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Variation in the size of synaptic contacts along developing and mature motor terminal branches
Developmental Brain Research, 5 (1982) 11-22
11
Elsevier Biomedical Press
Variation in the Size of Synaptic Contacts along Developing and Mature Motor Terminal Branches D. F. DAVEY and M. R. BENNETT*
The Neurobiology Laboratory, Department of Physiology, University of Sydney, N.S. IV. 2006 (Australia) (Accepted February 8th, 1982)
Key words: synaptic contacts - - development - - motor endplate
The secretion of a quantum from groups of release sites (ffie)declines along the length of terminal branches at the amphibian neuromuscular junction. The morphological basis of this decline in ffaohas been studied at neuromuscularjunctions in juvenile muscles (fibre length 4 ram) and adult muscles (fibre length 22 mm). Serial sections cut through the length of the junctions have been examined with both light and electron microscopy. Juvenile junctions consist of two short (< 50/~m) terminal branches; adult junctions often consist of 4 long (100-500/~m) terminal branches. Synaptic contacts are largest near the origins of terminal branches and decline in size towards the end of branches. The number of horseradish peroxidase-labelled synaptic vesicles at release sites, following stimulation in the presence of the enzyme, is largest for sites closest to the origin of the terminal branches. The results suggest that the decline in r~o along the length of terminal branches is due to decline in the size of release sites. INTRODUCTION The average number of quanta secreted at different recording sites along the length of neuromuscular junctions is not constantL Small groups of release sites along individual terminal branches secrete different numbers of quanta (l~e) : file is highest for sites close to the origin of the terminal branch near the last myelin segment of the parent axon; it is lowest for sites near the end of a branchl,6,1L Since the sites closest to the origin of the branch are laid down first during developmentS,z2, these observations also suggest the possibility that file declines along a terminal branch from sites first laid down during development to those last laid down. The morphological basis of this pattern of nonuniformity in file has been studied in the present work. Serial sections cut through the lengths of toad neuromuscular junctions have been examined with both light and electron microscopy. The architecture of the junction has been reconstructed with the aid of computer techniques, and the size of synaptic contacts along the length of terminal branches de* To whom all correspondence should be addressed. 0165-3806/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
termined. The results indicate that the size of these synaptic contacts declines from near the origin to the end of terminal branches. Preliminary reports of aspects of this work have been presented1,14. MATERIALS AND METHODS
Preparations lliofibularis muscles together with their sciatic nerve were dissected from the toad (Bufo marinus). Muscle fibres about 4 mm long were dissected from juvenile toads, whereas fibres of about 22 mm length were obtained from adult toads. These fibres allowed for a developmental study of terminal size. The muscles were pinned out on silicone rubber in the base of 10-ml perspex baths; the unipennate structure of the muscle was spread into a parallelogram in which the two tendons lay on the short sides. The muscle was oriented with what had been the lateral surface upwards. The structural analysis was restricted to a small region of muscle distal to the point of nerve entry (Fig. 2; this was the same region used in electrophysiological experiments re-
12 ported in the previous paperG). The muscles were bathed in a Ringer's solution which contained (mmol/I): K + 2.5; Na ~ 120; C1- 121; Ca 2E 1.8; HPO22- 2.15; HzPO4- 0.85 (pH 7.2).
Distribution of fibre types Whole muscles mounted as described above were fixed with formalin, frozen sectioned, and stained with Sudan black al. This allowed a comparison between the distribution of muscle types in Bufo marinus iliofibularis with that reported for Xenopus laevis (ref. 21). Localization of eholinesterase activity Iliofibularis muscles were fixed for 1 h in 1 0 ~ formalin-15 ~ sucrose. Cholinesterase activity was localized using 30-40 min incubation according to the method of Karnovsky and Roots 18. Labelling synaptic vesicles with horseradish peroxidase An estimate of synaptic vesicle participation in secretion at different release sites was made. Muscles were first bathed for 1 h in Krebs solution containing horseradish peroxidase (HRP, Sigma type VI; 2.5 mg/ml). The attached sciatic nerve was then stimulated at 1 Hz for 1 h using a suction electrode 7. The preparation was rinsed with Krebs solution devoid of H R P for 30 min prior to fixation. Fixation for electron microscopy Muscles were fixed overnight at 4 °C in glutaraldehyde (20 g/l) dissolved in a Ringer's solution. The fixed muscles were rinsed with several changes of the Ringer's solution, and then a single small block suitable for embedding purposes was cut from the edge & t h e muscle opposite to the nerve entry (see Fig. 2) in such a way that the endplate region was in one end of the block. In some experiments the block was then incubated to localize H R P (see below). The block was then post-fixed using osmium tetroxide (10 g/l, 2 h), stained with uranyl nitrate (0.5 g/l, pH 5.0, 45 min), dehydrated and embedded in Spurr's (1969) resin and oriented to enable transverse sectioning of the end of the block containing the endplate region. Locafization of HRP Blocks were incubated for H R P localization after
they were cut from the rinsed glutaraldehyde fixed muscles using the following schedule: four 10-min rinses with Tris-maleate buffer (0.15 mol/l, pH 7.6): 45 min infiltration of 3,3'-diaminobenzidine tetrahydrochloride (DAB, 0.06% in the Tris-maleate buffer); four 15-min incubations with DAB and 0.01 ~ HzOz dissolved in the Tris-maleate buffer; and finally three 10-rain rinses with the Tris-maleate buffer. The blocks were th.en osmium-fixed and further processed as described above.
Serial sectioning Sectioning was performed with a Cambridge Instruments (A. F. Huxley pattern) Ultramicrotome equipped with an orientating specimen holder. The holder was adjusted to give precisely transverse sections, after which the block was not removed from the microtome until the serial sectioning was completed. The block faces were too large to be cut with the available diamond knives, so glass knives had to be used. The procedure consisted of repeated cycles of thick and thin sectioning which we will refer to as cuts. One cut generally included twenty l-#m sections followed by about 10 th.in sections. (In some cases fifteen 1.5 /zm or forty 1 /zm sections were collected.) It was judged more important to obtain thin sections with a minimum of block refacing tban to obtain sections of the highest quality. The micrographs obtained from these sections reflect this decision. The thick sections were mounted serially on glass slides and stained for light microscopy using 1 To toluidine blue in 1 ~/oborax, followed after rinsing by 1 ~ safanine-O in 1 ~ borax. The thin sections were mounted on grids and stained with lead citrate for electron microscopy. The total thickness of each cut was recorded for analytical purposes. A muscle fibre was only considered for complete reconstruction and morphometric analysis after verifying that it could be observed in thin sections for all cuts containing endplates. Furthermore, it was necessary to have at least 3 cuts on either side of the endplates, to ensure against accidental underestimate of terminal length. Three-dimensional reconstructions Analysis of endplate structure, which required the use of as many as 700 thick sections, was carried out with the assistance of a computer-aided reconstruction technique. The outlines of muscle fibres to be
13 analyzed were obtained from single thick sections at about 10/~m intervals. The images produced with a Leitz carbon-arc microprojector at an enlargement of 1600 x were traced onto transparent sheets. The position of axons and all structures that might be nerve terminals were examined with the electron microscope to positively identify nerve endings and locate those not found with the light microscope. Electron micrographs were taken of all terminals found. The verified tracings were digitized for computer reconstruction using the principle employed by Bennett et al. 2, but using a digitizing graphics tablet for input to a P D P l l / 1 0 computer. Each line was traced using the digitizer pen and a series o f x - y coordinates of the traced line were temporarily stored on disk. Coordinate reading and transfer to disk proceeded at the maximum possible rate. The traced line could then be approximated by a series of straight lines joining these points and its length approximated by the lengths of these lines. This length was then divided into n - - 1 segments, where n is the number of points used to represent the line. Then co-ordinates were calculated for points at the end of each segment and lying on the approximation formed by the series of straight lines mentioned above. Thus for each structure traced, the final representation in each section was by the same number of points, equally spaced. This scheme has the advantage that the corresponding points in adjacent sections can be connected by lines on the computer drawing, giving visual cues to these structural representations. The 2-dimensional co-ordinates of the structures digitized from the serial sections, together with the separation between sections, were used to calculate a 3-dimensiona 1 reconstruction of the structure of interest. Through further calculations these observations could be rotated in the 3-dimensional projection and accounting for the perspective. Two such projections, with the viewpoints appropriately separated, enable the 3-dimensional appearance of the reconstruction to be examined with a stereoscope. The plots of structures have been represented in 3 ways. In some the digitized lines f r o m each section have been plotted as dashed lines in their calculated position. In others, solid lines have been used and the corresponding points in adjacent sections have
been connected with solid lines. (The use of a data acquisition scheme in which the number of points recorded for a structure is the same in every section makes this process very simple.) Structures depicted in this way are described in the legends as drawn with continuous lines. In the third, only the lines connecting corresponding sections have been included, and these are drawn with 2 breaks per section. Structures drawn in this way are described as drawn with broken lines.
