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Simultaneous cytochemical demonstration of muscle fiber types and acetylcholinesterase in muscle fibers of dystrophic chickens
EXPERIMENTAL
NEUROLOGY
Simultaneous
60,68-82
(1978)
Cytochemical
Types
Demonstration
and Acetylcholinesterase of Dystrophic
C. R. ASHMORE, Laboratory California,
in Muscle
Fiber
Fibers
Chickens
P. VIGNERON,
L. MARGER,
of Muscle Biology, Department of Animal Davis, California 95616, and INRA, Statiorz ENSA, 34060 Montpellier-Cedex, Received
of Muscle
November
AND L. DOERR
Scieme, University of de Physiologic Anirnale,
Fraace
29,1977
A cytochemical procedure is described which allows the simultaneous observation of different types of muscle fibers and their motor end plates. The procedure utilizes an assay for myofibrillar adenosine 5’-triphosphatase (ATPase) activity followed by an assay for acetylcholinesterase (AChE) activity. This combined assay eliminates the necessity for using serial sections for observation of these two parameters. This combined assay increases the apparent AChE activity such that sites of AChE activity are revealed which are not visualized when using the AChE assay alone. In muscles from chicks with hereditary muscular dystrophy, it is shown that initially dystrophic fibers contain nuclei which react strongly for AChE activity. Subsequently many fibers exhibit an intense reaction for AChE activity over a major portion of their cell surface. AChE activity is also found along the splits of fragmenting fibers and on the periphery of necrotic vacuoles.
INTRODUCTION Classification of muscle fiber types is useful for the diagnosis of some types of neuromuscular pathologies, for observing adaptive responses to neuromuscular training, and for following developmental patterns of muscle fiber distributions. Several cytochemical techniques have been described Abbreviations : AChE-acetylcholinesterase ; ATPase-adenosine 5’-triphosphatase ; BuCh ; butyrylthiocholine. 1 This work was supported in part by grants from the Muscular Dystrophy Association of America, U.S. Public Health Science Grant AM 16716, and Delegation G&r&ale a la Recherche Scientifique et Technique Grant 75 7 1641. 68 0014-4886/78/0601-0068$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
AChE
AND
FIBER
TYPES
IN
DYSTROPHIC
MUSCLE
69
which allow the identification of different types of muscle fibers (4, 7, 10). In addition, various histological and cytochemical techniques are available for demonstration of the motor end plates of muscle fibers and the axons which imlervate these end plates (9, 15, 16, 18, 23). These techniques were also successfully applied for studying pathologies of end plates and for calculation of the terminal innervation ratio (6, 8, 22, 26). The purpose of this report is to present a cytochemical procedure for the demonstration of AChE activity in different types of muscle fibers and to examine some results from the application of this procedure to dystrophic chick muscle. In normal adult muscle fibers, AChE activity is confined to the motor end plate and its immediate vicinity and to myotendinous junctions. Several reports showed that this normal pattern of enzyme location is altered in dystrophic muscles. Beckett and Bourne (6) noted higher activity of AChE in the sarcoplasm of muscle fibers from human dystrophic patients. Wilson rt al. (27) observed high sarcoplasmic AChE activity in more than 10% of fibers from patients having a variety of abnormal neuromuscular ailments. Harman et al. (12), Law and Atwood (17), and Tennyson it nl. (22) reported the occurrence of fragmented and shrunken motor end plates in dystrophic mouse muscle. Jedrzejczyk et al. (14) reported decreased AChE activity at the motor end plates of dystrophic mouse muscle. Wilson et al. (25) followed the distribution of AChE isoenzymes in developing muscle fibers of normal and dystrophic chicks. They found that, of three AChE isoenzymes which are present in normal embryo muscle, two disappear shortly after hatching. However, all three forms are found in homogenates of fast-twitch muscles of dystrophic chicks. These embryonic forms of the enzyme are distributed diffusely in the cytoplasm of embryo muscle fibers, but they are confined to the neuromuscular junctional area of dystrophic chick muscle fibers (19). 1Vhereas AChE activity was found to be localized within 50 pm of normal fiber end plates, activity was detectable beyond 1.50pm of dystrophic fiber end plates. Using the cytochemical procedure reported here, we describe additional abnormal cellular localizations of AGE activity in dystrophic chick muscle fibers and their temporal relation to the pathogenesis of the myopathy. For classification of different types of muscle fibers, we use the term a to denote fibers which exhibit a low activity of myofibrillar ATPase and the term p to denote fibers kvhicli exhibit a high activity of myofibrillar ATI’ase activity after preincubation in an acid medium (2 j.
