Structural Differences of Pale and Strongly Stained Motor End-plates of the Rat Diaphragm Y. C. WONG AND M. C. IP Department of Anatomy, Faculty of Medicine, University of Hong Kong, Hong Kong
ABSTRACT According to the staining intensities for AChE, the motor endplates of the rat diaphragm can be classified into strong ( S ) and pale (P) types. About 34%of the total end-plates of the rat diaphragm are of S type and 50%of P type. The P end-plates differ from S end-plates in two aspects. First, the secondary subneural clefts of the S end-plates are well developed. They are numerous, long, closely packed and often branched. On the other hand, the secondary subneural clefts of the P end-plates are short, sparse and usually unbranched. Secondly, there seems to be a variation in AChE activity in the P end-plates. Focal negative AChE areas are found in the subneural apparatus of some P end-plates. It is concluded that the less well developed secondary subneural clefts and focal areas of negative AChE activity contribute to the paler staining of the P end-plates. It has long been known that end-plates un- different ranges of diameter. Furthermore, dergo growth and degeneration in a wide Padykula and Gauthier ('70) have also demonrange of experimental and pathological condi- strated t h a t motor end-plates of the red, intertions (Cijers and Woolf, '59; Zacks, '64; Jir- mediate and white fibers are structurally difmanova, '75). On the other hand, Barker and ferent. One is, therefore, prompted to think Ip ('66) demonstrated that such processes oc- that P and S end-plates may be related to cur also in normal muscle. They suggested myoneural junctions of red and white fibers that there is a causal relationship between the respectively. On the other hand, Ip ('74) has growth and degeneration of end-plates such demonstrated that there is no direct correlathat the terminals have a limited life-span, at tion between the staining intensities for the end of which they degenerate and are re- AChE and the size of the end-plates or the placed by new terminals, thus new end-plates. fiber diameter, and that the P and S endTuffery ('711, however, showed that although plates are randomly distributed among the growth and degeneration of the myoneural whole range of fiber diameters and not segrejunctions occur in normal muscle, they are not gated to a particular fiber type. I t is well known that the presence of an causally related, and that there is a tendency for an increase in the number of more complex innervation is essential for the structural differentiation of the subneural apparatus (Couendings in aging. More recently, based on the staining inten- teaux, '73; Teravainen, '68) and that denervasities for acetylcholinesterase (AChE), Ip ('74) tion and re-innervation are followed by degenhas demonstrated that myoneural junctions eration and subsequent regeneration of the can be classified into pale (PI and strongly motor end-plates (Baur et al., '62; Saito and staining ( S ) types. He suggested that the dif- Zacks, '69). It is, therefore, possible that the ference in staining properties of myoneural difference in staining properties of the P and S junctions may reflect possible inherent varia- end-plates reflects a structural difference betions in the structure of the subneural ap- tween the myoneural junctions. This investigation was undertaken to determine the ultraparatus. It is well established t h a t in skeletal mus- structural differences between the P and S cles, including the diaphragm, there are three end-plates, as identified by their reaction for morphologically distinct types of fibers, i.e., AChE. red, intermediate and white fibers (Gauthier Accepted January 3, '78. and Padykula, '66; Gauthier, '691, which have AM.
J. ANAT. (1978) 152: 529-538.
529
530
Y. C. WONG AND M. C. IP MATERIALS AND METHODS
Young adult rats were killed by cervical dislocation and the diaphragm removed and fixed in chilled 2.5% glutaraldehyde in phosphate buffer a t pH 7.2. After fixing for four hours, the tissues were washed in several changes of phosphate buffer to remove excess fixative. The tissues were then transferred to normal saline. The modified thiocholine method of Coupland and Holmes ('57) was used for demonstrating AChE. The tissues were incubated in a medium containing the following substances: glycine, 1.5 mg/ml; copper sulphate, 1 mg/ml; magnesium chloride, 3.8 mg/ml; sodium sulphate, 203 mg/ml; and acetyl thiocholine (Edward Gurr, United Kingdom), 1.15 mg/ml in an acetic acid-sodium acetate buffer a t p H 4.8-5.2. After incubation the tissues were subsequently treated with a dilute (5%)aqueous solution of ammonium sulphide for five minutes to make the reaction product visible. It is generally believed that in this method the substrate is hydrolysed by enzymes, AChE and some nonspecific ChE, to liberate thiocholine a t the reaction site, although the point is now being debated (Brzin and Pucihar, '76). The thiocholine then reacts with copper ions in the incubation medium to form an insoluble precipitate of copper thiocholine, which is subsequently converted into a visible dark brown deposit of copper sulphide. A range of pH from 4.8 to 5.2 was employed and found to be suitable to give minimal diffusion. Incubation was carried out at room temperature from 30 minutes to 2.5 hours with constant agitation to ensure sufficient penetration. For light microscopy, some of the tissue was fixed either with or without added iso-OMPA M) as a ChE inhibitor, to block out the non-specific reaction. After incubation, the tissues were washed with dis-
