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Acetylcholine receptors and motor nerve terminals in developing chick skeletal muscles as revealed by fluorescence microscopy
111
DevelopmentalBrain Research, 8 (1983) 11 I- 118 Elsevier Biomedical Press
Acetylcholine Receptors and Motor Nerve Terminals in Developing Chick Skeletal Muscles as Revealed by Fluorescence Microscopy YUJ1 ISHIKAWA, SADAHIKO MASUKO and YUTAKA SHIMADA
Department of A natomy, School of Medicine, Chiba University, Chiba 280 (Japan) (Accepted November 2nd, 1982)
Key words: neuromuscular junction - acetylcholine receptor -- neuron-specific antibody - neurotoxin nerve terminal -development - fluorescence microscopy
The developmental changes in the distribution of acetylcholine receptors (AChRs) relative to developing nerve terminals were investigated in the posterior latissimus dorsi (PLD) muscle of the chicken by double fluorescent staining of AChRs and nerve terminals on the same muscle fibers. For this, fluorescence microscopy using rhodamine-labeled erabutoxin b (TMR-Eb) for AChRs and indirect immunofluorescenee (FITC) microscopy using an antibody against nerve membrane were applied. The AChR regions and the nerve terminals changed in their shapes and sizes during development. The AChR regions and the nerve terminals were usually similar in shape and location. However, differences in sizes and configurations of the two were observed at stage 5 (hatching) and at stage 7 (adult): at hatching, the AChR regions were usually doughnut-shaped. Most nerve terminals were perimeter-shaped under fluorescence microscopy and bulbous with scanning electron microscopy at this stage. Later, the nerve terminals began to exhibit doughnut- or double circle-shaped configurations similar to those shown by the AChR regions. Thus, the developmental changes in shape of the AChR regions appeared to precede at this stage. In adult neuromuscular junctions, the AChR regions were composed of a group of smaller AChR areas, while the nerve terminals possessed a tree-shaped configuration with many round-shaped expansions which were connected with fine threads. Only the expansive parts of the nerve terminals faced the AChR regions. INTRODUCTION
Precise knowledge of the relationships between the appearance of postsynaptic components and the development of presynaptic nerve terminals is an essential step in understanding the interactions between pre- and postsynaptic structures during development. In the previous paper s, we have described the correlation between the development of acetylcholine receptors (AChRs) and that of cholinesterase (ChE) activity in the skeletal muscle of chick embryos. Although the development of neuromuscular junctions in chick skeletal muscles has been studied several times with silver impregnation techniques and staining for ChE 1.6,jLl4,ts, the precise distribution of AChRs or ChE relative to the developing nerve terminals is still not well understood. One reason is that the nerve terminals and ChE have been stained on different muscle fibers; another is that the developmental correlation between AChRs and nerve terminals has ()165-3806/83/0000 0000/503.00 :";I983 Elsevier Science Publishers
not been investigated. The approach taken in this report is to combine staining procedures so that pre- and postsynaptic structures can be visualized simultaneously at the same junctional sites. The present study describes changes in the distribution of AChRs relative to the developing nerve terminals in chick posterior latissimus dorsi (PLD) muscles by staining AChRs and nerve terminals on the same muscle fibers, using rhodamine-labeled erabutoxin b (TMR-Eb) and fluorescence microscopy for AChRs 7 and using antibody against nerve membrane and indirect immunofluorescence (FITC) microscopy10. MATERIALS AND METHODS
Materials PLD muscles were obtained from chick embryos from 8 to 20 days of incubation, chicks of 1 to 40 days, and adult fowls. Tetramethyl rhodamine isothiocyanate (TMR)
112 was purchased from Research Organics (Cleveland. Ohio): fluorescein (FITC)-labeled rabbit anti-mouse IgG was a product of Miles-Yeda (Rehovot, Israeli. Erabutoxin b (Eb) was modified with TMR according to the method of Ishikawa and Shimada ~.
A ntibody against nerve membrane The preparation and characterization of antibody specific to nerve membrane will be published in detail elsewhere ~°. Briefly, a-motoneuron cultures were prepared from spinal cords of chick embryos at 60-66 h ofincubationL After 7 days, the neuronal cells were collected and injected intraperitoneally into BALB/c mice. After 5 weeks, antisera were prepared, and the IgG was fractionated from the sera by ammonium sulfate and absorbed with a homogenate of mesonephros of 10-day chick embryos.