Electron micrography Thin sections were photographed for morphometry using a Philips EM 201c, generally at an instrument magnification of 6000 × . Photographic prints of these micrographs were produced by a further 3 × enlargement.
Morphometry Measurements of terminal area and synaptic contact were made directly on electron micrographs using the graphics tablet and a computer programme which accounts for magnification of the micrographs.
Terminology The definition of terms used in the description of
~
terminal branch
last myelin segment /
c0ntact
y muscle fibre
cholinesterase
I
/
terminal size (cross-sectional area)
Fig. 1. Terminology used to describe the nerve endings at neuromuscular junctions on muscle fibres in the region of the iliofibularis shown in Fig. 2. A motor axon divides after the last myelin segment, and gives rise to two pairs of terminal branches. Each of the terminal branches of a pair runs in opposite directions along the long axis of the muscle fibre. One terminal branch possesses a secondary terminal branch. The synaptic contact is the length of the region of approximation (in transverse sections) between the nerve terminal and the muscle fibre.
14
Fig. 2. Transverse section through a whole toad iliofibularis muscle stained with Sudan black. This muscle is from the right leg viewed from the tibial end. The in vivo shape of the muscle has been distorted, because the muscle was pinned out in the manner usually used for electrophysiologyor ultrastructural analysis (see Materials and Methods). The superficial surface in vivo is to the left. The lateral surface in vivo is uppermost, consequently the nerve entry is on the right. The region enclosed within the rectangle has been used for electrophysiologicalinvestigations~ and was examined for the structural investigations in this report. Calibration : 1 mm. the neuromuscular junction are given in reference to Fig. 1. This schematic junction consists of 4 terminal branches which originate from 2 axon branches. The synaptic contact is indicated by arrows in a transverse section through a region of terminal branch which contains a high density of syaaptic vesicles (Figs. 4, 7); it is the length of terminal membrane between the arrows in close apposition to the muscle membrane. The section with the highest vesicle density over the 10 thin sections in each cut (see above) was used to determine the synaptic contact. Tb_e length of a release site is given by the length of terminal contact at an 'active zorte 'x2. This sometimes corresponds to a synaptic contact. It is delineated by the convergence of synaptic vesicles towards the pre-synaptic membrane in the transverse sections. These are clear following HRP-labelling of the synapt;c vesicles.
vealed a variation in staining of individual fibres (Fig. 2). This was similar to that described by Lhnnergren and Smith 21 for the iliofibularis of the African toad Xenopus laevis. They showed that pale cells are twitch fibres which fatigue during tetanic contractions, and would now be termed fast fatiguable 10 (type FF). The present study was restricted to pale cells in the regions shown by the rectangle in Fig. 2; in vivo these are located on the superficial surface of the muscle. Sections through this selected region reveal a population of cells of uniform appearance and fairly uniform size. In Xenopus, these fibres have a type of en plaque neuromuscular junction which is characterized by a number of long and fairly straight terminal branches that run parallel to the muscle fibre axis2L In Bufo, cholinesterase staining of neuromuscular junctions in the region indicated in Fig. 2 were of this kind.
RESULTS
Reconstruction of juvenile neuromuscular junctions
Distribution of fibre types
The neuromuscular junctions of 6 fibres from 4 mm long juvenile muscles were reconstructed. Five of these had a very simple structure consisting of
Sudan black staining of iliofibularis muscles re-
1 Ii
B
~ I ,i ~j
,
I
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Fig. 3. Stereoscopic 3-dimensional reconstructions from 2 juvenile (4 mm) muscle fibres. The myofibres are shown with dashed lines. A: the simplest neuromuscular junction observed, consisting of 2 short equal-length terminal branches. B: the most complex neuromuscular junction observed : this consists of 2 short and 3 very short terminal branches; the 2 short branches do not maintain synaptic contact along their whole length (broken lines). Calibration: 100 ym.
4mm
mvof" ib r e
Fig. 4. Thin sections from successive cuts taken from a single terminal branch in a juvenile (4 ram) muscle fibre. Each section is labelled with the distance separating it from the origin of the axon branch leading to the terminal. The first micrograph shows the first terminal contact observed. The terminal was not present in the cut after the last shown in this series. Note that this terminal was stimulated in the presence of horseradish peroxidase (HRP), some of the synaptic vesicles are filled with H R P reaction product. The combination of the H R P reaction and the serial section procedures used (see Materials and Methods) is responsible for the section quality.
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Fig. 5. Changes in terminal size (A, cross-sectional area) and synaptic contact (B) along the length of terminal branches on a juvenile muscle fibre. The zero reference indicates the point at which terminal branches originate from the axon. C gives the numbers of HRP-labelled synaptic vesicles at different sites along the length of terminal branches, stimulated in the presence of HRP. These results are for the neuromuscular junction in the stereoscopic pair of Fig. 3A.
two short ( < 100/~m) terminal branches of about equal length with the occasional addition of a very short branch; the total contact length between terminal and muscle was about 200 #m. An example of a simple junction is shown in stereoscopic Fig. 3A. The main pair of terminal branches arises as a consequence of the splitting of the axons near the last myelin segment. This gives rise to a discontinuity in
synaptic contact along the length of the muscle fibre, between the pair of terminal branches. The remaining juvenile junction of the 6 studied had a complex structure consisting of 2 short and 3 very short terminal branches, as shown in stereoscopic Fig. 3B : the short terminal branches do not maintain synaptic contact along their entire length. Nevertheless, even this complex juvenile endplate did not have a total synaptic contact in excess of 200 #m. Examination of serial electron micrographs of terminal branches indicated that the terminals decrease in size from their origins at the axon to their ends (Fig. 4). Two measurements were used to quantify these observations: terminal size, measured as the cross-sectional area of transverse sections through a terminal branch; and the synaptic contact, measured as the length of close 20 nm apposition between pre- and postsynaptic membranes in transverse sections. Fig. 5 shows plots of both terminal size and synaptic contact along the length of the terminal branches of a typical juvenile endplate; zero is taken as point of origin of terminal branches from their parent axon. Both the terminal size and synaptic contact decrease from high values near the branch origin to low values at its end. An estimate was also made of the participation of synaptic vesicles in secretion at different sites along the length of terminal branches. The number of horseradish peroxidase (HRP)-labelled vesicles at different sites was determined following stimulation of axons in the presence of HRP (see Materials and Methods). The quantitative results are shown in Fig. 5C: the number of HRP-labelled vesicles declines along the length of terminal branches in the same way as the length of the synaptic contact.