tilled water before they were transferred into glycerine. Muscle fibers containing P and S end-plates were teased and separated under a binocular dissecting microscope. The muscle fibers containing P or S end-plates were then processed separately. These specimens were postfixed in 1% osmium tetroxide for two hours, dehydrated and embedded in Epon for electron microscopy. Thin sections were cut with a Porter-Blum MT-IIB microtome and counter-stained with uranyl acetate and lead citrate solution, and examined with a Phillips 300 electron microscope. RESULTS
Light microscopy
Figures 1 and 2 show the pale (PI and strongly stained (S) end-plates in the rat diaphragm. As can be seen in figure 1, all the motor end-plates in the diaphragm were located in a zone near the costal margin. The P end-plates appeared paler and vague in outline while the S end-plates were stained strongly and the subneural apparatus was clearly delineated in sharp outline (fig. 2). Not all the end-plates could be classified into P or S types. Some end-plates were stained strongly in one area and rather palely in another area of the same end-plates (see also Ip, '74). That is to say, some end-plates had a mosaic of P and S characteristics. Some, however, had a staining intensity between the S and P endplates, thus constituting an intermediate type. There were still others which were not classifiable. The frequencies of distribution of the types of end-plates according to staining intensities are presented in table 1. From the above table it is clear that about 50% of the end-plates of the rat diaphragm were of the P type and 34%of the S type. In addition, there were some 14%of the mosaic and intermediate types and about 2%were not
TABLE 1
Random sampling of 200end-plates from each of the rats toshow thepercentage ofeach type ofend-plate according to their staining intensities for AChE ~
End-plates
S end-plates
P end-plates
Mosaic and intermediate
Animal
No
No
No
%
No
%
R1 R2 R3 R4 R5 5
70 59 61 80 70 340
35 27 25 27 28 142
17.5 13.5 12.5 13.5 14 14.2
3 5 8
1.5 2.5 4
2 18
1 1.8
%
35 29.5 30.5 40 35 34
92 109 106 93 100 500
%
46 54.5 53 46.5 50 50
Not classifiable
-
-
Total no of end-plates
200 200 200 200 200 1,000
MOTOR END-PLATES OF THE RAT DIAPHRAGM
classifiable. The staining intensities of these end-plates bore no direct relationship t o the size of the end-plates Or the diameter Of the muscle fibers on which they terminated, as reported previously Up, '74: figs. la,b).
Electron microscoDv 1 "
The fine structure of the myoneural junction has been studied very extensively (Zacks, '64; Couteaux, '73). Under the electron microscope a typical motor end-plate consists of a n axon terminal occupying a recess on the surface of the muscle. This terminal contains a collection of mitochondria and an aggregation of synaptic vesicles. The sarcolemma of the muscle is separated from the axon terminal by a gap, known as the primary subneural cleft. In addition, the sarcolemma in the area of neuromuscular contact forms secondary infoldings called secondary subneural clefts or folds. These sarcolemmal infoldings a r e known collectively as t h e subneural a p paratus visible under the light microscope. Cytochemical studies Strong (S) end-plates The S end-plates were identified and isolated from the other types of end-plates before embedding for EM. Therefore, any end-plates subsequently obtained from this material belonged to the S type. Under the electron microscope each S endplate contained a n axon terminal and a well developed subneural apparatus (fig. 3). The mitochondria and the synaptic vesicles were somewhat damaged as a result of the cytochemical t r e a t m e n t but these structures could still be identified (fig. 3). However, the muscle structure was well preserved as can be seen in the micrographs shown. A large number of mitochondria could be seen in the sarcoplasm of the sole plate (although not visible in fig. 3). The dense material in the subneural clefts, both the primary and secondary clefts, was the product of the AChE reaction. This product was seen uniformly and in even density throughout both subneural clefts (fig. 3). The secondary subneural clefts, as outlined by the dense reaction product, were very extensive, closely packed, often branched and extended deep into the sarcoplasm. On the average the secondary clefts of the S end-plates were about 1-1.5 p m in depth. Sometimes some reaction product was found between the axon terminals and teloglial cells.
