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The ultrastructure of normal and denervated neuromuscular synapses in mouse gastrocnemius muscle
662 THE ULTRASTRUCTURE OF NORMAL AND DENERVATED NEUROMUSCULAR SYNAPSES INMOUSEGASTROCNEMIUS MUSCLE' J. F. REGER Department
of Anatomy,
University
of Colorado Medical Received
March
Center, Denver, Colorado,
U.S.A.
4, 1956
STUDIES on the ultrastructure of the normal neuromuscular synapse [l, 5,7,8,9, lo] furnish a basis for experimental investigations using the electron microscope. Further investigation is needed in an attempt to learn more about the synaptic vesicles and membranes and their possible role in neuromuscular transmission. The following is a preliminary report of a study on the normal and denervated neuromuscular ultrastructure. Material and m&hods.-Gastrocnemius muscles of mice were denervated by sciatic section. Normal and denervated muscle were prepared identically. Tissue was fixed in buffered osmium tetroxide (pH 7.6) and subsequently dehydrated, embedded and sectioned for electron microscopy. Several hundred micrographs from 25 normal and 25 experimental preparations were made, of which the 2 included are representatives. Observations.-Normal neuromuscular junction. In the normal motor end-plate from gastrocnemius muscle of mice the synapse consists of closely apposed axolemma and sarcolemma (Fig. 1). The resulting synaptolemma is 500-600 A in thickness and composed of five differentially osmicated layers, best seen at arrows in Fig. 1. From the presynaptic axoplasmic to the postsynaptic sarcoplasmic side there are: (1) densely osmicated axolemma, 100 A in thickness, (2) an area of light osmification, 150 a in thickness, (3) a middle densely osmicated region, which appears beaded in certain regions (best seen at arrows, Fig. I), 100 A in thickness, (4) another light zone, 150 A in thickness, and (5) the dense inner layer of sarcolemma, 100 A in thickness. The postsynaptic membrane (sarcolemma) is deeply infolded at the synapse forming a series of lamellae. The middle dense layer of the synaptolemma may be seen to assume the same infolding as that of the postsynaptic membrane. The axoplasm contains mitochondria, synaptic vesicles and endoplasmic reticulum (Fig. 1). Synaptic vesicles are 300-500 A in size; they are found in the postsynaptic sarcoplasm as well, though fewer in number. The sarcoplasm of the end-plate also contains granules approximately 100 A in size. Denervated neuromuscular junction. After 48 hours denervation the axolemmal portion of the synaptolemma has almost completely retracted from the sarcolemma and lysis of the axolemma has begun (Fig. 2). A postsynaptic membrane free of axolemma remains, except in one area where a definitive axolemma may be seen. 1 Supported by grants from the Muscular Dystrophy Associations of America, Incorporated, and U.S. Public Health Grant No. B-605. Experimental
Cell Research 12
Ultrastructure of the neuromuscular synapse
663
The axon appears to have undergone considerable lysis and no recognizable mitochondria, synaptic vesicles, or endoplasmic reticulum are evident. Discussion.-The normal ultrastructure of the synaptolemma described here is similar to that reported by Robertson [lo]. The middle dense layer of the synaptolemma is probably sarcolemmal because of its continuation into the junctional folds of the postsynaptic membrane. Further support to this interpretation is given by the fact that after 48 hours denervation this layer is still observed intact as part of the postsynaptic membrane. The intervening dense layer of the synaptolemma may then be interpreted as an outer dense layer of postsynaptic membrane complex. The significance of its vesicular or beaded appearance is not known. The infolding of the sarcolemma might be thought of as increasing the available postsynaptic surface area. Such an infolding of postsynaptic membranes has not been observed in most other types of synapses studied with the electron microscope [3, 5, 61, although Luft [4] reported similar infoldings of the electroplaque membranes of eel electric organs. Couteaux [2] first described the laminated structure of the neuromuscular postsynaptic membrane and by cytochemical methods identified the presence of acetylcholinesterase. In view of the complex nature of neuromuscular transmission, further studies of this membrane by electron microscopy and cytochemistry are warranted. Synaptic vesicles of the type observed in the junctional axo- and sarcoplasm in this study have been reported for other synapses 13, 4, 5, 61. Their function at the synapse remains a subject of speculation, although suggestions have been made that they may contain small quantities of chemical transmitter substance [3, 6, lo]. Confirmation of this will have to await investigation of the biochemical properties of synaptic vesicles. The close association of synaptic vesicles and endoplasmic reticulum in the axoplasm may be significant. De Robertis and Bennett [3] have suggested that synaptic vesicles may be formed by the endoplasmic reticulum. Summary.-Normal and 48 hour denervated neuromuscular synapses of mouse gastrocnemius were studied. Axolemmal retraction from sarcolemma was observed after 48 hours denervation. The postsynaptic sarcolemma is described as a three layered membrane complex. This membrane is deeply infolded resulting in an increase in surface area. The normal axon contains mitochondria, endoplasmic reticulum and synaptic vesicles 300-500 A in size. After 48 hours denervation the axon and its contents display considerable lysis. The normal sarcoplasm contains synaptic ves icles and granules approximately 100 A in size.
