (if) are seen. Closer to the contact area, 400-600 A vesicles (v) are located. Interspersed among these vesicles and elsewhere, larger vesicles are found displaying a denser inner core (vertical arrows). At the contact area the axoplasm can be seen to protrude (horizontal arrows), especially opposite the junctional folds (J). Here 400-600 A vesicles and an increased density of the axon plasma membrane may be seen. Extracellular filamentous material (el)in the lower center is seen to be continuous with the basement membrane material. In the space between the plasma membranes of the muscle and the nerve cell, membrane-bound structures (S) (Schwann cell processes?) are associated with the axon plasma membrane. × 33,000. FIG. 6. High magnification of part of the motor myoneural junction in the frog muscle spindle. To the right of the muscle cell nucleus (Nu) the muscle plasma membrane exhibits several junctional folds (J). Opposite the entrances of these folds the axoplasm may be seen protruding (horizontal arrows), and in some places the axon plasma membrane displays higher electron density than elsewhere. In between each of these protrusions, membrane-bound cell processes (S) (Schwann cell processes?) can be located close to the axon plasma membrane. The oblique arrow in the lower part of the picture points to a part of the axon plasma membrane that appears triple-layered. The oblique arrow in the upper center points to a larger vesicle with a dense core. To the lower right, thin neurofilaments (if) and two mitochondria (Mi) are seen. × 70,000.
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at mammalian junctions (I), accumulations of tightly packed mitochondria located juxtanuclearly (Fig. 8 a) were often found. The individual mitochondria were seldom estimated to be less than 0.25 # in diameter. In specimens prefixed in formaldehyde, an electron opaque thick coating was observed associated with the sarcoplasmic side of the muscle plasma membrane in the region of the junctional folds (Fig. 7a, b, c). This material appeared more prominent at the plasma membrane between the junctional folds and at their sides than at the bottom of them. As many muscles had been fixed at different lengths, it was possible to observe the nerve-muscle relationship at different lengths of the sarcomeres. In the frog a frequency of 3-4 junctional folds per sarcomere has been estimated (3), which appears as a less complicated pattern than that found in mammalia (i). When analyzing the appearance of the motor myoneura] junction at different sarcomere lengths, it was observed that at rest length, or with stretched sarcomeres (for example, the specimens illustrated in Figs. 3 and 7), the typical frequency was 3-4 junctional folds per sarcomere. At shortened sarcomeres the differences observed were grossly a wavy appearance of the whole junctional area (Fig. 8a) and the number of the junctional folds per sarcomere changed (Figs. 8a and 8b). The sarcoplasmic characteristics of all muscle fibers were analyzed. In the transversely sectioned series it was found that all muscle fibers changed their relative content along their course, such as myofilaments, sarcotubular systems, and amount of mitochondria. Even their overall diameter changed (compare fiber I in Figs. 2a and 2 d). No apparent differencescould be found between different intrafusal muscle fibers which would characterize them as twitch or slow (15), although no high resolution analysis was made. DISCUSSION The present investigation has uncovered certain morphological features typical for motor myoneural junctions in muscle spindles of the frog. The intrafusal motor nerve ending differs from the extrafusal in that it is enclosed in the same capsule as are the sensory nerve endings. Thus it is clear that the criterion stated by Cajal (4) FIG. 7 a--c. Three micrographs showing different examples of the appearance of the motor myoneural junction areas when prefixed in 4% formaldehyde before postfixation in osmium tetroxide. All three, and especially the center one (b), display a thick coat of electron opaque material located at and associated with the inside of the muscle plasma membrane. This material is predominantly located in between and on the side walls of the junctional folds (arrows). Otherwise the morphology of the junctional area is similar to that illustrated in Figs. 5 and 6, except that very few 400-600 ,~ vesicles are observed in the axoplasm in the ending illustrated in (b). In Fig. 7a indications of crowding of the 400-600 /~ vesicles are seen in association with the axon plasma membrane opposite the entrances to the junctional folds. A frequency of 3-4 junctional folds per sarcomere is exhibited in (b). The sarcomere length in (b) was measured to about 2.7 #. x 40,000.