53 1
Pale (P) end-dates of the end-plates, the endAs in the plates were also similarly isolated and separately embedded and therefore would not be confused with the S type by EM. The P end-plates had the same basic structure a s the S end-plates. The most striking differences, however, was the less extensive development of the secondary subneural clefts (fig. 4). They were sparse, wider apart and fewer in number. The clefts were usually shorter and unbranched. The average depth of the junctional folds was about 1p m or less. In figure 4 the subneural clefts were filled with dense deposits of AChE reaction product just like t h a t of the S end-plates. There was no apparent difference in intensity of AChE reaction. However, in some other P end-plates the reaction was less uniform. This is shown in figures 5 and 6. In figure 5, in the areas indicated by arrows, there was no reaction product in the secondary clefts. The organelles of the axon terminal were well preserved in these end-plates. The subneural clefts appeared intact in the negative regions (fig. 5). Figure 6 shows a similar phenomenon, but in this endplate the negative area was larger. DISCUSSION
The existence of the S and P end-plates in normal muscle was first reported by Ip ('74). The S end-plates stain more strongly €or AChE while the P end-plates are less intensely stained. Padykula and Gauthier ('67a,b; '70) reported t h a t in the r a t diaphragm red, intermediate and white fibers are present in ratios of 60:20:20. They also demonstrated that the motor end-plates of these fiber types are structurally different from each other. They showed that the end-plates on the red fibers are generally smaller and the gutter is characterized by fewer and shorter synaptic folds, while the end-plates on the white fibers are larger, with more closely packed and deeper secondary subneural folds (Padykula and Gauthier, '70). In a separate study, based on the size and pattern of nerve terminals, Korneliussen and Waerhaug ('73) found that motor terminals can also be classified into three morphological types, i.e., types A, B and C. They showed, in addition, that the frequencies of occurrence of these three types of terminals (20.5% A, 20% B, 59.5% C) coincide with the frequencies of
532
Y. C. WONG AND M. C. IP
the fiber types in the rat diaphragm as given by Gauthier and Padykula ('66) and Padykula and Gauthier ('70) (20%white, 20%intermediate and 60% red). They, therefore, concluded that the A, B, and C types of terminals end on the white, intermediate and red fibers, respectively. In our present study the frequencies of the P, intermediate and S end-plates were 50%, 14%and 34%,respectively. The possible relationship of the P, intermediate and S endplates to the red, intermediate and white fibers, therefore, calls for comment. If the P and S end-plates, as reported here, correspond in fact to the end-plates of the red and white fibers (Padykula and Gauthier, '70), or type C and A terminals (Korneliussen and Waerhaug, '731, respectively, one would expect the P end-plates to be found on, and solely on small (red) fibers and the S endplates on, and solely on large (white) fibers. However, this was not the case in view of a previous report from this laboratory, arising from the same series of studies (Ip, '741, which showed that the staining intensities of the motor end-plates for AChE bear no direct relationship to their size and that both P and S end-plates are present in fibers having a wide range of diameter and are not confined to a particular size group. From the structural point of view, the morphology of the P end-plates does bear some resemblance to the end-plates on the red fibers (Padykula and Gauthier, '70), namely the P end-plates have a less well developed subneural apparatus, with sparse and short secondary subneural folds. But i t is equally pertinent to note that the P end-plates are also found in large, presumably white fibers and that the S end-plates are present also on small, presumably red fibers (Ip, '74). In addition, Ip also showed that some end-plates have mosaic characteristics of both P and S types, which further indicates that the two categories of end-plates can be present on the same fiber type and even on the same fiber. It is, therefore, unlikely that the P and Send-plates presented here are the same as the type C and A terminals (Korneliussen and Waerhaug, '73) or end-plates on the red and white fibers (Padykula and Gauthier, '701, respectively. The P end-plates as revealed in this study differ from S end-plates in two main aspects: First, the subneural clefts, more specifically the secondary clefts of the P end-plates, are less well developed. They are short, wider
apart and usually unbranched. On the other hand the subneural clefts of the S end-plates are well developed. The secondary subneural clefts are numerous, long and closely packed. In addition, the subneural folds are often branched. So the total area or space in the subneural clefts of the S end-plates is larger than in the P end-plates. When the muscle is stained for AChE, more reaction product can be collected in the subneural clefts of the S end-plates than the P end-plates. As a result, the S end-plates appear denser or darker as seen under the light microscope. Secondly, there seems to be a variation in AChE activity in the P end-plates. AChE may be as concentrated as in the S type in one region while completely lacking ke., negative) or very much lower in activity in other regions of the P end-plate. The negative area also varies from one P end-plate to another. Some have a small negative area while others have rather extensive negative regions. These variations may further lower the staining intensities of the P end-plates. The reason behind this negative reaction is not certain. There are several factors which may account for the negative areas in the P endplates. First, there is the possibility of a lack of uniformity in diffusion of the substrate into the subneural cleft, so that the substrate may not be available for reaction in certain areas of the end-plates, thus negative in reaction. Although in the present study the incubation time had been increased considerably (up to 2.5 hours), one cannot rule out the possibility of an irregularity of diffusion of the substrate into the cleft. The second possibility is that the negative areas may be the result of a fixation artefact. It has been demonstrated that after fixation by glutaraldehyde, about three-quarters of the original AChE activity of the tissue is lost (Koelle, Davis and Koelle, '74). It is, therefore, possible that this inhibitory property of glutaraldehyde on the activity of AChE may be a factor contributing to the negative areas in the P end-plates. In this connexion it may be of significance to note that in all the S endplates examined no similar negative areas were observed. Thirdly, there is still the possibility that the negative areas may represent actual regions which are lacking AChE or too low to be detected by our present cytochemical method. If this is the case, then it would imply that in