Reconstruction of mature neuromuscular junctions The mature neuromuscular junctions of 7 fibres from 22 mm long mature muscles were reconstructed. Only one of these had the very simple juvenile structure consisting of two short terminal branches (see Fig. 3A). The remaining junctions were all more complex, with a considerably greater total length of terminal branches. Examples of these are shown in stereoscopic Fig. 6. In one case (Fig. 6A) the myelinated axon divides to form two pairs of terminal branches, one on either side of the muscle fibre. This division of the axon to form multiple pairs of termi-
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Fig. 6. Stereoscopic 3-dimensional reconstructions from 3 mature (22 ram) muscle fibres. The myofibres are shown with dashed lines. A: a simple neuromuscular junction, consisting of 2 pairs of relatively short terminal branches. B: a typical neuromuscular junction; this consists of one very long pair of terminal branches, and another relatively short pair; one of the long terminal branches does not maintain synaptic contact along its entire length (broken lines). C: a complex neuromuscular junction; the parent axon gave rise to a pair of long terminal branches making variable synaptic contact (broken lines) and a short terminal branch which divided into a terminal branch pair; these in turn divided to form secondary terminal pairs. Calibration: 100/zm.
hal branches was frequently observed; it lead to a substantial number of terminal branches at mature endplates compared with juvenile endplates. Some terminal branches do not maintain synaptic contact along their entire length. In the case shown in stereoscopic Fig. 6B, one terminal branch lifted off the muscle fibre for a short distance before once more forming synaptic contact (see also ref. 24). The most complex endplate reconstructed is shown in stereoscopic Fig. 6C. The parent axort divided to form a single pair of terminal branches and a short terminal branch which itself divided to form a pair of terminal branches; these in turrt divided to form a further pair of terminal branches. Some of these terminal branches did not maintain synaptic contact along their entire length. Most terminal branches at mature endplates decrease in size along their length. Serial electron micrographs show that both the terminal size and
synaptic contact decrease from near the origin of a terminal branch to its end (Fig. 7). Quantitative results for two endplates are shown in Fig. 8: the terminal size and synaptic corttact decrease in the proximo-distal direction along individual branches, after they reach their maximum values near the last myelin segment. Transverse sections through terminal branches occasionally passed through release sites (see Fig. 7); the size of these tapered along the length of mature terminal branches as they did along juvenile branches (see above). One mature junction studied showed remarkable constancy in synaptic contact length along each of its terminal branches. This junction was exceptional. Table I gives the synaptic contact along the length of terminal branches of 6 mature and 6 juvenile junctions. These branches are each of about the same length in the mature and juvenile cases respectively. Table II gives terminal sizes along the length
18 22mm m y o f i b r ~
Fig. 7. Thin sections from successivecuts taken from a single terminal branch in a mature (22 ram) muscle fibre (compare with Fig. 4). Each section is labelled with the distance separating it from the origin of the axon branch leading to the terminal. The first micrograph shows the first terminal contact observed. The terminal was not present in the cut after the last shown in this series.
of these branches. The average synaptic contact and terminal size decline after they reach their maximum size near the last myelin segment. DISCUSSION
Changes in terminal size and synaptic contact along terminal branches Both the cross-sectional area of transverse sections through terminal branches and the length of apposition between pre- and post-synaptic membranes varied along the length o f terminal branches. For most branches this took the form of an increase in terminal area and contact length near the last myelin segment, followed by a gradual decrease towards the ends of the branches (Figs. 5, 8). The
accuracy of this work is limited by the serial sampling for the electron microscope occurring at intervals of about 20 # m (see Materials and Methods); this interval was necessitated by the amount of work involved in the reconstructions of mature endplates some of which were over 500/~m long. Small fingerlike processes off the terminal branches may have been missed at this sampling distance of 20 ,um. However, zinc-iodide staining of terminal branches did not reveal any such processes (see Fig. 3 in ref. 6). The question arises as to whether the tapering of the terminal branches is due to the fixation procedures used in the present work. Terminal branches viewed with the light microscope, after a variety of fixation and staining procedures, show the same pattern of tapering as described in the present work
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Fig. 8. Changes in terminal size and synaptic contact along the length of terminal branches on 2 mature myofibres (a and b). a: gives the results for the two terminal branches of the neuromuscular junction of Fig. 6A. b" gives the results for the several terminal branches of the neuromuscular junction of Fig. 6B. The zero reference indicates the point at which terminal branches originate from the axon.
20 (see, for example, Figs. 11 a n d 12 in ref. 24). Either
# m . This length o f terminal branch is p r o b a b l y
all the fixatives an d stains used in these w o r k s lead
insufficient to show the tapering in size o f release
to terminal tapering or this is a true feature o f the
sites at m a t u r e endplates. It should be emphasized in
a n a t o m y o f the n e u r o m u s c u l a r ju n c ti o n . F u r t h e r -
this context that not all m a t u r e terminal branches
more, other electron m i c r o s c o p e studies o f m a t u r e
tapered c o n t i n u o u s l y f r o m near their origins to their
terminal branches 24, using fixatives o f different ionic
ends: a few branches reached a fairly constant size
c o m p o s i t i o n f r o m that in the present w o r k (see ref.
and tapered abruptly t o w ar d s their ends; others
13) have described terminal branches with similar
tapered to a very small size and then a b r u p t l y in-
characteristics:
creased again to finally taper once m o r e (Fig. 8).
these
include
well-differentiated and
These variations show that e r r o n e o u s conclusions
synaptic gutters only partially occupied by the ter-
can be reached f r o m the sampling o f a small length
synaptic
gutters
without
terminal
contacts
o f t e r m i n a l b r a n c h rather t h a n r e c o n s t r u c t i e n o f t he
mi na l branch (see Fig. 8 above). Some observations suggest that the active zones
whole n e u r o m u s c u l a r junction.
or release sites are o f c o n s t a n t length along t e r m i n a l branches (see Fig. 15 in ref. 16). A m e a n o f 24 conse-
Variation in s y n a p t i c contact length and the secretion
cutive release sites were studied by these authors. As release sites are at m o s t 2 / ~ m apart, these consecu-
o f quanta Th e average n u m b e r o f q u a n t a secreted at differ-
tive release sites co v er e d on average a distance o f 48
ent recording sites al o n g the length o f an endplate
TABLE I The length of synaptic contact at different distances along the length of 6 terminal branches Each branch is from a different junction. The largest values are nearly always at a distance of 40/zm; other values have been normalized to those found at 40 #m. In column P are given the results of Student's t-test for significant differences between the normalized values at 40 #m and those at other distances. Distance along branch
Synaptic contact (ttm)
Mean ~ S.E.M.
Mature terminal branches 0 0 20 0.8 0.28 40 2.7 0.93 60 2.9 1.0 80 1.6 0.56 120 1.3 0.45 160 0
0 1.7 0.68 2.5 1.0] 2.4 0.96 1.8 0.72 0.8 0.32 0
0 0.6 0.27 2.1 0.96 2.2 1.0 2.2 1.0 1.1 0.5 0
0 1.9 0.73 2.6 1.0 2.4 0.92 ----0
0 2.2 0.96 2.3 1.0 2.3 1.0 --0.9 0.39 0
0 2.1 1.0 2.1 1.0 1.4 0.67 0.57 1.3 0.62 0
0 1.55 ± 0.26 0.65 ± 0.12 2.38 ± 0.10 0.98 ± 0.01 2.27 dz 0.18 0.93 ~ 0.05 1.70 ± 0.18 0.71 ± 0.07 1.10 t 0.10 0.46 ± 0.04 0
Juvenile terminal branches 0 0 20 1.70 0.49 40 3.50 1.00 60 2.50 0.71 80 1.40 0.40 100 0
0 2.30 1.00 1.40 0.61 0.70 0.30 --0
0 2.30 1.00 --1.60 0.70 --0
0 2.90 1.00 2.60 0.90 2.50 0.86 1.4 0.54 0
0 0.90 0.64 1.40 1.00 0.60 0.43 0.30 0.21 0
0 1.30 0.57 2.30 1.00 --1.60 0.70 0
0 1.9o 4- 0.27 0.78 ± 0.09 2.10 =t= 0.34 0.90 ± 0.07 1.58 ± 0.37 0.60 4- 0.09 1.18 4- 0.26 0.46 ± 0.10 0
* P < 0.05.