Double staining of A ChRs and nerves PLD muscles of various developmental stages were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10-15 min, and cut longitudinally into several pieces with razors while immersed in the fixative. Then the specimens were washed in phosphate-buffered saline (PBS) for 30 min, and soaked in PBS containing 0.2% bovine serum albumin for 15 30 min. They were incubated in 10 7 M TMR-Eb in PBS which contained anti-nerve IgG for 2 4 h at room temperature. The specimens were washed in PBS for 30 min and incubated in FITC-labeled rabbit anti-mouse IgG (diluted 800 times with PBS) which also contained 10 ~ M TMREb for 2-3 h. The muscle fibers were washed in PBS, teased gently on microscopic slides, mounted in 40% glycerol, and examined under a Zeiss standard microscope equipped with filters BP 546/12, FT 580 LP 590 for TMR fluorescence and BP 450-490, FT 510, LP 520 and KP 560 for F1TC fluorescence.
Scanning electron microscopy The motor nerve endings of PLD muscles from 2-day-old chicks were observed with the scanning electron microscope according to the
method of Desaki and Uehara 5. The muscles were fixed in 3% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) for 5 h, cut into small pieces, and post-fixed for 30..45 min in 2% OsO4. The specimens were rinsed in water, treated with 8 N HC1 for 30--45 min at 60~C, and washed again in water. The specimens were dehydrated through ethanol and immersed in isoamyl acetate. After critical point drying and sputter-coating with gold, they were examined with a Joel JSM-25 SII scanning electron microscope operated at 25 kV.
RESULTS
The development of neuromuscular junctions of chick PLD muscles was classified into 7 stages in a previous paper by simultaneous staining for AChRs and ChE 8. In this article, we examine the correlation between the distribution of AChRs and the structure of nerve terminals on developing muscles at each stage. Stage 1 (8 10 day embryos). The AChR regions were small (Fig. la), and usually accompanied by small nerve terminals (Fig. lb). The configuration of the nerve terminals was very simple at this stage, and it was difficult to identify terminals that were not associated with the AChR regions. Stage 2 (8-11 day embryos). The AChR regions were spindle-shaped (Fig. 2a). Thin nerves branched from the nerve bundles and formed small terminal swellings, which were oriented along the longitudinal axis of the myotubes, at the same sites as those of the AChR regions (Fig. 2b). The size of the nerve terminals was almost the same as those of the AChR regions. Stage 3 (10-12 day embryos). The AChR regions were increased in length (Fig. 3a), and terminal swellings of the nerves were observed associated with them (Fig. 3b). The size of the nerve terminals was almost equal to that of the AChR regions. Stage 4 (12-17 day embryos). The AChR regions were increased in width and oval-shaped in appearance (Fig. 4a). These regions were accompanied by the terminal swellings of the
Fig. 1. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the PLD muscle fibers from a 9-day embryo (stage 1). a: a photograph in the rhodamine channel, b: the same field in the fluore~ein channel. Note the presence of the small AChR regions and the corresponding nerve terminals (1 6). Scale: 20/tm. Fig. 2. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an I I-day embryo (stage 2). a: the neuromuscular junctions (I-3) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 ttm. Fig. 3. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an I I-day embryo (stage 3). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Elongated neuromuscular junctions (! and 2) are seen. Scale: 20 #m. Fig. 4. Double-staining with TMR-Eb (a) and antibody against nerve membrane (bl of the muscle fibers from an 1l-day embryo (stage 4). a: oval-shaped neuromuscular junctions (1 and 2) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 p.m.
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Fig. 5. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (early phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Thin nerves branch from the nerve bundle and immediately form the terminal swellings ( I 4). The face-on view C1 and 2) and the lateral view (3 and 4)of the neuromuscular junctions. Scale: 20 ~m. Fig. 6. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (middle phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note that the AChR regions are doughnut-shaped, whereas the corresponding nerve terminals are still perimeter-shaped in their face-on view (I and 2). Scale: 20 ~tm.
nerves (Fig. 4b). C o m p o u n d structures were observed within them. Stage 5 (15-day embryos to 3-day-oM chicks). Significant changes in the structures of the AChR regions and of the nerve terminals occurred during this period. The AChR regions were disc-shaped early in this period (Fig. 5a). The nerve terminals were usually enlarged, and their contours were perimeter-shaped (Fig. 5b). The AChR regions became doughnut-shaped in the middle of the period (Fig. 6a), while the structure of the nerve terminals was unchanged (Fig. 6b). The doughnut-shaped perforation in the AChR regions was nearly completed by hatching8. Scanning electron microscopy revealed that the nerve terminals were usually bulbous at this phase (Fig. 7), although they were mainly perimeter-shaped when examined by fluorescent microscopy. At the end of this stage, the
nerve terminals began to exhibit either a doughnut-shaped or a double circle-shaped configuration (Figs. 8 and 9). Stage 6 (3-day to 2-week chicks). The doughnut-shaped AChR regions became disconnected at one point, resulting in a C-shaped configuration (Fig. 10a). The contours of the nerve terminals also showed a C-shaped structure (Fig. lOb). Stage 7 (2-week to adult chicken). The C-shaped A C h R regions increased in size, branched and split into smaller areas (Fig. I la). The contours of the nerve terminals branched also but, instead of splitting, they constricted at several points. Thus, several expansions were formed along the length of the terminal arborizations (Fig. lib), the sites of which corresponded with those of the AChR areas. In adult neuromuscular junctions, the AChR
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Fig. 7. A scanning electron micrograph of the neuromuscular junctions in the PLD muscle fibers from a 2-day chick (middle phase of stage 5). Note that the nerve terminals (I-4) are bulbous. The synaptic depression of neuromuscular junction (3) is partially seen (arrow). A nerve terminal (1) appears to be covered with a flattened process from a Schwann cell (S). A nerve terminal (4) is possibly detached from the muscle fibers showing the surface which has directly faced the synaptic depression. On the surface of muscle fibers (M), the swelling due to the presence of nuclei is seen (N). Scale: 10/~m.