MOTOR END-PLATES OF THE RAT DIAPHRAGM
addition to the known variations in end-plate AChE activity in different muscles (Brzin and Zajicek, '58) and the structural dimorphism of the motor end-plates as demonstrated in the present study, there may also be an irregularity in concentration and distribution of AChE in each motor end-plate. Again this is pure speculation with no evidence to support the hypothesis. ACKNOWLEDGMENTS
This study was supported by a Grant 1581 254 to YCW from the Committee on Higher Degrees and Research Grants, University of Hong Kong. The authors wish to express their sincere gratitude to Professor F. P. Lisowski for reading the manuscript. Technical assistance of Mr. Y. S. Tong and Miss L. S. Kung is gratefully acknowledged. LITERATURE CITED Barker, D., and M. C. Ip 1966 Sprouting and degeneration of mammalian motor axons in normal and deafferentated skeletal muscle. Proc. R. SOC.London, Series B, 163: 538-554. Bauer, W. C., J. M. BlumbergandS. I. Zacks 1962 Short and long term ultrastructure changes in denervated mouse motor end-plates. Proc. IV Inter. Congress of Neuropathol., Munich, Georg. Thieme Verlag, Stuttgart, pp. 16-18. Brzin, M., and P. S. Pucihar 1976 Iodine, thiocyanate and cyanide ions as capturing reagents in one-step copperthiocholine method for cytochemical localization of cholinesterase activity. Histochemistry, 48: 283-292. Brzin, M., and J. Zajicek 1958 Quantitative determination of cholinesterase activity in individual end-plates of normal and denervated gastrocnemius muscle. Nature, 181: 626. Cijers, C., and A. L. Woolf 1959 The Innervation of Muscle: A Biopsy Study. Blackwell, Oxford. Coupland, R. E., and R. L. Holmes 1957 The use of cholinesterase technique for t h e demonstration of peripheral nervous structures. Quart. J. Micro. Sci., 98: 327-330. Couteaux, R. 1973 Motor end-plate structure. In: The
533
Structure and Function of Muscle. Vol. I, part 2. G. H. Bourne, ed. Second ed. Academic Press, New York, pp. 483-530. Gauthier, G. F. 1969 On the relationship of ultrastructural and cytological features to colour in mammalian skeletal muscle. 2. Zellforsch. mikrosk. Anat., 95; 462-482. Gauthier, G. F., and H. A. Padykula 1966 Cytological studies of fiber types in skeletal muscle. A comparative study of the mammalian diaphragm. J. Cell Biol., 28: 333-354. Ip, M. C. 1974 Some morphological features of the myoneural junctions in certain normal muscles of t h e rat. Anat. Rec., 180: 605-616. Jirmanova, I. 1975 Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle. J. Neurol., 4: 141-155. Koelle, G. B., R. Davis and W. A. Koelle 1974 Effects of aldehyde fixation and of preganglionic denervation on acetykholinesterase and butyrocholinesterase of cat autonomic ganglia. J. Histochem. Cytochem., 22: 244-251. Korneliussen, H., and 0.Waerhaug 1973 Three morphological types of motor nerve terminals in the rat diaphragm, and thin possible innervation of different muscle fiber types. 2.Anat. Entwickl-Gesch., 140: 73-84. Padykula, H. A,, and G. F. Gauthier 1967a Ultrastructural features of three fiber types in t h e rat diaphragm. Anat. Rec., 157: 296-297. 1967b Morphological and cytochemical characteristics of fiber types in normal mammalian skeletal muscle. In: Exploratory Concepts in Muscular Dystrophy and Related Disorders. A. T. Milhorat, ed. Internat. Congr. Series No. 147. Amsterdam: Excerpta Medica Foundation, pp. 117-131. 1970 The ultrastructure of the neuromuscular junctions of mammalian red, white and intermediate skeletal muscle fibers. J. Cell Biol., 46: 27-41. Saito, A., and S. I. Zacks 1969 Fine structure of neuromuscular junction after nerve section and implantation of nerve in denervated muscle. Expl. and Molec. Pathol., 10: 256-273. Teravainen, H. 1968 Development of the myoneural junction in the rat. 2. Zellforsch., 87: 247-265. Tuffery, A. R. 1971 Growth and degeneration of motor end-plates in normal cat hind limb muscles. J. Anat., 110: 221-247. Zacks, S. I. 1964 The Motor End-plate. W. B. Saunders Co.,Philadelphia and London.
PLATE I EXPLANATION O F FIGURES
1 Rat diaphragm stained for AChE to show the S and P end-plates. Note that P endplates are stained less intensely than the S end-plates. Incubation time, one and one-half hours. X 50. 2
High-magnification micrograph to show the S and P end-plates. The S end-plates are dark and have a sharp outline, while the P end-plates are paler and have a vague outline. Incubation time, one and one-half hours. x 300.
3 Low-magnification electron micrograph of a n S end-plate. The section was cut perpendicular to the surface of the muscle fiber. The organelles such as mitochondria and synaptic vesicles of the axon terminal (AT) are somewhat damaged. The subneural clefts, both the primary and secondary synaptic clefts, are delineated by dense deposits of copper sulphide. Note that the secondary synaptic clefts are deep, closely packed and usually branched. Sole plate nuclei (SN) and nucleus of teloglial cell (TN) are also shown. my, myofibrils. Incubated for one and one-half hours. X 17,400.
534
MOTOR END-PLATES OF THE RAT DIAPHRAGM Y. C. Wong and M. C. Ip
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
4
Electron micrograph of a P end-plate. Note t h a t the secondary subneural clefts are sparse, short and usually unbranched. The mitochondria (M)in the axon terminal (AT) are somewhat damaged. TC, teloglial cell. Incubated for one hour. X 14,160.
5 High-magnification electron micrograph of a P end-plate t o show the more extensive area of negative reaction (arrows) for AChE. In t h e region indicated by empty arrows a negative reaction is also seen in t h e primary subneural cleft. Incubated for one and one-half hours. AT, axon terminal. X 47,550. 6
536
P end-plate. Note t h e extensive negative area in t h e middle of this micrograph. Incubated for two hours. AT, axon terminal. x 30,400.