REFERENCES 1. BEAMS, H. W. and EVANS, T. C., Proe. Sot. Expfl. Hal. Med. 82, 344 (1953). 2. COUTEAUX, R., Rev. Can. Biol. 6, 563 (1947). 3. DE ROBERTIS, E. D. P. and BENNETT, H. S., J. Biophys. Biochem. Cytol. 1, 47 (1955). 4. LUFT, J. H., J. Biophys. Biochem. Cytol. 2 (Supplement), 229 (1956). 5. PALADE, G. E., Anat. Rec. 118, 335 (1954). Abstract. 6. PALAY, S. L., J. Biophys. Biochem. Cytof. 2 (Supplement), 193 (1956). 7. REGER, J. F., Anaf. Rec. 118, 344 (1954). Abstract. 8. ibid. 122, 1 (1955). 9. ROBERTSON, J. D., Anaf. Rec. 116,346 (1954). Abstract. 10. __ J. Biophys. Bioehem. Cyfof. 2, 381 (1956).
Experimental
Cell Research 12
J. F. Reger
Fig. l.-Normal neuromuscular synapse. Axon (Ao) in upper fourth of figure contains a mitochondrion (M), synaptic vesicles (SV) and endoplasmic reticulum (ER). Axolemma (AI) is closely apposed to sarcolemma (SI). Arrows delimit synaptolemma. The sarcoplasm (So) contains granules and synaptic vesicles (SV). A portion of a myofibril (My) appears in the lower right. Experimental
Cell Research 12
Ultrastructure of the neuromuscular synapse
665
Fig. 2.--Neuromuscular synapse after 48 hours’ denervation. The axon (Ao) mldergoing lysis is seen retracted from sarcolemma (3). Small portions of axolemma (Al) may be seen. Arrows indicate middle dense layer of synaptolemma. Postsynaptic sarcoplasm (So) is in the lower left. Experimental
Cell Research 12
of Anatomy,
University
of Colorado Medical Received
March
Center, Denver, Colorado,
U.S.A.
4, 1956
STUDIES on the ultrastructure of the normal neuromuscular synapse [l, 5,7,8,9, lo] furnish a basis for experimental investigations using the electron microscope. Further investigation is needed in an attempt to learn more about the synaptic vesicles and membranes and their possible role in neuromuscular transmission. The following is a preliminary report of a study on the normal and denervated neuromuscular ultrastructure. Material and m&hods.-Gastrocnemius muscles of mice were denervated by sciatic section. Normal and denervated muscle were prepared identically. Tissue was fixed in buffered osmium tetroxide (pH 7.6) and subsequently dehydrated, embedded and sectioned for electron microscopy. Several hundred micrographs from 25 normal and 25 experimental preparations were made, of which the 2 included are representatives. Observations.-Normal neuromuscular junction. In the normal motor end-plate from gastrocnemius muscle of mice the synapse consists of closely apposed axolemma and sarcolemma (Fig. 1). The resulting synaptolemma is 500-600 A in thickness and composed of five differentially osmicated layers, best seen at arrows in Fig. 1. From the presynaptic axoplasmic to the postsynaptic sarcoplasmic side there are: (1) densely osmicated axolemma, 100 A in thickness, (2) an area of light osmification, 150 a in thickness, (3) a middle densely osmicated region, which appears beaded in certain regions (best seen at arrows, Fig. I), 100 A in thickness, (4) another light zone, 150 A in thickness, and (5) the dense inner layer of sarcolemma, 100 A in thickness. The postsynaptic membrane (sarcolemma) is deeply infolded at the synapse forming a series of lamellae. The middle dense layer of the synaptolemma may be seen to assume the same infolding as that of the postsynaptic membrane. The axoplasm contains mitochondria, synaptic vesicles and endoplasmic reticulum (Fig. 1). Synaptic vesicles are 300-500 A in size; they are found in the postsynaptic sarcoplasm as well, though fewer in number. The sarcoplasm of the end-plate also contains granules approximately 100 A in size. Denervated neuromuscular junction. After 48 hours denervation the axolemmal portion of the synaptolemma has almost completely retracted from the sarcolemma and lysis of the axolemma has begun (Fig. 2). A postsynaptic membrane free of axolemma remains, except in one area where a definitive axolemma may be seen. 1 Supported by grants from the Muscular Dystrophy Associations of America, Incorporated, and U.S. Public Health Grant No. B-605. Experimental
Cell Research 12
Ultrastructure of the neuromuscular synapse