1.2
P
<0.05*
-<0.25 <0.01" <0.001"
<0.25
<0.05* <0.02*
21 TABLE I! The terminal size at different distances along the length o f 6 terminal branches
Each branch is from a different junction. The largest values are nearly always at a distance of 40/~m; other values have been normalized to those found at 40/zm. In column P are given the results of Student's t-test for significant differences between the normalized values at 40/zm and those at other distances. Distance along branch (t~m)
Terminal size (#m ~)
Mature terminal branches 0 0
20 40 60
80 120 160
40 60 80 100
P
0
0
0
0
0
0
1.6 0.52 3.1
1.0 0.43 2.3
1.0 0.71 1.4
1.60 4- 0.25 0.64 i 0.10 2.50 ± 0.24
<0.001 * --
1.0
1.0
1.0
1.0
1.00 4- 0
--
1.4 0.61 2.2 0.96 0.5 0.22 0
1.4 1.0 1.3 0.93 0.5 0.36 0
1.2 0.38 3.2 1.0 1.9 0.59 --0.4 0.13 0
2.5 1.0 2.5
3.0 0.97 1.5 0.48 1.3 0.42 0
2.3 0.85 2.7 1.0 1.7 0.63 ----0
2.5 1.0 1.8 0.72 1.6 0.64 0
1.98 4- 0.24 0.80 4- 0.08 1.70 4- 0.17 0.77 ± 0.10 0.86 4- 0.22 0.35 ± 0.08 0
0
0
0
0
0
0
1.9 1.0 0.6 0.32 0.5 0.26 --0
2.1 1.0 --1.3 0.62 --0
2.5 1.0 1.3 0.52 1.2 0.48 0.5 0.38 0
0.3 0.32 0.9 1.0 0.1 0.11 0.1 0.11 0
0.5 0.42 1.2 1.0 --0.7 0.58 0
1.33 ± 0.35 0.67 4- 0.14 1.30 4- 0.28 0.75 4- 0.13 1.16 -4- 0.39 0.49 4- 0.14 0.40 -- 0.11 0.30 ± 0.10 0
Juvenile terminal branches 0 0
20
Mean 4- S.E.M.
0.7 0.26 2.5 0.93 2.7 1.0 0.3 0.11 0
<0.05* <0.05* <0.001"
<0.25 --<0.10 <0.05*
* P < 0.05. varies 5 because the probability of secretion declines along the length of individual terminal branches 6. This is possibly due to the decline in size of the synaptic contact along the length o f t e r m i n a l branches described in the present w o r k ; it is unlikely to result from the complete breaks in synaptic contact along t e r m i n a l branches (see Fig. 6) as these occurred infrequently. T h e size of release sites does n o t determine the size of q u a n t a as these do n o t vary along the length of terminal branches 6. The development of terminal branches
Synaptic t r a n s m i s s i o n begins d u r i n g development w h e n contact is made between small enlargements at the ends o f u n m y e l i n a t e d m o t o r axons and myotubesa,9,19. These small enlargements g r o ~ i n length, parallel to the long axis of the myotube, a n d f o r m a pair o f terminal branches 22. I n the case of the
a m p h i b i a n iliofibularis, most of the fibres have lost their multiple i n n e r v a t i o n when the muscle reaches a length of 4 m m (see text a n d Figs. 2 a n d 5B in ref. 9). This is confirmed i n the present work, i n which n o multiply innervated synaptic sites were observed on 4 m m long muscle fibres. The r e m a i n i n g j u n c t i o n s at this time still consist of a pair of short terminal branches. F u r t h e r development of the endplate consists primarily of a n increase in the n u m b e r of termin a l - b r a n c h pairs, in the length of terminal branches a n d in the secondary b r a n c h i n g o f some t e r m i n a l branches. A n increase in the average q u a n t a l c o n t e n t o f the e.p.p. (fin) accompanies the growth of terminals d u r i n g either the r e i n n e r v a t i o n or development of synapses i n muscles 4,23. According to the arguments above 1~ increases due to a n increase i n the n u m b e r of t e r m i n a l - b r a n c h pairs rather t h a n to a n increase
22 in the length of individual terminal branches; only a small increase in ffa would be expected from the a d d i t i o n of low-probability release sites at the ends of terminal branches, unless this is a c c o m p a n i e d by a n increase in ffi at the existing release sites. M a t u r e synapses consisting o f quite different total lengths of terminal branches have been described above. A correlation has been sought 2° between ffl a n d the total length of terminal branches at a m p h i b i a n synapses in low [Ca]0. I n the first group of 20 endplates f r o m two muscles, n o correlation was f o u n d between r~ a n d terminal length. However, in a
correlation was observed. Nevertheless, endplates of a given terminal length varied over 20-fold in their value (see Fig. 4 in ref. 20). This poor correlation is to be expected if release sites closest to the last myelin segment have the highest probability for secretion: ffa is then largely i n d e p e n d e n t of the r e m a i n i n g length of t e r m i n a l a n d only increases substantially if new terminal branches are laid down. Release sites towards the end of terminal branches do secrete with a n increasingly higher probability d u r i n g the facilitation that accompanies a short high-frequency train of impulses G.
second group of 32 endplates from 3 muscles a linear REFERENCES 1 Bennett, M. R., Davey, D. F. and Lavidis, N. A., Variation in the numbers of quanta secreted at different sites along developing nerve terminals: correlation with release site ultrastructure, Proc. Aust. PhysioL PharmacoL Soc., 11 (1980) 36P. 2 Bennett, M. R., Davey, D. F. and Uebel, K., The growth of segmental nerves from the brachial myotomes into the proximal muscles of the chick forelimb during development, J. comp. Neurol., 188 (1980) 335-357. 3 Bennett, M. R., Fisher, C., Florin, T., Quine, M. and Robinson, J., The effect of calcium ions and temperature on the binomial parameters that control acetylcholine release by a nerve impulse at amphibian neuromuscular synapses, J. Physiol. (Lond.), 271 (1977) 641-672. 4 Bennett, M. R. and Florin, T., A statistical analysis of the release of acetylcholine at newly formed synapses in striated muscle, J. Physiol. (Lond.), 238 (1974) 93-107. 5 Bennett, M. R. and Lavidis, N. A., The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites, J. gen. Physiol., 74 (1979) 429-456. 6 Bennett, M. R. and Lavidis, N. A., Variation in quantal secretion at different release sites along developing and mature motor terminal branches, Develop. Brain Res., 5 (1982) 1-9. 7 Bennett, M. R., McGrath, P. and Davey, D. F., The regression of synapses formed by a foreign nerve in a mature axolotl striated muscle, Brain Res., 173 (1979) 451-469. 8 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515-545. 9 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in amphibian striated muscle during development, J. PhysioL (Lond.), 265 (1975) 261. 10 Burke, R. E., Levine, D. N., Tsairis, P. and Zajac, F. E., III, Physiological types and histochemical profiles in motor units of the cat gastrocnemius, J. Physiol. (Lond.), 234 (1972) 723. 11 Carleton, H. M., Histological Technique, 4th Edn., Oxford University Press, London, 1967. 12 Couteaux, R. E. and Pecot-Dechavassine, M., V6sicles synaptiques et poches au niveau des 'zones actives' de la
jonction neuromusculaire', C.R. Gebd, Seances Acad. Sci., 271 (1970) 2346-2349. 13 Davey, D. F., The effect of fixative tonicity on the myosin filament lattice volume of frog muscle fixed following exposure to normal or hypertonic Ringer, Histochem. J., 5 (1973) 87-104. 14 Davey, D. F. and Bennett, M. R., Variation in the size of release sites along nerve terminal branches in developing and adult toad iliofibularis muscle, Canad. Physiol., 11 (1981) 74. 15 De Cino, P. A., Reinnervation of frog muscle, Neurosci. Abstr., 6 (1980) 92. 16 Heuser, J. E., Reese, T. S., Dennis, M. J., Jan, Y. and Evans, L., Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release, J. Cell Biol., 81 (1979) 275-300. 17 Heuser, J. E., Reese, T. S. and Landis, D. M. D., Functional changes in frog neuromuscular junctions studied with freeze-fracture, J. Neurocytol., 3 (1974) 109-131. 18 Karnovsky, J. J. and Roots, L., A 'direct-colouring' thiocholine method for cholinesterases, J. Histochem. Cytochem., 12 (1964) 219-221. 19 Kullberg, R. W., Lentz, T. L. and Cohen, M. W., Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and fine-structural study, Develop. Biol., 60 (1977) 101-129. 20 Kuno, M., Turkanis, S. A. and Weakly, J. M., Correlation between nerve terminal size and transmitter release at the neuromuscular junction of the frog, J. Physiol. (Lond.), 213 (1971) 545-556. 21 L~innergren,J. and Smith, R. S., Types of muscle fibres in toad skeletal muscles, ActaphysioL scand., 68 (1966) 263274. 22 Letinsky, M. S. and Morrison-Graham, K., Structure of developing frog neuromuscular junctions, J. Neurocytol., 9 (1980) 321-342. 23 McGrath, P. A. and Bennett, M. R., The development of synaptic connections between different segmental motoneurons and striated muscles in an axolotl limb, Develop. Biol., 69 (1979) 133-146. 24 Wernig, A., Pecto-Dechavassine, M. and St6ver, H.. Sprouting and regression of the nerve at the frog neuromuscular junction in normal conditions and after prolonged paralysis with curare, J. Areurocytol., 9 (1980) 277 303.