MOTOR END-PLATES OF THE RAT DIAPHRAGM Y. C. Wong and M. C. Ip
PLATE 2
ABSTRACT According to the staining intensities for AChE, the motor endplates of the rat diaphragm can be classified into strong ( S ) and pale (P) types. About 34%of the total end-plates of the rat diaphragm are of S type and 50%of P type. The P end-plates differ from S end-plates in two aspects. First, the secondary subneural clefts of the S end-plates are well developed. They are numerous, long, closely packed and often branched. On the other hand, the secondary subneural clefts of the P end-plates are short, sparse and usually unbranched. Secondly, there seems to be a variation in AChE activity in the P end-plates. Focal negative AChE areas are found in the subneural apparatus of some P end-plates. It is concluded that the less well developed secondary subneural clefts and focal areas of negative AChE activity contribute to the paler staining of the P end-plates. It has long been known that end-plates un- different ranges of diameter. Furthermore, dergo growth and degeneration in a wide Padykula and Gauthier ('70) have also demonrange of experimental and pathological condi- strated t h a t motor end-plates of the red, intertions (Cijers and Woolf, '59; Zacks, '64; Jir- mediate and white fibers are structurally difmanova, '75). On the other hand, Barker and ferent. One is, therefore, prompted to think Ip ('66) demonstrated that such processes oc- that P and S end-plates may be related to cur also in normal muscle. They suggested myoneural junctions of red and white fibers that there is a causal relationship between the respectively. On the other hand, Ip ('74) has growth and degeneration of end-plates such demonstrated that there is no direct correlathat the terminals have a limited life-span, at tion between the staining intensities for the end of which they degenerate and are re- AChE and the size of the end-plates or the placed by new terminals, thus new end-plates. fiber diameter, and that the P and S endTuffery ('711, however, showed that although plates are randomly distributed among the growth and degeneration of the myoneural whole range of fiber diameters and not segrejunctions occur in normal muscle, they are not gated to a particular fiber type. I t is well known that the presence of an causally related, and that there is a tendency for an increase in the number of more complex innervation is essential for the structural differentiation of the subneural apparatus (Couendings in aging. More recently, based on the staining inten- teaux, '73; Teravainen, '68) and that denervasities for acetylcholinesterase (AChE), Ip ('74) tion and re-innervation are followed by degenhas demonstrated that myoneural junctions eration and subsequent regeneration of the can be classified into pale (PI and strongly motor end-plates (Baur et al., '62; Saito and staining ( S ) types. He suggested that the dif- Zacks, '69). It is, therefore, possible that the ference in staining properties of myoneural difference in staining properties of the P and S junctions may reflect possible inherent varia- end-plates reflects a structural difference betions in the structure of the subneural ap- tween the myoneural junctions. This investigation was undertaken to determine the ultraparatus. It is well established t h a t in skeletal mus- structural differences between the P and S cles, including the diaphragm, there are three end-plates, as identified by their reaction for morphologically distinct types of fibers, i.e., AChE. red, intermediate and white fibers (Gauthier Accepted January 3, '78. and Padykula, '66; Gauthier, '691, which have AM.
J. ANAT. (1978) 152: 529-538.
529
530
Y. C. WONG AND M. C. IP MATERIALS AND METHODS
Young adult rats were killed by cervical dislocation and the diaphragm removed and fixed in chilled 2.5% glutaraldehyde in phosphate buffer a t pH 7.2. After fixing for four hours, the tissues were washed in several changes of phosphate buffer to remove excess fixative. The tissues were then transferred to normal saline. The modified thiocholine method of Coupland and Holmes ('57) was used for demonstrating AChE. The tissues were incubated in a medium containing the following substances: glycine, 1.5 mg/ml; copper sulphate, 1 mg/ml; magnesium chloride, 3.8 mg/ml; sodium sulphate, 203 mg/ml; and acetyl thiocholine (Edward Gurr, United Kingdom), 1.15 mg/ml in an acetic acid-sodium acetate buffer a t p H 4.8-5.2. After incubation the tissues were subsequently treated with a dilute (5%)aqueous solution of ammonium sulphide for five minutes to make the reaction product visible. It is generally believed that in this method the substrate is hydrolysed by enzymes, AChE and some nonspecific ChE, to liberate thiocholine a t the reaction site, although the point is now being debated (Brzin and Pucihar, '76). The thiocholine then reacts with copper ions in the incubation medium to form an insoluble precipitate of copper thiocholine, which is subsequently converted into a visible dark brown deposit of copper sulphide. A range of pH from 4.8 to 5.2 was employed and found to be suitable to give minimal diffusion. Incubation was carried out at room temperature from 30 minutes to 2.5 hours with constant agitation to ensure sufficient penetration. For light microscopy, some of the tissue was fixed either with or without added iso-OMPA M) as a ChE inhibitor, to block out the non-specific reaction. After incubation, the tissues were washed with dis-
tilled water before they were transferred into glycerine. Muscle fibers containing P and S end-plates were teased and separated under a binocular dissecting microscope. The muscle fibers containing P or S end-plates were then processed separately. These specimens were postfixed in 1% osmium tetroxide for two hours, dehydrated and embedded in Epon for electron microscopy. Thin sections were cut with a Porter-Blum MT-IIB microtome and counter-stained with uranyl acetate and lead citrate solution, and examined with a Phillips 300 electron microscope. RESULTS