663
The axon appears to have undergone considerable lysis and no recognizable mitochondria, synaptic vesicles, or endoplasmic reticulum are evident. Discussion.-The normal ultrastructure of the synaptolemma described here is similar to that reported by Robertson [lo]. The middle dense layer of the synaptolemma is probably sarcolemmal because of its continuation into the junctional folds of the postsynaptic membrane. Further support to this interpretation is given by the fact that after 48 hours denervation this layer is still observed intact as part of the postsynaptic membrane. The intervening dense layer of the synaptolemma may then be interpreted as an outer dense layer of postsynaptic membrane complex. The significance of its vesicular or beaded appearance is not known. The infolding of the sarcolemma might be thought of as increasing the available postsynaptic surface area. Such an infolding of postsynaptic membranes has not been observed in most other types of synapses studied with the electron microscope [3, 5, 61, although Luft [4] reported similar infoldings of the electroplaque membranes of eel electric organs. Couteaux [2] first described the laminated structure of the neuromuscular postsynaptic membrane and by cytochemical methods identified the presence of acetylcholinesterase. In view of the complex nature of neuromuscular transmission, further studies of this membrane by electron microscopy and cytochemistry are warranted. Synaptic vesicles of the type observed in the junctional axo- and sarcoplasm in this study have been reported for other synapses 13, 4, 5, 61. Their function at the synapse remains a subject of speculation, although suggestions have been made that they may contain small quantities of chemical transmitter substance [3, 6, lo]. Confirmation of this will have to await investigation of the biochemical properties of synaptic vesicles. The close association of synaptic vesicles and endoplasmic reticulum in the axoplasm may be significant. De Robertis and Bennett [3] have suggested that synaptic vesicles may be formed by the endoplasmic reticulum. Summary.-Normal and 48 hour denervated neuromuscular synapses of mouse gastrocnemius were studied. Axolemmal retraction from sarcolemma was observed after 48 hours denervation. The postsynaptic sarcolemma is described as a three layered membrane complex. This membrane is deeply infolded resulting in an increase in surface area. The normal axon contains mitochondria, endoplasmic reticulum and synaptic vesicles 300-500 A in size. After 48 hours denervation the axon and its contents display considerable lysis. The normal sarcoplasm contains synaptic ves icles and granules approximately 100 A in size.
REFERENCES 1. BEAMS, H. W. and EVANS, T. C., Proe. Sot. Expfl. Hal. Med. 82, 344 (1953). 2. COUTEAUX, R., Rev. Can. Biol. 6, 563 (1947). 3. DE ROBERTIS, E. D. P. and BENNETT, H. S., J. Biophys. Biochem. Cytol. 1, 47 (1955). 4. LUFT, J. H., J. Biophys. Biochem. Cytol. 2 (Supplement), 229 (1956). 5. PALADE, G. E., Anat. Rec. 118, 335 (1954). Abstract. 6. PALAY, S. L., J. Biophys. Biochem. Cytof. 2 (Supplement), 193 (1956). 7. REGER, J. F., Anaf. Rec. 118, 344 (1954). Abstract. 8. ibid. 122, 1 (1955). 9. ROBERTSON, J. D., Anaf. Rec. 116,346 (1954). Abstract. 10. __ J. Biophys. Bioehem. Cyfof. 2, 381 (1956).
Experimental
Cell Research 12
J. F. Reger
Fig. l.-Normal neuromuscular synapse. Axon (Ao) in upper fourth of figure contains a mitochondrion (M), synaptic vesicles (SV) and endoplasmic reticulum (ER). Axolemma (AI) is closely apposed to sarcolemma (SI). Arrows delimit synaptolemma. The sarcoplasm (So) contains granules and synaptic vesicles (SV). A portion of a myofibril (My) appears in the lower right. Experimental
Cell Research 12
Ultrastructure of the neuromuscular synapse
665
Fig. 2.--Neuromuscular synapse after 48 hours’ denervation. The axon (Ao) mldergoing lysis is seen retracted from sarcolemma (3). Small portions of axolemma (Al) may be seen. Arrows indicate middle dense layer of synaptolemma. Postsynaptic sarcoplasm (So) is in the lower left. Experimental
Cell Research 12