Normal chicks ailtl chicks with lieretlitarx muscular dystrophy from lilies maintai~~ctl at this station \sere usc~l in this study. The snrtorius,
70
ASHMORE
ET
AL,
anterior latissimus dorsi, posterior latissimus dorsi, superficial pectoralis, and m. complexus were examined at hatching and at weekly intervals for 10 weeks. Chicks were killed by exsanguination. Muscle samples were removed, pinned to neoprene boards, and quick-frozen in dry ice and isopentane. Pieces of muscle were cut with an International cryostat, floated on a water solution of 0.1% serum albumin at 4°C picked up on coverslips, and dried at 4°C. This allowed the muscle sections to stick to the coverslips during subsequent incubations for enzyme activities. After drying, the sections were fixed 12 min at 4°C in 0.5 N formic acid adjusted to pH 4.20 with 1 M NaOH. The optimum pH may vary slightly according to species, as well as the thickness of the muscle sections. For the results described below, we found 16 pm to be the optimal thickness of the sections. After fixation, the sections were rinsed twice for 1 min in 0.1 M Tris-HCl and 18 mM CaCla, pH 7.8, and then rinsed again with distilled Hz0 for 2 min. Next the sections were incubated for myofibrillar ATPase by a method adapted from Pearse (20). The medium was 0.1 M sodium barbiturate, 0.18 M CaClz adjusted to pH 9.4 with 0.1 M NaOH, and containing 5 mM ATP. The sections were incubated 10 min at 37°C. Although the incubation time does not allow for completion of the reaction, it is sufficient for identification of the fiber types. Longer periods of incubation may make some fibers too dark for observation of the motor end plates. After incubation for ATPase activity, the sections were rinsed three times in 1% CaClz during 10 min, transferred to 2% CaC13 for 3 min, washed 10 min with distilled water, and developed 2 min in 1% ammonium sulfide. After washing 10 min in running tap water, the sections were incubated for acetylcholinesterase ( AChE) activity and stained to reveal axons by a method adapted from Toop (23). The incubation medium for AChE activity was according to Page (18). Sections were placed in 10 ml stock solution and 20 mg acetylthiocholine adjusted to pH 5.5 with 1 N HCl. (Stock solution: CuSO4 5Hs0, 0.3 g; maleic acid, 1.75 g ; glycine, 0.375 g ; MgClz*6Hz0, 1.0 g ; 1 N NaOH, 30 ml; 20-25s Na2SOI, 170 ml.) Incubation was carried out for 10 min, then the sections were rinsed three times in distilled water. Next the sections were placed 5 to 10 min in fresh 0.5% KaFe (CN)s at room temperature, rinsed three times in distilled water, and fixed 3 to 5 min, again at room temperature, in buffered formol saline, pH 7.0. After washing in several changes of distilled water (15 min), the sections were placed 30 min in fresh 20% aqueous AgNOj with 0.1% CuS04*5H20 at 37°C. Sections were subsequently rinsed in distilled water and developed 10 s in 1 g quinol and 5 g NaZSOA in 100 ml distilled
AchE
FIG. AChE
AND
FIRER
TYPES
IN
DYSTROPIIIC
1. Longitudinal section of sartorius muscle and with terminal axon staining. X 1250.
MPSXE
of a Z-week-old
chick,
reacted
water at room temperature. Sections were then rinsed in distilled fixed 2 min in 5% sodium thiosulfate, rinsed again in distilled dehydrated with alcohol, and mounted.
for
water, water,
RESULTS Figure 1 shows motor end plates from a section of the sartorius muscle from a Z-week-old normal chick reacted for AChE activity alone by a method modified from Toop (23), and Fig. 2 shows a section from the same muscle reacted for AChE activity after preincubation for myofihrillar ATPase activity according to the procedure descril)ed above. The terminal axon is clearly visil)le in Kg. 1, -\vhereas preinculmtion for iuyofihrillar (Fig. 2). It is th(JUght that ,4TPase activity results in no as011 staining
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AL.