11
Elsevier Biomedical Press
Variation in the Size of Synaptic Contacts along Developing and Mature Motor Terminal Branches D. F. DAVEY and M. R. BENNETT*
The Neurobiology Laboratory, Department of Physiology, University of Sydney, N.S. IV. 2006 (Australia) (Accepted February 8th, 1982)
Key words: synaptic contacts - - development - - motor endplate
The secretion of a quantum from groups of release sites (ffie)declines along the length of terminal branches at the amphibian neuromuscular junction. The morphological basis of this decline in ffaohas been studied at neuromuscularjunctions in juvenile muscles (fibre length 4 ram) and adult muscles (fibre length 22 mm). Serial sections cut through the length of the junctions have been examined with both light and electron microscopy. Juvenile junctions consist of two short (< 50/~m) terminal branches; adult junctions often consist of 4 long (100-500/~m) terminal branches. Synaptic contacts are largest near the origins of terminal branches and decline in size towards the end of branches. The number of horseradish peroxidase-labelled synaptic vesicles at release sites, following stimulation in the presence of the enzyme, is largest for sites closest to the origin of the terminal branches. The results suggest that the decline in r~o along the length of terminal branches is due to decline in the size of release sites. INTRODUCTION The average number of quanta secreted at different recording sites along the length of neuromuscular junctions is not constantL Small groups of release sites along individual terminal branches secrete different numbers of quanta (l~e) : file is highest for sites close to the origin of the terminal branch near the last myelin segment of the parent axon; it is lowest for sites near the end of a branchl,6,1L Since the sites closest to the origin of the branch are laid down first during developmentS,z2, these observations also suggest the possibility that file declines along a terminal branch from sites first laid down during development to those last laid down. The morphological basis of this pattern of nonuniformity in file has been studied in the present work. Serial sections cut through the lengths of toad neuromuscular junctions have been examined with both light and electron microscopy. The architecture of the junction has been reconstructed with the aid of computer techniques, and the size of synaptic contacts along the length of terminal branches de* To whom all correspondence should be addressed. 0165-3806/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
termined. The results indicate that the size of these synaptic contacts declines from near the origin to the end of terminal branches. Preliminary reports of aspects of this work have been presented1,14. MATERIALS AND METHODS
Preparations lliofibularis muscles together with their sciatic nerve were dissected from the toad (Bufo marinus). Muscle fibres about 4 mm long were dissected from juvenile toads, whereas fibres of about 22 mm length were obtained from adult toads. These fibres allowed for a developmental study of terminal size. The muscles were pinned out on silicone rubber in the base of 10-ml perspex baths; the unipennate structure of the muscle was spread into a parallelogram in which the two tendons lay on the short sides. The muscle was oriented with what had been the lateral surface upwards. The structural analysis was restricted to a small region of muscle distal to the point of nerve entry (Fig. 2; this was the same region used in electrophysiological experiments re-
12 ported in the previous paperG). The muscles were bathed in a Ringer's solution which contained (mmol/I): K + 2.5; Na ~ 120; C1- 121; Ca 2E 1.8; HPO22- 2.15; HzPO4- 0.85 (pH 7.2).
Distribution of fibre types Whole muscles mounted as described above were fixed with formalin, frozen sectioned, and stained with Sudan black al. This allowed a comparison between the distribution of muscle types in Bufo marinus iliofibularis with that reported for Xenopus laevis (ref. 21). Localization of eholinesterase activity Iliofibularis muscles were fixed for 1 h in 1 0 ~ formalin-15 ~ sucrose. Cholinesterase activity was localized using 30-40 min incubation according to the method of Karnovsky and Roots 18. Labelling synaptic vesicles with horseradish peroxidase An estimate of synaptic vesicle participation in secretion at different release sites was made. Muscles were first bathed for 1 h in Krebs solution containing horseradish peroxidase (HRP, Sigma type VI; 2.5 mg/ml). The attached sciatic nerve was then stimulated at 1 Hz for 1 h using a suction electrode 7. The preparation was rinsed with Krebs solution devoid of H R P for 30 min prior to fixation. Fixation for electron microscopy Muscles were fixed overnight at 4 °C in glutaraldehyde (20 g/l) dissolved in a Ringer's solution. The fixed muscles were rinsed with several changes of the Ringer's solution, and then a single small block suitable for embedding purposes was cut from the edge & t h e muscle opposite to the nerve entry (see Fig. 2) in such a way that the endplate region was in one end of the block. In some experiments the block was then incubated to localize H R P (see below). The block was then post-fixed using osmium tetroxide (10 g/l, 2 h), stained with uranyl nitrate (0.5 g/l, pH 5.0, 45 min), dehydrated and embedded in Spurr's (1969) resin and oriented to enable transverse sectioning of the end of the block containing the endplate region. Locafization of HRP Blocks were incubated for H R P localization after
they were cut from the rinsed glutaraldehyde fixed muscles using the following schedule: four 10-min rinses with Tris-maleate buffer (0.15 mol/l, pH 7.6): 45 min infiltration of 3,3'-diaminobenzidine tetrahydrochloride (DAB, 0.06% in the Tris-maleate buffer); four 15-min incubations with DAB and 0.01 ~ HzOz dissolved in the Tris-maleate buffer; and finally three 10-rain rinses with the Tris-maleate buffer. The blocks were th.en osmium-fixed and further processed as described above.