regions were composed of a group of smaller AChR areas (Fig. 12a), whereas the contours of the nerve terminals possessed a tree-shaped configuration with 30-100 round-shaped expansions which were connected with fine threads (Fig. 12b). The expansions of the nerve terminals corresponded with the split AChR areas. DISCUSSION
In the present study, we used an anti-nerve membrane antibody to visualize nerve terminals. The antibody stains the surface of the nerves ~°, enabling observation of the entire nerve terminals from the early developmental
stages. However, the antibody stain tended to reveal only the contours of the nerve terminals at later stages in development. One possibility is that the intensity of the fluorescence is enhanced at the periphery of the terminals where the membranes are folded, although the entire membrane is uniformly stained with antibody. Another explanation is that the antigen is preferentially localized at peripheral portions of the nerve terminals at later stages of development. The observation that the entire surface of the nerve terminals were occasionally stained at later stages make the former possibility likely. The double fluorescence microscopy using the antibody and TMR-Eb revealed a correla-
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Fig. 8. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (late phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note that both AChR region and the nerve terminal are doughnut-shaped in their face-on view (I). Scale: 20 #m. Fig. 9. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 5-day chick (late phase of stage 5 stage 6). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note the presence of doughnut- or C-shaped AChR regions and the corresponding nerve terminals of double circle-shape (I- 3). Scale: 20 #m.
tion between the development of the AChR regions and that of the nerve terminals. Both were usually present with similar shapes at the same sites throughout embryonic development. However, the size and configuration of the two were not always identical. For example, the nerve terminals were perimeter-shaped, while the AChR regions were doughnut-shaped at the middle of stage 5. The scanning electron microscopy revealed that the nerve terminals were usually bulbous at this phase. Therefore, it is possible that, although the bulbous terminals cover the entire regions of the postsynaptic membrane, only the central portion of the terminal swelling faces the membrane lacking in AChRs. A discrepancy was also observed in the adult neuromuscular junction. Only the expansive parts of the nerve terminals face the AChR regions, and connecting threads between these expansions are not accompanied with them. In the
previous paper, we reported that ChE activity was positive in the form of branching trees which contained negative areas in the center of each expansion of thc adult neuromuscular junctionL These observations suggest that the functional a n d / o r structural specialization(s) occurred at different portions within a single neuromuscular junction. The development of neuromuscular junctions in chick skeletal muscles has been studied by silver impregnation ~.6.~H4,~5. A comparison of our results with those reports indicates some differences concerning configurations of the nerve terminals: near hatching, we usually observed perimeter-shaped nerve terminals by antibody staining; these were morphologically different from those of adult chicken. This observation seems to agree with that of Wake ~5 but not with those of Hirano ~ and Atsumi ~. Atsumi ~ stressed the presence of ramificated nerve terminals with
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Fig. 10. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 10-day chick (stage 6). a: the face-on view of a C-shaped neuromuscular junction (1) seen in the rhodamine channel, b:the same field in the fluorescein channel. Scale: 20 am. Fig. 1i. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a young chicken 37 days after hatching (stage 7). a: neuromuscular junctions (1-4) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 p.m. Fig. 12. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an adult chicken. a: a neuromuscular junction seen in the rhodamine channel, b: the same field in the fluorescein channel. Note that the AChR region is composed of a group of smaller AChR areas, whereas the contours of the nerve terminal posseses a tree-shaped configuration. Scale: 20 p,m.