FIG. 2. Longitudinal section from the same muscle as in Fig. 1, but incubated for ATPase activity prior to assay for AChE and axon staining. Note the absence of terminal axon and increased intensity of AChE activity. X 1250. some aspect of the myofibrillar ATPase reaction destroys the structural integrity of the terminal axon. In many sections reacted first for ATPase activity and then for AChE activity, a thread-like structure with a light, beaded staining reaction was visible, which appeared to be the remnant of an axon. Preliminary experinlents suggest that it is the interaction with ammonium sulfide in the ATPase reaction which provokes the disappearance of the axon. A second effect of the combined reaction can be seen by comparing the end plates shown in Figs. 1 and 2. Preincubation with myofibrillar ATPase appears to intensify the area of AChE activity. Using Toop’s method for AChE activity, less than half the amount of reaction product was present at the site of end plates when butyrylthiocholine (BuCh) was substituted for AChE. The end product of BuChE
A&E
AiVD
FIBER
TYPES
Ii’G
FIG. 3. Longitudinal section from l-week-old and AChE activities, but with butyrylthiocholine Note the doughnut-shaped end plates. X 1250.
DYSTROPHIC
sartorius muscle substituted
73
MCSCLE
incubated for ACh
for ATPase as substrate.
activity is associated with the synaptic gutter, but not with the junctional folds (22). Figure 3 demonstrates the end-plate reaction when BuCh was used as substrate and the sections were reacted both for AChE activity and for myofibrillar ATPase activity. The amount of reaction product was considerably diminished when BuChE was the substrate compared to the amount of product when ACh was the substrate, and many end plates appeared to have central areas which were void of reaction product, producing a “doughnut” appearance. No reaction product was visible when both ACh and BuCh were omitted for the assay. Figures 2 and 4 demonstrate the value of using the combined ATPase and AChE reaction on the sanie section. Broth (Y ant1 /I muscle film types are clearly visil)le, along with their motor end plates. Because the sections
74
FIG. 4. Longitudinal myofibrillar ATPase bands on the multiply
ASHMORE
ET
AL.
section from the same muscle as in Figs. and AChE activities. Note the irregular innervated p fibers. X 800.
1 and 2, reacted for spacing of end-plate
were preincubated in an acid medium, the /3 fibers stained darkly, whereas the (Y fibers showed an absence of ATPase activity (2). In Fig. 2 it is also clearly shown that end plates on the (Y fibers are considerably larger
FIG. 5. Longitudinal section from pectoralis muscle of chick, reacted for myofibrillar ATPase and AChE activities. staining of some internally placed nuclei. X 800.
a 3-week-old dystrophic Note the intense nuclear
FIG. 6. Longitudinal section from hereditary muscular dystrophy, reacted Note the AChE reactive sites along of activity were not visible when the The motor end plates appear fragmented.
pectoralis muscle of a J-week-old chick with for myofibrillar XTPase and AChE activities. the sarcolemmae of some fibers. These sites muscle was assayed for AChE activity alone. X 875.