Serial sectioning Sectioning was performed with a Cambridge Instruments (A. F. Huxley pattern) Ultramicrotome equipped with an orientating specimen holder. The holder was adjusted to give precisely transverse sections, after which the block was not removed from the microtome until the serial sectioning was completed. The block faces were too large to be cut with the available diamond knives, so glass knives had to be used. The procedure consisted of repeated cycles of thick and thin sectioning which we will refer to as cuts. One cut generally included twenty l-#m sections followed by about 10 th.in sections. (In some cases fifteen 1.5 /zm or forty 1 /zm sections were collected.) It was judged more important to obtain thin sections with a minimum of block refacing tban to obtain sections of the highest quality. The micrographs obtained from these sections reflect this decision. The thick sections were mounted serially on glass slides and stained for light microscopy using 1 To toluidine blue in 1 ~/oborax, followed after rinsing by 1 ~ safanine-O in 1 ~ borax. The thin sections were mounted on grids and stained with lead citrate for electron microscopy. The total thickness of each cut was recorded for analytical purposes. A muscle fibre was only considered for complete reconstruction and morphometric analysis after verifying that it could be observed in thin sections for all cuts containing endplates. Furthermore, it was necessary to have at least 3 cuts on either side of the endplates, to ensure against accidental underestimate of terminal length. Three-dimensional reconstructions Analysis of endplate structure, which required the use of as many as 700 thick sections, was carried out with the assistance of a computer-aided reconstruction technique. The outlines of muscle fibres to be
13 analyzed were obtained from single thick sections at about 10/~m intervals. The images produced with a Leitz carbon-arc microprojector at an enlargement of 1600 x were traced onto transparent sheets. The position of axons and all structures that might be nerve terminals were examined with the electron microscope to positively identify nerve endings and locate those not found with the light microscope. Electron micrographs were taken of all terminals found. The verified tracings were digitized for computer reconstruction using the principle employed by Bennett et al. 2, but using a digitizing graphics tablet for input to a P D P l l / 1 0 computer. Each line was traced using the digitizer pen and a series o f x - y coordinates of the traced line were temporarily stored on disk. Coordinate reading and transfer to disk proceeded at the maximum possible rate. The traced line could then be approximated by a series of straight lines joining these points and its length approximated by the lengths of these lines. This length was then divided into n - - 1 segments, where n is the number of points used to represent the line. Then co-ordinates were calculated for points at the end of each segment and lying on the approximation formed by the series of straight lines mentioned above. Thus for each structure traced, the final representation in each section was by the same number of points, equally spaced. This scheme has the advantage that the corresponding points in adjacent sections can be connected by lines on the computer drawing, giving visual cues to these structural representations. The 2-dimensional co-ordinates of the structures digitized from the serial sections, together with the separation between sections, were used to calculate a 3-dimensiona 1 reconstruction of the structure of interest. Through further calculations these observations could be rotated in the 3-dimensional projection and accounting for the perspective. Two such projections, with the viewpoints appropriately separated, enable the 3-dimensional appearance of the reconstruction to be examined with a stereoscope. The plots of structures have been represented in 3 ways. In some the digitized lines f r o m each section have been plotted as dashed lines in their calculated position. In others, solid lines have been used and the corresponding points in adjacent sections have
been connected with solid lines. (The use of a data acquisition scheme in which the number of points recorded for a structure is the same in every section makes this process very simple.) Structures depicted in this way are described in the legends as drawn with continuous lines. In the third, only the lines connecting corresponding sections have been included, and these are drawn with 2 breaks per section. Structures drawn in this way are described as drawn with broken lines.
Electron micrography Thin sections were photographed for morphometry using a Philips EM 201c, generally at an instrument magnification of 6000 × . Photographic prints of these micrographs were produced by a further 3 × enlargement.
Morphometry Measurements of terminal area and synaptic contact were made directly on electron micrographs using the graphics tablet and a computer programme which accounts for magnification of the micrographs.
Terminology The definition of terms used in the description of
~
terminal branch
last myelin segment /
c0ntact
y muscle fibre
cholinesterase
I
/
terminal size (cross-sectional area)
Fig. 1. Terminology used to describe the nerve endings at neuromuscular junctions on muscle fibres in the region of the iliofibularis shown in Fig. 2. A motor axon divides after the last myelin segment, and gives rise to two pairs of terminal branches. Each of the terminal branches of a pair runs in opposite directions along the long axis of the muscle fibre. One terminal branch possesses a secondary terminal branch. The synaptic contact is the length of the region of approximation (in transverse sections) between the nerve terminal and the muscle fibre.
14
Fig. 2. Transverse section through a whole toad iliofibularis muscle stained with Sudan black. This muscle is from the right leg viewed from the tibial end. The in vivo shape of the muscle has been distorted, because the muscle was pinned out in the manner usually used for electrophysiologyor ultrastructural analysis (see Materials and Methods). The superficial surface in vivo is to the left. The lateral surface in vivo is uppermost, consequently the nerve entry is on the right. The region enclosed within the rectangle has been used for electrophysiologicalinvestigations~ and was examined for the structural investigations in this report. Calibration : 1 mm. the neuromuscular junction are given in reference to Fig. 1. This schematic junction consists of 4 terminal branches which originate from 2 axon branches. The synaptic contact is indicated by arrows in a transverse section through a region of terminal branch which contains a high density of syaaptic vesicles (Figs. 4, 7); it is the length of terminal membrane between the arrows in close apposition to the muscle membrane. The section with the highest vesicle density over the 10 thin sections in each cut (see above) was used to determine the synaptic contact. Tb_e length of a release site is given by the length of terminal contact at an 'active zorte 'x2. This sometimes corresponds to a synaptic contact. It is delineated by the convergence of synaptic vesicles towards the pre-synaptic membrane in the transverse sections. These are clear following HRP-labelling of the synapt;c vesicles.
vealed a variation in staining of individual fibres (Fig. 2). This was similar to that described by Lhnnergren and Smith 21 for the iliofibularis of the African toad Xenopus laevis. They showed that pale cells are twitch fibres which fatigue during tetanic contractions, and would now be termed fast fatiguable 10 (type FF). The present study was restricted to pale cells in the regions shown by the rectangle in Fig. 2; in vivo these are located on the superficial surface of the muscle. Sections through this selected region reveal a population of cells of uniform appearance and fairly uniform size. In Xenopus, these fibres have a type of en plaque neuromuscular junction which is characterized by a number of long and fairly straight terminal branches that run parallel to the muscle fibre axis2L In Bufo, cholinesterase staining of neuromuscular junctions in the region indicated in Fig. 2 were of this kind.
RESULTS
Reconstruction of juvenile neuromuscular junctions
Distribution of fibre types
The neuromuscular junctions of 6 fibres from 4 mm long juvenile muscles were reconstructed. Five of these had a very simple structure consisting of
Sudan black staining of iliofibularis muscles re-
1 Ii
B
~ I ,i ~j
,
I
'
Fig. 3. Stereoscopic 3-dimensional reconstructions from 2 juvenile (4 mm) muscle fibres. The myofibres are shown with dashed lines. A: the simplest neuromuscular junction observed, consisting of 2 short equal-length terminal branches. B: the most complex neuromuscular junction observed : this consists of 2 short and 3 very short terminal branches; the 2 short branches do not maintain synaptic contact along their whole length (broken lines). Calibration: 100 ym.
4mm
mvof" ib r e
Fig. 4. Thin sections from successive cuts taken from a single terminal branch in a juvenile (4 ram) muscle fibre. Each section is labelled with the distance separating it from the origin of the axon branch leading to the terminal. The first micrograph shows the first terminal contact observed. The terminal was not present in the cut after the last shown in this series. Note that this terminal was stimulated in the presence of horseradish peroxidase (HRP), some of the synaptic vesicles are filled with H R P reaction product. The combination of the H R P reaction and the serial section procedures used (see Materials and Methods) is responsible for the section quality.
16
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Fig. 5. Changes in terminal size (A, cross-sectional area) and synaptic contact (B) along the length of terminal branches on a juvenile muscle fibre. The zero reference indicates the point at which terminal branches originate from the axon. C gives the numbers of HRP-labelled synaptic vesicles at different sites along the length of terminal branches, stimulated in the presence of HRP. These results are for the neuromuscular junction in the stereoscopic pair of Fig. 3A.
two short ( < 100/~m) terminal branches of about equal length with the occasional addition of a very short branch; the total contact length between terminal and muscle was about 200 #m. An example of a simple junction is shown in stereoscopic Fig. 3A. The main pair of terminal branches arises as a consequence of the splitting of the axons near the last myelin segment. This gives rise to a discontinuity in
synaptic contact along the length of the muscle fibre, between the pair of terminal branches. The remaining juvenile junction of the 6 studied had a complex structure consisting of 2 short and 3 very short terminal branches, as shown in stereoscopic Fig. 3B : the short terminal branches do not maintain synaptic contact along their entire length. Nevertheless, even this complex juvenile endplate did not have a total synaptic contact in excess of 200 #m. Examination of serial electron micrographs of terminal branches indicated that the terminals decrease in size from their origins at the axon to their ends (Fig. 4). Two measurements were used to quantify these observations: terminal size, measured as the cross-sectional area of transverse sections through a terminal branch; and the synaptic contact, measured as the length of close 20 nm apposition between pre- and postsynaptic membranes in transverse sections. Fig. 5 shows plots of both terminal size and synaptic contact along the length of the terminal branches of a typical juvenile endplate; zero is taken as point of origin of terminal branches from their parent axon. Both the terminal size and synaptic contact decrease from high values near the branch origin to low values at its end. An estimate was also made of the participation of synaptic vesicles in secretion at different sites along the length of terminal branches. The number of horseradish peroxidase (HRP)-labelled vesicles at different sites was determined following stimulation of axons in the presence of HRP (see Materials and Methods). The quantitative results are shown in Fig. 5C: the number of HRP-labelled vesicles declines along the length of terminal branches in the same way as the length of the synaptic contact.