118 'boutons' in chick intercostal muscles near hatching. These terminals are essentially similar to those of the adult. This disagreement may be due to incomplete silver impregnations of the nerve terminals. The stain of the silver methods tends to be restricted to the central portion of the nerves where there are numerous neurofibrils. However, differences of the muscles used and/ or other explanations cannot be ruled out. Disc- and doughnut-shaped nerve terminals also were found during the postnatal development of nerve terminals of the cat by the gold chloride method ~2. Nystr6m a2 reported that the doughnut-shaped nerve terminals were differentiated from the primitive disc-like ones by 'perforations' at i-2 places in their center. This finding is consistent with our present result that the doughnut- or double circle-shaped nerve terminals of the chicken PLD muscle developed from the perimeter-shaped ones at stage 5. However. we observed that this change in the nerve terminals appeared to lag behind that in the
REFERENCES I Atsumi. S., The histogenesis of motor neurons with special reference to the correlation of their endplate formation. I, The development of endplates in the intercostal muscle in the chick embryo, Acta Anat,. 80 (1971) 161 182. 2 Braithwaite, A. W. and Harris, A. J.. Neural influence on acetylcholine receptor clusters in emb~onic development of skeletal muscles, Nature (Lond.). 279 (1979) 549 55 I. 3 (hangeux, J.-P. and Danchin, A., Selective stabilization of developing synapses as a mechanism tbr the specification of neuronal networks, Nature tl, ond). 264 (1976) 705 712, 4 Christian, ('. N., I)anicls, M. P.. Sugiyama. H.. Vo~el, Z., Jacques. L. and Nelson, P. G., A factor from neurons incrca>c:, the number of acet~ Icholinc receptor aggrcgatc~ t)n cultured mr.sole cells, Proc. mlt..lead. ,S'(z. I '.S...I.. 75 (1978)4011 4015 5 Desaki. J. and Uehara, Y., The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy, J. Neurocvtol.. 10(1981) 101- 110. 6 Hirano, H.. A histochemical study of the cholinesterase activity in the neuromuscular junction in developing chick skeletal muscles, Arch. Histol. Jap.. 28 (1967) 89 101. 7 Ishikawa. Y. and Shimada, Y., Fluorescent staining of acetylcholine receptors at the neuromuscular junction by means of rhodamine-labeled erabutoxin b. In M. Ito. N. Tsukahara, K. Kubota and K. Yagi (Eds.). Integrutive
AChR regions. This result seems inconsistent with the hypothesis that the distribution of junctional AChRs is controlled by neural influences throughout the development of the neuromuscular synapses 2- 4.~3. ACKNOWLEDGEMENTS
We are grateful to Professor N. Tamiya, Tohoku University, for his kind gift of erabutoxin b, and to Professor B. Wilson, University of California, Davis, for his critical reading of the manuscript. We also wish to thank Mr. N. Nakamura and Mrs. K. Shimizu for their technical assistance. This research was supported by grants from the following: the Japanese Ministry of Education, Science and Culture, the National Center for Nervous, Mental and Muscular Disorders ( N C N M D D 82-03) of the Japanese Ministry of Health and Welfare, and the Muscular Dystrophy Association of America.
Control Functions o f the Brain. Vol. 3, Kodansha Scientific, Tokyo/Elsevier, Amsterdam. 1980, pp. 29 31. 8 Ishikawa, Y. and Shimada. Y.. Acetylcholine receptors and cholinesterase in developing chick skeletal muscle fibers. Develop. Brain Res.. 5 (1982) 187-- 197. 9 Masuko, S., Kuromi, H. and Shimada, Y., Isolation and culture of motoneurons from embryonic chicken spinal cords, Proc. nat. A cad. Sci. U.S.A., 76 (1979) 3537- 3541. 10 Masuko. S. and Shimada. Y., Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts, Develop. Biol., in press. 11 Mumenthaler, M. and Engel, W. K.,Cytological localization of cholinesterase in developing chick embryo skeletal muscle. A cta A nat.. 47 ( 1961 ) 274 299. 12 NystrOm, B., Postnatal development of motor nerve terminals in 'slow-red' and 'fast-white' cat muscles. Acta neurol. S t a n d , 44 (1968) 363 383. 13 Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, I., Hohnson, M. M. and Salpeter, M. M., Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc. nat. Acad Sci. I,(S.A.. 75 (1978) 2035-2039. 14 Tello, J. F.. Die Einstehung der motorischen und sensiblen Nervenendigungen. I. In dem Iokomotorischen Systome der hOheren Wirbehiere. Muskul~ire Histogenese. Z.A nat. Entw.-Gesch.. 64 (1922) 348 440. 15 Wake, K., Motor endplates in developing chick embryo skeletal muscle: histological structure and histochemical localization of cholinesterase activit','. A rch. ttistoL Jap. 25(1964)23 41.