Light microscopy
Figures 1 and 2 show the pale (PI and strongly stained (S) end-plates in the rat diaphragm. As can be seen in figure 1, all the motor end-plates in the diaphragm were located in a zone near the costal margin. The P end-plates appeared paler and vague in outline while the S end-plates were stained strongly and the subneural apparatus was clearly delineated in sharp outline (fig. 2). Not all the end-plates could be classified into P or S types. Some end-plates were stained strongly in one area and rather palely in another area of the same end-plates (see also Ip, '74). That is to say, some end-plates had a mosaic of P and S characteristics. Some, however, had a staining intensity between the S and P endplates, thus constituting an intermediate type. There were still others which were not classifiable. The frequencies of distribution of the types of end-plates according to staining intensities are presented in table 1. From the above table it is clear that about 50% of the end-plates of the rat diaphragm were of the P type and 34%of the S type. In addition, there were some 14%of the mosaic and intermediate types and about 2%were not
TABLE 1
Random sampling of 200end-plates from each of the rats toshow thepercentage ofeach type ofend-plate according to their staining intensities for AChE ~
End-plates
S end-plates
P end-plates
Mosaic and intermediate
Animal
No
No
No
%
No
%
R1 R2 R3 R4 R5 5
70 59 61 80 70 340
35 27 25 27 28 142
17.5 13.5 12.5 13.5 14 14.2
3 5 8
1.5 2.5 4
2 18
1 1.8
%
35 29.5 30.5 40 35 34
92 109 106 93 100 500
%
46 54.5 53 46.5 50 50
Not classifiable
-
-
Total no of end-plates
200 200 200 200 200 1,000
MOTOR END-PLATES OF THE RAT DIAPHRAGM
classifiable. The staining intensities of these end-plates bore no direct relationship t o the size of the end-plates Or the diameter Of the muscle fibers on which they terminated, as reported previously Up, '74: figs. la,b).
Electron microscoDv 1 "
The fine structure of the myoneural junction has been studied very extensively (Zacks, '64; Couteaux, '73). Under the electron microscope a typical motor end-plate consists of a n axon terminal occupying a recess on the surface of the muscle. This terminal contains a collection of mitochondria and an aggregation of synaptic vesicles. The sarcolemma of the muscle is separated from the axon terminal by a gap, known as the primary subneural cleft. In addition, the sarcolemma in the area of neuromuscular contact forms secondary infoldings called secondary subneural clefts or folds. These sarcolemmal infoldings a r e known collectively as t h e subneural a p paratus visible under the light microscope. Cytochemical studies Strong (S) end-plates The S end-plates were identified and isolated from the other types of end-plates before embedding for EM. Therefore, any end-plates subsequently obtained from this material belonged to the S type. Under the electron microscope each S endplate contained a n axon terminal and a well developed subneural apparatus (fig. 3). The mitochondria and the synaptic vesicles were somewhat damaged as a result of the cytochemical t r e a t m e n t but these structures could still be identified (fig. 3). However, the muscle structure was well preserved as can be seen in the micrographs shown. A large number of mitochondria could be seen in the sarcoplasm of the sole plate (although not visible in fig. 3). The dense material in the subneural clefts, both the primary and secondary clefts, was the product of the AChE reaction. This product was seen uniformly and in even density throughout both subneural clefts (fig. 3). The secondary subneural clefts, as outlined by the dense reaction product, were very extensive, closely packed, often branched and extended deep into the sarcoplasm. On the average the secondary clefts of the S end-plates were about 1-1.5 p m in depth. Sometimes some reaction product was found between the axon terminals and teloglial cells.
53 1
Pale (P) end-dates of the end-plates, the endAs in the plates were also similarly isolated and separately embedded and therefore would not be confused with the S type by EM. The P end-plates had the same basic structure a s the S end-plates. The most striking differences, however, was the less extensive development of the secondary subneural clefts (fig. 4). They were sparse, wider apart and fewer in number. The clefts were usually shorter and unbranched. The average depth of the junctional folds was about 1p m or less. In figure 4 the subneural clefts were filled with dense deposits of AChE reaction product just like t h a t of the S end-plates. There was no apparent difference in intensity of AChE reaction. However, in some other P end-plates the reaction was less uniform. This is shown in figures 5 and 6. In figure 5, in the areas indicated by arrows, there was no reaction product in the secondary clefts. The organelles of the axon terminal were well preserved in these end-plates. The subneural clefts appeared intact in the negative regions (fig. 5). Figure 6 shows a similar phenomenon, but in this endplate the negative area was larger. DISCUSSION