Reconstruction of mature neuromuscular junctions The mature neuromuscular junctions of 7 fibres from 22 mm long mature muscles were reconstructed. Only one of these had the very simple juvenile structure consisting of two short terminal branches (see Fig. 3A). The remaining junctions were all more complex, with a considerably greater total length of terminal branches. Examples of these are shown in stereoscopic Fig. 6. In one case (Fig. 6A) the myelinated axon divides to form two pairs of terminal branches, one on either side of the muscle fibre. This division of the axon to form multiple pairs of termi-
7
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Fig. 6. Stereoscopic 3-dimensional reconstructions from 3 mature (22 ram) muscle fibres. The myofibres are shown with dashed lines. A: a simple neuromuscular junction, consisting of 2 pairs of relatively short terminal branches. B: a typical neuromuscular junction; this consists of one very long pair of terminal branches, and another relatively short pair; one of the long terminal branches does not maintain synaptic contact along its entire length (broken lines). C: a complex neuromuscular junction; the parent axon gave rise to a pair of long terminal branches making variable synaptic contact (broken lines) and a short terminal branch which divided into a terminal branch pair; these in turn divided to form secondary terminal pairs. Calibration: 100/zm.
hal branches was frequently observed; it lead to a substantial number of terminal branches at mature endplates compared with juvenile endplates. Some terminal branches do not maintain synaptic contact along their entire length. In the case shown in stereoscopic Fig. 6B, one terminal branch lifted off the muscle fibre for a short distance before once more forming synaptic contact (see also ref. 24). The most complex endplate reconstructed is shown in stereoscopic Fig. 6C. The parent axort divided to form a single pair of terminal branches and a short terminal branch which itself divided to form a pair of terminal branches; these in turrt divided to form a further pair of terminal branches. Some of these terminal branches did not maintain synaptic contact along their entire length. Most terminal branches at mature endplates decrease in size along their length. Serial electron micrographs show that both the terminal size and
synaptic contact decrease from near the origin of a terminal branch to its end (Fig. 7). Quantitative results for two endplates are shown in Fig. 8: the terminal size and synaptic corttact decrease in the proximo-distal direction along individual branches, after they reach their maximum values near the last myelin segment. Transverse sections through terminal branches occasionally passed through release sites (see Fig. 7); the size of these tapered along the length of mature terminal branches as they did along juvenile branches (see above). One mature junction studied showed remarkable constancy in synaptic contact length along each of its terminal branches. This junction was exceptional. Table I gives the synaptic contact along the length of terminal branches of 6 mature and 6 juvenile junctions. These branches are each of about the same length in the mature and juvenile cases respectively. Table II gives terminal sizes along the length
18 22mm m y o f i b r ~
Fig. 7. Thin sections from successivecuts taken from a single terminal branch in a mature (22 ram) muscle fibre (compare with Fig. 4). Each section is labelled with the distance separating it from the origin of the axon branch leading to the terminal. The first micrograph shows the first terminal contact observed. The terminal was not present in the cut after the last shown in this series.
of these branches. The average synaptic contact and terminal size decline after they reach their maximum size near the last myelin segment. DISCUSSION
Changes in terminal size and synaptic contact along terminal branches Both the cross-sectional area of transverse sections through terminal branches and the length of apposition between pre- and post-synaptic membranes varied along the length o f terminal branches. For most branches this took the form of an increase in terminal area and contact length near the last myelin segment, followed by a gradual decrease towards the ends of the branches (Figs. 5, 8). The
accuracy of this work is limited by the serial sampling for the electron microscope occurring at intervals of about 20 # m (see Materials and Methods); this interval was necessitated by the amount of work involved in the reconstructions of mature endplates some of which were over 500/~m long. Small fingerlike processes off the terminal branches may have been missed at this sampling distance of 20 ,um. However, zinc-iodide staining of terminal branches did not reveal any such processes (see Fig. 3 in ref. 6). The question arises as to whether the tapering of the terminal branches is due to the fixation procedures used in the present work. Terminal branches viewed with the light microscope, after a variety of fixation and staining procedures, show the same pattern of tapering as described in the present work
19 b
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Fig. 8. Changes in terminal size and synaptic contact along the length of terminal branches on 2 mature myofibres (a and b). a: gives the results for the two terminal branches of the neuromuscular junction of Fig. 6A. b" gives the results for the several terminal branches of the neuromuscular junction of Fig. 6B. The zero reference indicates the point at which terminal branches originate from the axon.
20 (see, for example, Figs. 11 a n d 12 in ref. 24). Either
# m . This length o f terminal branch is p r o b a b l y
all the fixatives an d stains used in these w o r k s lead
insufficient to show the tapering in size o f release
to terminal tapering or this is a true feature o f the
sites at m a t u r e endplates. It should be emphasized in
a n a t o m y o f the n e u r o m u s c u l a r ju n c ti o n . F u r t h e r -
this context that not all m a t u r e terminal branches
more, other electron m i c r o s c o p e studies o f m a t u r e
tapered c o n t i n u o u s l y f r o m near their origins to their
terminal branches 24, using fixatives o f different ionic
ends: a few branches reached a fairly constant size
c o m p o s i t i o n f r o m that in the present w o r k (see ref.
and tapered abruptly t o w ar d s their ends; others
13) have described terminal branches with similar
tapered to a very small size and then a b r u p t l y in-
characteristics:
creased again to finally taper once m o r e (Fig. 8).
these
include
well-differentiated and
These variations show that e r r o n e o u s conclusions
synaptic gutters only partially occupied by the ter-
can be reached f r o m the sampling o f a small length
synaptic
gutters
without
terminal
contacts
o f t e r m i n a l b r a n c h rather t h a n r e c o n s t r u c t i e n o f t he
mi na l branch (see Fig. 8 above). Some observations suggest that the active zones
whole n e u r o m u s c u l a r junction.
or release sites are o f c o n s t a n t length along t e r m i n a l branches (see Fig. 15 in ref. 16). A m e a n o f 24 conse-
Variation in s y n a p t i c contact length and the secretion
cutive release sites were studied by these authors. As release sites are at m o s t 2 / ~ m apart, these consecu-
o f quanta Th e average n u m b e r o f q u a n t a secreted at differ-
tive release sites co v er e d on average a distance o f 48
ent recording sites al o n g the length o f an endplate
TABLE I The length of synaptic contact at different distances along the length of 6 terminal branches Each branch is from a different junction. The largest values are nearly always at a distance of 40/zm; other values have been normalized to those found at 40 #m. In column P are given the results of Student's t-test for significant differences between the normalized values at 40 #m and those at other distances. Distance along branch
Synaptic contact (ttm)
Mean ~ S.E.M.
Mature terminal branches 0 0 20 0.8 0.28 40 2.7 0.93 60 2.9 1.0 80 1.6 0.56 120 1.3 0.45 160 0
0 1.7 0.68 2.5 1.0] 2.4 0.96 1.8 0.72 0.8 0.32 0
0 0.6 0.27 2.1 0.96 2.2 1.0 2.2 1.0 1.1 0.5 0
0 1.9 0.73 2.6 1.0 2.4 0.92 ----0
0 2.2 0.96 2.3 1.0 2.3 1.0 --0.9 0.39 0
0 2.1 1.0 2.1 1.0 1.4 0.67 0.57 1.3 0.62 0
0 1.55 ± 0.26 0.65 ± 0.12 2.38 ± 0.10 0.98 ± 0.01 2.27 dz 0.18 0.93 ~ 0.05 1.70 ± 0.18 0.71 ± 0.07 1.10 t 0.10 0.46 ± 0.04 0
Juvenile terminal branches 0 0 20 1.70 0.49 40 3.50 1.00 60 2.50 0.71 80 1.40 0.40 100 0
0 2.30 1.00 1.40 0.61 0.70 0.30 --0
0 2.30 1.00 --1.60 0.70 --0
0 2.90 1.00 2.60 0.90 2.50 0.86 1.4 0.54 0
0 0.90 0.64 1.40 1.00 0.60 0.43 0.30 0.21 0
0 1.30 0.57 2.30 1.00 --1.60 0.70 0
0 1.9o 4- 0.27 0.78 ± 0.09 2.10 =t= 0.34 0.90 ± 0.07 1.58 ± 0.37 0.60 4- 0.09 1.18 4- 0.26 0.46 ± 0.10 0
* P < 0.05.