DevelopmentalBrain Research, 8 (1983) 11 I- 118 Elsevier Biomedical Press
Acetylcholine Receptors and Motor Nerve Terminals in Developing Chick Skeletal Muscles as Revealed by Fluorescence Microscopy YUJ1 ISHIKAWA, SADAHIKO MASUKO and YUTAKA SHIMADA
Department of A natomy, School of Medicine, Chiba University, Chiba 280 (Japan) (Accepted November 2nd, 1982)
Key words: neuromuscular junction - acetylcholine receptor -- neuron-specific antibody - neurotoxin nerve terminal -development - fluorescence microscopy
The developmental changes in the distribution of acetylcholine receptors (AChRs) relative to developing nerve terminals were investigated in the posterior latissimus dorsi (PLD) muscle of the chicken by double fluorescent staining of AChRs and nerve terminals on the same muscle fibers. For this, fluorescence microscopy using rhodamine-labeled erabutoxin b (TMR-Eb) for AChRs and indirect immunofluorescenee (FITC) microscopy using an antibody against nerve membrane were applied. The AChR regions and the nerve terminals changed in their shapes and sizes during development. The AChR regions and the nerve terminals were usually similar in shape and location. However, differences in sizes and configurations of the two were observed at stage 5 (hatching) and at stage 7 (adult): at hatching, the AChR regions were usually doughnut-shaped. Most nerve terminals were perimeter-shaped under fluorescence microscopy and bulbous with scanning electron microscopy at this stage. Later, the nerve terminals began to exhibit doughnut- or double circle-shaped configurations similar to those shown by the AChR regions. Thus, the developmental changes in shape of the AChR regions appeared to precede at this stage. In adult neuromuscular junctions, the AChR regions were composed of a group of smaller AChR areas, while the nerve terminals possessed a tree-shaped configuration with many round-shaped expansions which were connected with fine threads. Only the expansive parts of the nerve terminals faced the AChR regions. INTRODUCTION
Precise knowledge of the relationships between the appearance of postsynaptic components and the development of presynaptic nerve terminals is an essential step in understanding the interactions between pre- and postsynaptic structures during development. In the previous paper s, we have described the correlation between the development of acetylcholine receptors (AChRs) and that of cholinesterase (ChE) activity in the skeletal muscle of chick embryos. Although the development of neuromuscular junctions in chick skeletal muscles has been studied several times with silver impregnation techniques and staining for ChE 1.6,jLl4,ts, the precise distribution of AChRs or ChE relative to the developing nerve terminals is still not well understood. One reason is that the nerve terminals and ChE have been stained on different muscle fibers; another is that the developmental correlation between AChRs and nerve terminals has ()165-3806/83/0000 0000/503.00 :";I983 Elsevier Science Publishers
not been investigated. The approach taken in this report is to combine staining procedures so that pre- and postsynaptic structures can be visualized simultaneously at the same junctional sites. The present study describes changes in the distribution of AChRs relative to the developing nerve terminals in chick posterior latissimus dorsi (PLD) muscles by staining AChRs and nerve terminals on the same muscle fibers, using rhodamine-labeled erabutoxin b (TMR-Eb) and fluorescence microscopy for AChRs 7 and using antibody against nerve membrane and indirect immunofluorescence (FITC) microscopy10. MATERIALS AND METHODS
Materials PLD muscles were obtained from chick embryos from 8 to 20 days of incubation, chicks of 1 to 40 days, and adult fowls. Tetramethyl rhodamine isothiocyanate (TMR)
112 was purchased from Research Organics (Cleveland. Ohio): fluorescein (FITC)-labeled rabbit anti-mouse IgG was a product of Miles-Yeda (Rehovot, Israeli. Erabutoxin b (Eb) was modified with TMR according to the method of Ishikawa and Shimada ~.
A ntibody against nerve membrane The preparation and characterization of antibody specific to nerve membrane will be published in detail elsewhere ~°. Briefly, a-motoneuron cultures were prepared from spinal cords of chick embryos at 60-66 h ofincubationL After 7 days, the neuronal cells were collected and injected intraperitoneally into BALB/c mice. After 5 weeks, antisera were prepared, and the IgG was fractionated from the sera by ammonium sulfate and absorbed with a homogenate of mesonephros of 10-day chick embryos.
Double staining of A ChRs and nerves PLD muscles of various developmental stages were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 10-15 min, and cut longitudinally into several pieces with razors while immersed in the fixative. Then the specimens were washed in phosphate-buffered saline (PBS) for 30 min, and soaked in PBS containing 0.2% bovine serum albumin for 15 30 min. They were incubated in 10 7 M TMR-Eb in PBS which contained anti-nerve IgG for 2 4 h at room temperature. The specimens were washed in PBS for 30 min and incubated in FITC-labeled rabbit anti-mouse IgG (diluted 800 times with PBS) which also contained 10 ~ M TMREb for 2-3 h. The muscle fibers were washed in PBS, teased gently on microscopic slides, mounted in 40% glycerol, and examined under a Zeiss standard microscope equipped with filters BP 546/12, FT 580 LP 590 for TMR fluorescence and BP 450-490, FT 510, LP 520 and KP 560 for F1TC fluorescence.