The existence of the S and P end-plates in normal muscle was first reported by Ip ('74). The S end-plates stain more strongly €or AChE while the P end-plates are less intensely stained. Padykula and Gauthier ('67a,b; '70) reported t h a t in the r a t diaphragm red, intermediate and white fibers are present in ratios of 60:20:20. They also demonstrated that the motor end-plates of these fiber types are structurally different from each other. They showed that the end-plates on the red fibers are generally smaller and the gutter is characterized by fewer and shorter synaptic folds, while the end-plates on the white fibers are larger, with more closely packed and deeper secondary subneural folds (Padykula and Gauthier, '70). In a separate study, based on the size and pattern of nerve terminals, Korneliussen and Waerhaug ('73) found that motor terminals can also be classified into three morphological types, i.e., types A, B and C. They showed, in addition, that the frequencies of occurrence of these three types of terminals (20.5% A, 20% B, 59.5% C) coincide with the frequencies of
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Y. C. WONG AND M. C. IP
the fiber types in the rat diaphragm as given by Gauthier and Padykula ('66) and Padykula and Gauthier ('70) (20%white, 20%intermediate and 60% red). They, therefore, concluded that the A, B, and C types of terminals end on the white, intermediate and red fibers, respectively. In our present study the frequencies of the P, intermediate and S end-plates were 50%, 14%and 34%,respectively. The possible relationship of the P, intermediate and S endplates to the red, intermediate and white fibers, therefore, calls for comment. If the P and S end-plates, as reported here, correspond in fact to the end-plates of the red and white fibers (Padykula and Gauthier, '70), or type C and A terminals (Korneliussen and Waerhaug, '731, respectively, one would expect the P end-plates to be found on, and solely on small (red) fibers and the S endplates on, and solely on large (white) fibers. However, this was not the case in view of a previous report from this laboratory, arising from the same series of studies (Ip, '741, which showed that the staining intensities of the motor end-plates for AChE bear no direct relationship to their size and that both P and S end-plates are present in fibers having a wide range of diameter and are not confined to a particular size group. From the structural point of view, the morphology of the P end-plates does bear some resemblance to the end-plates on the red fibers (Padykula and Gauthier, '70), namely the P end-plates have a less well developed subneural apparatus, with sparse and short secondary subneural folds. But i t is equally pertinent to note that the P end-plates are also found in large, presumably white fibers and that the S end-plates are present also on small, presumably red fibers (Ip, '74). In addition, Ip also showed that some end-plates have mosaic characteristics of both P and S types, which further indicates that the two categories of end-plates can be present on the same fiber type and even on the same fiber. It is, therefore, unlikely that the P and Send-plates presented here are the same as the type C and A terminals (Korneliussen and Waerhaug, '73) or end-plates on the red and white fibers (Padykula and Gauthier, '701, respectively. The P end-plates as revealed in this study differ from S end-plates in two main aspects: First, the subneural clefts, more specifically the secondary clefts of the P end-plates, are less well developed. They are short, wider
apart and usually unbranched. On the other hand the subneural clefts of the S end-plates are well developed. The secondary subneural clefts are numerous, long and closely packed. In addition, the subneural folds are often branched. So the total area or space in the subneural clefts of the S end-plates is larger than in the P end-plates. When the muscle is stained for AChE, more reaction product can be collected in the subneural clefts of the S end-plates than the P end-plates. As a result, the S end-plates appear denser or darker as seen under the light microscope. Secondly, there seems to be a variation in AChE activity in the P end-plates. AChE may be as concentrated as in the S type in one region while completely lacking ke., negative) or very much lower in activity in other regions of the P end-plate. The negative area also varies from one P end-plate to another. Some have a small negative area while others have rather extensive negative regions. These variations may further lower the staining intensities of the P end-plates. The reason behind this negative reaction is not certain. There are several factors which may account for the negative areas in the P endplates. First, there is the possibility of a lack of uniformity in diffusion of the substrate into the subneural cleft, so that the substrate may not be available for reaction in certain areas of the end-plates, thus negative in reaction. Although in the present study the incubation time had been increased considerably (up to 2.5 hours), one cannot rule out the possibility of an irregularity of diffusion of the substrate into the cleft. The second possibility is that the negative areas may be the result of a fixation artefact. It has been demonstrated that after fixation by glutaraldehyde, about three-quarters of the original AChE activity of the tissue is lost (Koelle, Davis and Koelle, '74). It is, therefore, possible that this inhibitory property of glutaraldehyde on the activity of AChE may be a factor contributing to the negative areas in the P end-plates. In this connexion it may be of significance to note that in all the S endplates examined no similar negative areas were observed. Thirdly, there is still the possibility that the negative areas may represent actual regions which are lacking AChE or too low to be detected by our present cytochemical method. If this is the case, then it would imply that in
MOTOR END-PLATES OF THE RAT DIAPHRAGM
addition to the known variations in end-plate AChE activity in different muscles (Brzin and Zajicek, '58) and the structural dimorphism of the motor end-plates as demonstrated in the present study, there may also be an irregularity in concentration and distribution of AChE in each motor end-plate. Again this is pure speculation with no evidence to support the hypothesis. ACKNOWLEDGMENTS