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and accepted by Katz (13) concerning the exclusive extracapsular existence of m o t o r m y o n e u r a l junctions o n intrafusal muscle fibers must be reconsidered. The close proximity of the m o t o r nerve endings to the sensory nerve endings therefore can be t h o u g h t of as shortening the distance for the contraction wave influencing the sensory nerve endings and thus facilitating the subsequent afferent impulse. The mere fact of encapsulation also brings up the question of pharmacological effect on the m o t o r nerve endings. It has been stated that curare is less effective for intrafusal than for extrafusal nerve endings (6, 12). It m a y be justifiable to assume that the spindle capsule would act as a general diffusion barrier and as a specific one if it is considered to contribute to maintaining a special environment for the sensory nerve endings (10). Electrophysiological data (6, 7, 12) have disclosed the presence of two different types of afferent responses related to twitch and slow activation of the muscle spindle. The anatomical correlate for this p h e n o m e n o n has been assigned partly to the existence of Endbuschel- and grape-type endings occurring on the intrafusal muscle fibers (8). I n the present investigation no definite qualitative differences could be f o u n d between several m o t o r junctions studied. With respect to the m o r p h o l o g y of the muscle fibers, no apparent differences could be f o u n d which would characterize them as twitch or slow (15). The differences observed were related quantitatively to the length of individual m o t o r m y o n e u r a l contacts, the spread of the innervation areas along the muscle fibers, the dimensions of the nerve endings and extent of ramification of one single nerve fiber. A l t h o u g h the size of the nerve supplying the innervation areas analyzed was n o t observed, the nerve fiber constituting endings in the transversely sectioned specimen (Fig. 2) was quite thin. It also had extensive ramifications and f o r m e d relatively short contact regions with all the intrafusal muscle fibers concerned. Only in exceptional cases did the thickness of the nerve endings exceed 3/~. However, in two longitudinally sectioned specimens where lengthy contacts were observed (Figs: 3 and 4), a thickness of 3-4 ,u was measured. With respect to the spread over one intrafusal muscle fiber, the former specimen exhibited lengths maximally estimated to 80 #, while the latter
FIG. 8. This micrograph demonstrates the morphology of the motor myoneural junction in a s p e c i where the sarcomere length was measured to an average of 1.8 #. The appearance should be compared with the one exhibited in Fig. 7b where the sarcomere length was measured to 2.7 #. (a) The whole motor nerve ending (ME) can be observed folded and making an impression into the muscle surface. The Schwann cell cover (SC) is seen to follow the folding of the nerve ending. In the center of the axoplasm a 900 A granulated vesicle can be seen (vertical arrow). To the lower right, a small part of a 15 #-long and 3/z-wide accumulation of mitochondria (Mi) can be seen. Very few junctional folds are observed in the muscle plasma membrane in contact with the motor nerve ending, x 27,500. (b) Another part of the same junctional area as illustrated above. Here the sarcomeres can be observed to be about 1.8 /z long. The frequency of junctional folds is about 6 per sarcomere (vertical arrows), x 43,500. men
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specimens amounted to a minimal spread of 150-200/J. Therefore, the present authors are inclined to conclude that there is a differentiation occurring in the motor myoneural junctions of the frog muscle spindles with one type of ending displaying long contacts over great lengths of the fiber with relatively thick nerve endings (long type) and the other type having shorter contact areas, thinner nerve endings and an extensive arborescent pattern on a relatively short length of the muscle fiber (short type). The relative absence of junctional folds observed in the short type specimen might be of significance (5) but must be interpreted with caution as it is possible to section through fold-free areas at the amphibian motor myoneural junction (13, 18). The interpretation mentioned with respect to different types of nerve endings conforms with light microscopical data (8), thus classifying the long type as Endbuschel and the short type as grape-type ending. Functional interpretations will necessarily be speculation even if morphological considerations may be applied. In that case, however, a similar neuromuscular transmission of the two types of