Scanning electron microscopy The motor nerve endings of PLD muscles from 2-day-old chicks were observed with the scanning electron microscope according to the
method of Desaki and Uehara 5. The muscles were fixed in 3% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) for 5 h, cut into small pieces, and post-fixed for 30..45 min in 2% OsO4. The specimens were rinsed in water, treated with 8 N HC1 for 30--45 min at 60~C, and washed again in water. The specimens were dehydrated through ethanol and immersed in isoamyl acetate. After critical point drying and sputter-coating with gold, they were examined with a Joel JSM-25 SII scanning electron microscope operated at 25 kV.
RESULTS
The development of neuromuscular junctions of chick PLD muscles was classified into 7 stages in a previous paper by simultaneous staining for AChRs and ChE 8. In this article, we examine the correlation between the distribution of AChRs and the structure of nerve terminals on developing muscles at each stage. Stage 1 (8 10 day embryos). The AChR regions were small (Fig. la), and usually accompanied by small nerve terminals (Fig. lb). The configuration of the nerve terminals was very simple at this stage, and it was difficult to identify terminals that were not associated with the AChR regions. Stage 2 (8-11 day embryos). The AChR regions were spindle-shaped (Fig. 2a). Thin nerves branched from the nerve bundles and formed small terminal swellings, which were oriented along the longitudinal axis of the myotubes, at the same sites as those of the AChR regions (Fig. 2b). The size of the nerve terminals was almost the same as those of the AChR regions. Stage 3 (10-12 day embryos). The AChR regions were increased in length (Fig. 3a), and terminal swellings of the nerves were observed associated with them (Fig. 3b). The size of the nerve terminals was almost equal to that of the AChR regions. Stage 4 (12-17 day embryos). The AChR regions were increased in width and oval-shaped in appearance (Fig. 4a). These regions were accompanied by the terminal swellings of the
Fig. 1. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the PLD muscle fibers from a 9-day embryo (stage 1). a: a photograph in the rhodamine channel, b: the same field in the fluore~ein channel. Note the presence of the small AChR regions and the corresponding nerve terminals (1 6). Scale: 20/tm. Fig. 2. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an I I-day embryo (stage 2). a: the neuromuscular junctions (I-3) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 ttm. Fig. 3. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an I I-day embryo (stage 3). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Elongated neuromuscular junctions (! and 2) are seen. Scale: 20 #m. Fig. 4. Double-staining with TMR-Eb (a) and antibody against nerve membrane (bl of the muscle fibers from an 1l-day embryo (stage 4). a: oval-shaped neuromuscular junctions (1 and 2) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 p.m.
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Fig. 5. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (early phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Thin nerves branch from the nerve bundle and immediately form the terminal swellings ( I 4). The face-on view C1 and 2) and the lateral view (3 and 4)of the neuromuscular junctions. Scale: 20 ~m. Fig. 6. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (middle phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note that the AChR regions are doughnut-shaped, whereas the corresponding nerve terminals are still perimeter-shaped in their face-on view (I and 2). Scale: 20 ~tm.
nerves (Fig. 4b). C o m p o u n d structures were observed within them. Stage 5 (15-day embryos to 3-day-oM chicks). Significant changes in the structures of the AChR regions and of the nerve terminals occurred during this period. The AChR regions were disc-shaped early in this period (Fig. 5a). The nerve terminals were usually enlarged, and their contours were perimeter-shaped (Fig. 5b). The AChR regions became doughnut-shaped in the middle of the period (Fig. 6a), while the structure of the nerve terminals was unchanged (Fig. 6b). The doughnut-shaped perforation in the AChR regions was nearly completed by hatching8. Scanning electron microscopy revealed that the nerve terminals were usually bulbous at this phase (Fig. 7), although they were mainly perimeter-shaped when examined by fluorescent microscopy. At the end of this stage, the
nerve terminals began to exhibit either a doughnut-shaped or a double circle-shaped configuration (Figs. 8 and 9). Stage 6 (3-day to 2-week chicks). The doughnut-shaped AChR regions became disconnected at one point, resulting in a C-shaped configuration (Fig. 10a). The contours of the nerve terminals also showed a C-shaped structure (Fig. lOb). Stage 7 (2-week to adult chicken). The C-shaped A C h R regions increased in size, branched and split into smaller areas (Fig. I la). The contours of the nerve terminals branched also but, instead of splitting, they constricted at several points. Thus, several expansions were formed along the length of the terminal arborizations (Fig. lib), the sites of which corresponded with those of the AChR areas. In adult neuromuscular junctions, the AChR
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Fig. 7. A scanning electron micrograph of the neuromuscular junctions in the PLD muscle fibers from a 2-day chick (middle phase of stage 5). Note that the nerve terminals (I-4) are bulbous. The synaptic depression of neuromuscular junction (3) is partially seen (arrow). A nerve terminal (1) appears to be covered with a flattened process from a Schwann cell (S). A nerve terminal (4) is possibly detached from the muscle fibers showing the surface which has directly faced the synaptic depression. On the surface of muscle fibers (M), the swelling due to the presence of nuclei is seen (N). Scale: 10/~m.