1.2
P
<0.05*
-<0.25 <0.01" <0.001"
<0.25
<0.05* <0.02*
21 TABLE I! The terminal size at different distances along the length o f 6 terminal branches
Each branch is from a different junction. The largest values are nearly always at a distance of 40/~m; other values have been normalized to those found at 40/zm. In column P are given the results of Student's t-test for significant differences between the normalized values at 40/zm and those at other distances. Distance along branch (t~m)
Terminal size (#m ~)
Mature terminal branches 0 0
20 40 60
80 120 160
40 60 80 100
P
0
0
0
0
0
0
1.6 0.52 3.1
1.0 0.43 2.3
1.0 0.71 1.4
1.60 4- 0.25 0.64 i 0.10 2.50 ± 0.24
<0.001 * --
1.0
1.0
1.0
1.0
1.00 4- 0
--
1.4 0.61 2.2 0.96 0.5 0.22 0
1.4 1.0 1.3 0.93 0.5 0.36 0
1.2 0.38 3.2 1.0 1.9 0.59 --0.4 0.13 0
2.5 1.0 2.5
3.0 0.97 1.5 0.48 1.3 0.42 0
2.3 0.85 2.7 1.0 1.7 0.63 ----0
2.5 1.0 1.8 0.72 1.6 0.64 0
1.98 4- 0.24 0.80 4- 0.08 1.70 4- 0.17 0.77 ± 0.10 0.86 4- 0.22 0.35 ± 0.08 0
0
0
0
0
0
0
1.9 1.0 0.6 0.32 0.5 0.26 --0
2.1 1.0 --1.3 0.62 --0
2.5 1.0 1.3 0.52 1.2 0.48 0.5 0.38 0
0.3 0.32 0.9 1.0 0.1 0.11 0.1 0.11 0
0.5 0.42 1.2 1.0 --0.7 0.58 0
1.33 ± 0.35 0.67 4- 0.14 1.30 4- 0.28 0.75 4- 0.13 1.16 -4- 0.39 0.49 4- 0.14 0.40 -- 0.11 0.30 ± 0.10 0
Juvenile terminal branches 0 0
20
Mean 4- S.E.M.
0.7 0.26 2.5 0.93 2.7 1.0 0.3 0.11 0
<0.05* <0.05* <0.001"
<0.25 --<0.10 <0.05*
* P < 0.05. varies 5 because the probability of secretion declines along the length of individual terminal branches 6. This is possibly due to the decline in size of the synaptic contact along the length o f t e r m i n a l branches described in the present w o r k ; it is unlikely to result from the complete breaks in synaptic contact along t e r m i n a l branches (see Fig. 6) as these occurred infrequently. T h e size of release sites does n o t determine the size of q u a n t a as these do n o t vary along the length of terminal branches 6. The development of terminal branches
Synaptic t r a n s m i s s i o n begins d u r i n g development w h e n contact is made between small enlargements at the ends o f u n m y e l i n a t e d m o t o r axons and myotubesa,9,19. These small enlargements g r o ~ i n length, parallel to the long axis of the myotube, a n d f o r m a pair o f terminal branches 22. I n the case of the
a m p h i b i a n iliofibularis, most of the fibres have lost their multiple i n n e r v a t i o n when the muscle reaches a length of 4 m m (see text a n d Figs. 2 a n d 5B in ref. 9). This is confirmed i n the present work, i n which n o multiply innervated synaptic sites were observed on 4 m m long muscle fibres. The r e m a i n i n g j u n c t i o n s at this time still consist of a pair of short terminal branches. F u r t h e r development of the endplate consists primarily of a n increase in the n u m b e r of termin a l - b r a n c h pairs, in the length of terminal branches a n d in the secondary b r a n c h i n g o f some t e r m i n a l branches. A n increase in the average q u a n t a l c o n t e n t o f the e.p.p. (fin) accompanies the growth of terminals d u r i n g either the r e i n n e r v a t i o n or development of synapses i n muscles 4,23. According to the arguments above 1~ increases due to a n increase i n the n u m b e r of t e r m i n a l - b r a n c h pairs rather t h a n to a n increase
22 in the length of individual terminal branches; only a small increase in ffa would be expected from the a d d i t i o n of low-probability release sites at the ends of terminal branches, unless this is a c c o m p a n i e d by a n increase in ffi at the existing release sites. M a t u r e synapses consisting o f quite different total lengths of terminal branches have been described above. A correlation has been sought 2° between ffl a n d the total length of terminal branches at a m p h i b i a n synapses in low [Ca]0. I n the first group of 20 endplates f r o m two muscles, n o correlation was f o u n d between r~ a n d terminal length. However, in a
correlation was observed. Nevertheless, endplates of a given terminal length varied over 20-fold in their value (see Fig. 4 in ref. 20). This poor correlation is to be expected if release sites closest to the last myelin segment have the highest probability for secretion: ffa is then largely i n d e p e n d e n t of the r e m a i n i n g length of t e r m i n a l a n d only increases substantially if new terminal branches are laid down. Release sites towards the end of terminal branches do secrete with a n increasingly higher probability d u r i n g the facilitation that accompanies a short high-frequency train of impulses G.
second group of 32 endplates from 3 muscles a linear REFERENCES 1 Bennett, M. R., Davey, D. F. and Lavidis, N. A., Variation in the numbers of quanta secreted at different sites along developing nerve terminals: correlation with release site ultrastructure, Proc. Aust. PhysioL PharmacoL Soc., 11 (1980) 36P. 2 Bennett, M. R., Davey, D. F. and Uebel, K., The growth of segmental nerves from the brachial myotomes into the proximal muscles of the chick forelimb during development, J. comp. Neurol., 188 (1980) 335-357. 3 Bennett, M. R., Fisher, C., Florin, T., Quine, M. and Robinson, J., The effect of calcium ions and temperature on the binomial parameters that control acetylcholine release by a nerve impulse at amphibian neuromuscular synapses, J. Physiol. (Lond.), 271 (1977) 641-672. 4 Bennett, M. R. and Florin, T., A statistical analysis of the release of acetylcholine at newly formed synapses in striated muscle, J. Physiol. (Lond.), 238 (1974) 93-107. 5 Bennett, M. R. and Lavidis, N. A., The effect of calcium ions on the secretion of quanta evoked by an impulse at nerve terminal release sites, J. gen. Physiol., 74 (1979) 429-456. 6 Bennett, M. R. and Lavidis, N. A., Variation in quantal secretion at different release sites along developing and mature motor terminal branches, Develop. Brain Res., 5 (1982) 1-9. 7 Bennett, M. R., McGrath, P. and Davey, D. F., The regression of synapses formed by a foreign nerve in a mature axolotl striated muscle, Brain Res., 173 (1979) 451-469. 8 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in striated muscle during development, J. Physiol. (Lond.), 241 (1974) 515-545. 9 Bennett, M. R. and Pettigrew, A. G., The formation of synapses in amphibian striated muscle during development, J. PhysioL (Lond.), 265 (1975) 261. 10 Burke, R. E., Levine, D. N., Tsairis, P. and Zajac, F. E., III, Physiological types and histochemical profiles in motor units of the cat gastrocnemius, J. Physiol. (Lond.), 234 (1972) 723. 11 Carleton, H. M., Histological Technique, 4th Edn., Oxford University Press, London, 1967. 12 Couteaux, R. E. and Pecot-Dechavassine, M., V6sicles synaptiques et poches au niveau des 'zones actives' de la
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