junctions can be assumed and the differences considered quantitative. On purely physical grounds the probability of efficient transmission might be higher at a longer contact area supplied by a thicker nerve ending than that of a shorter contact area supplied by a thinner nerve ending. In one of the motor junctions of the long type a peculiar membrane relationship was found, essentially showing evaginations of the axoplasm into dilated junctional folds which contacted the muscle fiber without interposition of a basement membrane (Fig. 4). The meaning of this is obscured by the fact that it was observed in only one specimen and that it may represent a possible aberration in normal morphology. The axon plasma membrane opposite the entrances of the junctional folds was found to have increased electron opacity and a tendency for 400-600 A vesicles to accumulate in the axoplasm at these condensations. This is in agreement with previous observations (3). The cell processes interposed between the axon plasma membrane and the basement membrane (3, 17, 18) in this investigation were found to predominantly occupy locations between the entrances of the junctional folds (Fig. 6). The topographical localization of the axon protrusions, the axon plasma membrane opacities, the small vesicles, and the above-mentioned cell processes may indicate a functional relationship with respect to synaptic transmission. The content of the nerve endings described in this investigation was found not to deviate from previous investigations of extrafusal muscle fibers (3). To our knowledge the granular vesicle found has not been described previously in motor nerve endings but can be found in published pictures of endings on extrafusal muscle fibers (3). However, identical vesicles have been described as present elsewhere in the frog peripheral nervous system (2, 16, 19). The prevailing interpretation of the contents of these granular, 900 A vesicles has been to identify them with similar vesicles in the
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central nervous system referred to as "neurosecretory granules" (14). However, conclusions with respect to its functional relationship to the m o t o r myoneural junction can presently not be made since the nature of the contents of '~neurosecretory granules" has not yet been established. When subjecting the muscle to formaldehyde as a prefixative, as compared to conventional primary osmium tetroxide fixation, an apparent difference in amount of electron dense material was observed associated with the muscle plasma membrane at the sarcoplasmic side in the junctional area (Figs. 4-6 compared to Fig. 7). This would suggest the existence of proteinaceous material at this location. It could be that formaldehyde is a good preservative for proteins such as acetylcholinesterase, which would be anticipated to occur at this location. The results presented regarding differences in morphology of junctional areas at different sarcomere lengths of the muscle fiber indicate that mechanical forces in the process of contraction exert influence at this location. The authors are deeply indebted to Professor F. S. Sj6strand for support and valuable criticism and for the privilege of using his laboratory facilities. Thanks are also due to Mr. Herman Kabe for skillful photographic assistance, and to Dr. R. L. Schultz and Mrs. Kay McQuaid for correcting the English in the manuscript. REFERENCES 1. 2. 3. 4. 5. 6. 7.
8. 9. 10. 1 1.
12. 13. 14. 15. 16. 17. 18. 19.
ANDERSSON-CEDERGREN,E., Y. Ultrastruet. Res. Suppl. 1 (1959). ANDERSSON-CEDERGREN,E. and KARLSSON, U. (in preparation). BIRKS,R., HUXLEY, H. E. and KATZ, B., J. Physiol. (London) 150, 134 (1960). CAJAL, S. R. Y, Rev. Trim. Histol. Norm. y Patol., No. 1, !888. Quoted from Cajal, S. R. y, Histologie du Syst6me Nerveux, Vol. 1, pp. 485 seq. Madrid, 1952. COTEUX,R., Exptl. Cell Res. Suppl. 5, 294 (1958). EYZAGUIRRE,C., J. Neurophysiol. 20, 523 (1957). -J. Neurophysiol. 21, 465 (1958). GRAY, E. G., Proc. Roy. Soc. B 146, 416 (1957). KARLSSON,U., Proc. Intern. Congr. Electron Microscopy, 5th, Philadelphia, 1962, Vol. 2, U-4. Academic Press, New York, 1962. KARLSSON,U. and ANDERSSON-CEDERGREN,E. (in preparation). KARLSSON, U., ANDERSSON-CEDERGREN,E. and OTTOSON, O., J. Ultrastruct. Res. in press. KATZ, B., J. Exptl. Biol. 26, 201 (1949). - - - - Phil. Trans. Roy. Soc. London, B 243, 221 (1960). PALAY, S. L., in Progress in Neurobiology, Vol. 2, p. 31. Harper (Hoeber), New York, 1957. PEACHEY,L. D. and HUXLEY, A. F., Y. Ceil Biol. 13, 177 (1962). PICK, J., Anat. Record 144, 295 (1962). REGER, J. F., Anat. Record133, 327 (1959). ROBERTSON,J. D., Am. J. Phys. Med. 39, 1 (1960). TAxi, J., Compt. Rend. Acad. 252, 174 (1961).