This study was supported by a Grant 1581 254 to YCW from the Committee on Higher Degrees and Research Grants, University of Hong Kong. The authors wish to express their sincere gratitude to Professor F. P. Lisowski for reading the manuscript. Technical assistance of Mr. Y. S. Tong and Miss L. S. Kung is gratefully acknowledged. LITERATURE CITED Barker, D., and M. C. Ip 1966 Sprouting and degeneration of mammalian motor axons in normal and deafferentated skeletal muscle. Proc. R. SOC.London, Series B, 163: 538-554. Bauer, W. C., J. M. BlumbergandS. I. Zacks 1962 Short and long term ultrastructure changes in denervated mouse motor end-plates. Proc. IV Inter. Congress of Neuropathol., Munich, Georg. Thieme Verlag, Stuttgart, pp. 16-18. Brzin, M., and P. S. Pucihar 1976 Iodine, thiocyanate and cyanide ions as capturing reagents in one-step copperthiocholine method for cytochemical localization of cholinesterase activity. Histochemistry, 48: 283-292. Brzin, M., and J. Zajicek 1958 Quantitative determination of cholinesterase activity in individual end-plates of normal and denervated gastrocnemius muscle. Nature, 181: 626. Cijers, C., and A. L. Woolf 1959 The Innervation of Muscle: A Biopsy Study. Blackwell, Oxford. Coupland, R. E., and R. L. Holmes 1957 The use of cholinesterase technique for t h e demonstration of peripheral nervous structures. Quart. J. Micro. Sci., 98: 327-330. Couteaux, R. 1973 Motor end-plate structure. In: The
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Structure and Function of Muscle. Vol. I, part 2. G. H. Bourne, ed. Second ed. Academic Press, New York, pp. 483-530. Gauthier, G. F. 1969 On the relationship of ultrastructural and cytological features to colour in mammalian skeletal muscle. 2. Zellforsch. mikrosk. Anat., 95; 462-482. Gauthier, G. F., and H. A. Padykula 1966 Cytological studies of fiber types in skeletal muscle. A comparative study of the mammalian diaphragm. J. Cell Biol., 28: 333-354. Ip, M. C. 1974 Some morphological features of the myoneural junctions in certain normal muscles of t h e rat. Anat. Rec., 180: 605-616. Jirmanova, I. 1975 Ultrastructure of motor end-plates during pharmacologically-induced degeneration and subsequent regeneration of skeletal muscle. J. Neurol., 4: 141-155. Koelle, G. B., R. Davis and W. A. Koelle 1974 Effects of aldehyde fixation and of preganglionic denervation on acetykholinesterase and butyrocholinesterase of cat autonomic ganglia. J. Histochem. Cytochem., 22: 244-251. Korneliussen, H., and 0.Waerhaug 1973 Three morphological types of motor nerve terminals in the rat diaphragm, and thin possible innervation of different muscle fiber types. 2.Anat. Entwickl-Gesch., 140: 73-84. Padykula, H. A,, and G. F. Gauthier 1967a Ultrastructural features of three fiber types in t h e rat diaphragm. Anat. Rec., 157: 296-297. 1967b Morphological and cytochemical characteristics of fiber types in normal mammalian skeletal muscle. In: Exploratory Concepts in Muscular Dystrophy and Related Disorders. A. T. Milhorat, ed. Internat. Congr. Series No. 147. Amsterdam: Excerpta Medica Foundation, pp. 117-131. 1970 The ultrastructure of the neuromuscular junctions of mammalian red, white and intermediate skeletal muscle fibers. J. Cell Biol., 46: 27-41. Saito, A., and S. I. Zacks 1969 Fine structure of neuromuscular junction after nerve section and implantation of nerve in denervated muscle. Expl. and Molec. Pathol., 10: 256-273. Teravainen, H. 1968 Development of the myoneural junction in the rat. 2. Zellforsch., 87: 247-265. Tuffery, A. R. 1971 Growth and degeneration of motor end-plates in normal cat hind limb muscles. J. Anat., 110: 221-247. Zacks, S. I. 1964 The Motor End-plate. W. B. Saunders Co.,Philadelphia and London.
PLATE I EXPLANATION O F FIGURES
1 Rat diaphragm stained for AChE to show the S and P end-plates. Note that P endplates are stained less intensely than the S end-plates. Incubation time, one and one-half hours. X 50. 2
High-magnification micrograph to show the S and P end-plates. The S end-plates are dark and have a sharp outline, while the P end-plates are paler and have a vague outline. Incubation time, one and one-half hours. x 300.
3 Low-magnification electron micrograph of a n S end-plate. The section was cut perpendicular to the surface of the muscle fiber. The organelles such as mitochondria and synaptic vesicles of the axon terminal (AT) are somewhat damaged. The subneural clefts, both the primary and secondary synaptic clefts, are delineated by dense deposits of copper sulphide. Note that the secondary synaptic clefts are deep, closely packed and usually branched. Sole plate nuclei (SN) and nucleus of teloglial cell (TN) are also shown. my, myofibrils. Incubated for one and one-half hours. X 17,400.
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MOTOR END-PLATES OF THE RAT DIAPHRAGM Y. C. Wong and M. C. Ip
PLATE 1
PLATE 2 EXPLANATION OF FIGURES
4
Electron micrograph of a P end-plate. Note t h a t the secondary subneural clefts are sparse, short and usually unbranched. The mitochondria (M)in the axon terminal (AT) are somewhat damaged. TC, teloglial cell. Incubated for one hour. X 14,160.
5 High-magnification electron micrograph of a P end-plate t o show the more extensive area of negative reaction (arrows) for AChE. In t h e region indicated by empty arrows a negative reaction is also seen in t h e primary subneural cleft. Incubated for one and one-half hours. AT, axon terminal. X 47,550. 6
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P end-plate. Note t h e extensive negative area in t h e middle of this micrograph. Incubated for two hours. AT, axon terminal. x 30,400.
MOTOR END-PLATES OF THE RAT DIAPHRAGM Y. C. Wong and M. C. Ip
PLATE 2