regions were composed of a group of smaller AChR areas (Fig. 12a), whereas the contours of the nerve terminals possessed a tree-shaped configuration with 30-100 round-shaped expansions which were connected with fine threads (Fig. 12b). The expansions of the nerve terminals corresponded with the split AChR areas. DISCUSSION
In the present study, we used an anti-nerve membrane antibody to visualize nerve terminals. The antibody stains the surface of the nerves ~°, enabling observation of the entire nerve terminals from the early developmental
stages. However, the antibody stain tended to reveal only the contours of the nerve terminals at later stages in development. One possibility is that the intensity of the fluorescence is enhanced at the periphery of the terminals where the membranes are folded, although the entire membrane is uniformly stained with antibody. Another explanation is that the antigen is preferentially localized at peripheral portions of the nerve terminals at later stages of development. The observation that the entire surface of the nerve terminals were occasionally stained at later stages make the former possibility likely. The double fluorescence microscopy using the antibody and TMR-Eb revealed a correla-
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Fig. 8. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 19-day embryo (late phase of stage 5). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note that both AChR region and the nerve terminal are doughnut-shaped in their face-on view (I). Scale: 20 #m. Fig. 9. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 5-day chick (late phase of stage 5 stage 6). a: a photograph in the rhodamine channel, b: the same field in the fluorescein channel. Note the presence of doughnut- or C-shaped AChR regions and the corresponding nerve terminals of double circle-shape (I- 3). Scale: 20 #m.
tion between the development of the AChR regions and that of the nerve terminals. Both were usually present with similar shapes at the same sites throughout embryonic development. However, the size and configuration of the two were not always identical. For example, the nerve terminals were perimeter-shaped, while the AChR regions were doughnut-shaped at the middle of stage 5. The scanning electron microscopy revealed that the nerve terminals were usually bulbous at this phase. Therefore, it is possible that, although the bulbous terminals cover the entire regions of the postsynaptic membrane, only the central portion of the terminal swelling faces the membrane lacking in AChRs. A discrepancy was also observed in the adult neuromuscular junction. Only the expansive parts of the nerve terminals face the AChR regions, and connecting threads between these expansions are not accompanied with them. In the
previous paper, we reported that ChE activity was positive in the form of branching trees which contained negative areas in the center of each expansion of thc adult neuromuscular junctionL These observations suggest that the functional a n d / o r structural specialization(s) occurred at different portions within a single neuromuscular junction. The development of neuromuscular junctions in chick skeletal muscles has been studied by silver impregnation ~.6.~H4,~5. A comparison of our results with those reports indicates some differences concerning configurations of the nerve terminals: near hatching, we usually observed perimeter-shaped nerve terminals by antibody staining; these were morphologically different from those of adult chicken. This observation seems to agree with that of Wake ~5 but not with those of Hirano ~ and Atsumi ~. Atsumi ~ stressed the presence of ramificated nerve terminals with
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tr
!
I
Fig. 10. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a 10-day chick (stage 6). a: the face-on view of a C-shaped neuromuscular junction (1) seen in the rhodamine channel, b:the same field in the fluorescein channel. Scale: 20 am. Fig. 1i. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from a young chicken 37 days after hatching (stage 7). a: neuromuscular junctions (1-4) seen in the rhodamine channel, b: the same field in the fluorescein channel. Scale: 20 p.m. Fig. 12. Double-staining with TMR-Eb (a) and antibody against nerve membrane (b) of the muscle fibers from an adult chicken. a: a neuromuscular junction seen in the rhodamine channel, b: the same field in the fluorescein channel. Note that the AChR region is composed of a group of smaller AChR areas, whereas the contours of the nerve terminal posseses a tree-shaped configuration. Scale: 20 p,m.
118 'boutons' in chick intercostal muscles near hatching. These terminals are essentially similar to those of the adult. This disagreement may be due to incomplete silver impregnations of the nerve terminals. The stain of the silver methods tends to be restricted to the central portion of the nerves where there are numerous neurofibrils. However, differences of the muscles used and/ or other explanations cannot be ruled out. Disc- and doughnut-shaped nerve terminals also were found during the postnatal development of nerve terminals of the cat by the gold chloride method ~2. Nystr6m a2 reported that the doughnut-shaped nerve terminals were differentiated from the primitive disc-like ones by 'perforations' at i-2 places in their center. This finding is consistent with our present result that the doughnut- or double circle-shaped nerve terminals of the chicken PLD muscle developed from the perimeter-shaped ones at stage 5. However. we observed that this change in the nerve terminals appeared to lag behind that in the
REFERENCES I Atsumi. S., The histogenesis of motor neurons with special reference to the correlation of their endplate formation. I, The development of endplates in the intercostal muscle in the chick embryo, Acta Anat,. 80 (1971) 161 182. 2 Braithwaite, A. W. and Harris, A. J.. Neural influence on acetylcholine receptor clusters in emb~onic development of skeletal muscles, Nature (Lond.). 279 (1979) 549 55 I. 3 (hangeux, J.-P. and Danchin, A., Selective stabilization of developing synapses as a mechanism tbr the specification of neuronal networks, Nature tl, ond). 264 (1976) 705 712, 4 Christian, ('. N., I)anicls, M. P.. Sugiyama. H.. Vo~el, Z., Jacques. L. and Nelson, P. G., A factor from neurons incrca>c:, the number of acet~ Icholinc receptor aggrcgatc~ t)n cultured mr.sole cells, Proc. mlt..lead. ,S'(z. I '.S...I.. 75 (1978)4011 4015 5 Desaki. J. and Uehara, Y., The overall morphology of neuromuscular junctions as revealed by scanning electron microscopy, J. Neurocvtol.. 10(1981) 101- 110. 6 Hirano, H.. A histochemical study of the cholinesterase activity in the neuromuscular junction in developing chick skeletal muscles, Arch. Histol. Jap.. 28 (1967) 89 101. 7 Ishikawa. Y. and Shimada, Y., Fluorescent staining of acetylcholine receptors at the neuromuscular junction by means of rhodamine-labeled erabutoxin b. In M. Ito. N. Tsukahara, K. Kubota and K. Yagi (Eds.). Integrutive
AChR regions. This result seems inconsistent with the hypothesis that the distribution of junctional AChRs is controlled by neural influences throughout the development of the neuromuscular synapses 2- 4.~3. ACKNOWLEDGEMENTS
We are grateful to Professor N. Tamiya, Tohoku University, for his kind gift of erabutoxin b, and to Professor B. Wilson, University of California, Davis, for his critical reading of the manuscript. We also wish to thank Mr. N. Nakamura and Mrs. K. Shimizu for their technical assistance. This research was supported by grants from the following: the Japanese Ministry of Education, Science and Culture, the National Center for Nervous, Mental and Muscular Disorders ( N C N M D D 82-03) of the Japanese Ministry of Health and Welfare, and the Muscular Dystrophy Association of America.
Control Functions o f the Brain. Vol. 3, Kodansha Scientific, Tokyo/Elsevier, Amsterdam. 1980, pp. 29 31. 8 Ishikawa, Y. and Shimada. Y.. Acetylcholine receptors and cholinesterase in developing chick skeletal muscle fibers. Develop. Brain Res.. 5 (1982) 187-- 197. 9 Masuko, S., Kuromi, H. and Shimada, Y., Isolation and culture of motoneurons from embryonic chicken spinal cords, Proc. nat. A cad. Sci. U.S.A., 76 (1979) 3537- 3541. 10 Masuko. S. and Shimada. Y., Neuronal cell-surface specific antigen(s) is expressed during the terminal mitosis of cells destined to become neuroblasts, Develop. Biol., in press. 11 Mumenthaler, M. and Engel, W. K.,Cytological localization of cholinesterase in developing chick embryo skeletal muscle. A cta A nat.. 47 ( 1961 ) 274 299. 12 NystrOm, B., Postnatal development of motor nerve terminals in 'slow-red' and 'fast-white' cat muscles. Acta neurol. S t a n d , 44 (1968) 363 383. 13 Podleski, T. R., Axelrod, D., Ravdin, P., Greenberg, I., Hohnson, M. M. and Salpeter, M. M., Nerve extract induces increase and redistribution of acetylcholine receptors on cloned muscle cells. Proc. nat. Acad Sci. I,(S.A.. 75 (1978) 2035-2039. 14 Tello, J. F.. Die Einstehung der motorischen und sensiblen Nervenendigungen. I. In dem Iokomotorischen Systome der hOheren Wirbehiere. Muskul~ire Histogenese. Z.A nat. Entw.-Gesch.. 64 (1922) 348 440. 15 Wake, K., Motor endplates in developing chick embryo skeletal muscle: histological structure and histochemical localization of cholinesterase activit','. A rch. ttistoL Jap. 25(1964)23 41.