than those on the /3 fibers. Characteristically, there is a transverse band of end plates through each fasciculus, but p fibers have additional end plates outside the band (Fig. 4) (5). 0 n a single p fiber, there may be two or three end plates separated by only a few micrometers with another more than lOO/pm distant (Fig. 4 j (5). During the early stages of the myopathy in chicks with hereditary muscular dystrophy, only LYfibers are obviously affected ( 1). In the present study the first visible abnormality in AChE activity x-as observed at about 10 days of age. At that time a few fibers in the pectoralis muscle could be observed in longitudinal sections to have a few nuclei which exhibited intense nuclear and perinuclear staining. These were con~nonly found in a row near the center of the fiber. Sections reacted with Toop’s method alone failed to reveal these nuclear reactions. Sections cut transversely failed to show any morphologic abnormalities, with the exception of a few fibers which were beginnin g to hypertrophy. An occasional nucleus could be seen which had AChE activity. With increasing age the numbers of positive nuclei increased, as did the number of fibers containing positive nuclei (Fig. 5). By 2 to 3 weeks of age fiber hypertrophy was prevalent and AChE activity was also observed along the extrajunctional sarcolemma of several fibers, and by 4 weeks of age it was easily observed in both longitudinal and transverse sections (Figs. 6, 7). By 4 weeks of age some
ASIIMORE
ET
AL.
FIG. 7. Cross section from the same muscle as in Fig. 6, reacted for myofibrillar ATPase and AChE activities. Note the AChE activity on the sarcolemmae of some fibers, as well as in splits of fragmenting fibers. X 1250.
fibers were observed to be splitting and AChE activity was observed to be associated with the invaginating, or newly developing, membrane (Fig. 7). some fibers were observed to have vacuoles which were In cross sections devoid of stainable material, but were surrounded with AChE activity (Fig. 8). By 6 to 8 weeks of age the majority of fibers were clearly abnormal morphologically and many exhibited necrotic “holes” surrounded by AChE activity as well as sarcolemmal AChE activity (Figs. 7, 8). In some cases the entire fiber sarcoplasm had been removed, apparently by autolysis and phayocytosis, leaving only the sarcolemmal sheath and its associated AChE activity (Fig. 9). The majority of dystrophic fibers contained some nuclei which were positively stained for AChE activity, but no fiber was observed where all nuclei were stained. Figure 10 show-s
FIG.
fibrillar on their
8. Cross section from the same muscle as in Figs. 6 and 7, reacted for myoATPase and AChE activities. Note the necrotic vacuoles with AChE activity peripheries. X 1’250.
a remarkable demonstration of a dystrophic muscle fiber cut longitudinally through a segment which apparently comtains a necrotic hole that has opened to the cell surface and is lined with AChE activity. The myofibrillar material in the interior appears to be dissolved. In the sartorius muscle of dystrophic chicks, (Y fibers exhibited abnormalities in AChE activity similar to those described above for the pectoralis muscle. However, in these muscles the onset was obviously slower, with only a small percentage of the fibers showing nuclear or sarcolemmal AChE activity by 6 weeks of age. This muscle has a high percentage of p fibers and aerobic (Y fibers, both of which are relatively resistant to the disease process (2). The posterior latissimus dorsi of dystrophic chicks exhibited similar ahnormalities in AChE activity to the pectoralis muscle.
78
ASHMORE
ET
AL.
FIG. 9. Cross section from pectoralis muscle of a 6-week-old dystrophic chick, reacted for myofibrillar ATPase and AChE activities. Note the nuclear AChE reaction in some fibers and the single fiber where the sarcoplasm has been entirely removed by necrosis, leaving only sarcolemma and sarcolemmal AChE activity. X 1250.
The posterior l.atissimus dorsi is composed entirely of (Y fibers, as is the pectoralis. Although no measurements of end plates were made in this study, the only apparent abnormality was that many of the end plates on dystrophic fibers appeared to be fragmented. However, this was not seen prior to 6 weeks of age, and in longitudinal sections it was difficult to ascertain whether or not the fibers with fragmented end plates were also fibers which had undergone other substantial morphologic alterations. DISCUSSION The data presented here show that cytochemical display of myofibrillar ATPase activity and AChE activity in the same sections of muscle tissue
FIG. 10. Longitudinal section from pectoralis muscle of a 6-week-old dystrophic chick, reacted for myofibrillar ATPase and AChE activities. The fiber has been cut through the necrotic vacuole, which is lined with AChE activity. Internally, the vacuole is devoid of cross-striated material. X 1250.
will provide valuable information which is difficult to obtain by performing these assays separately. Because of the thickness of the muscle fibers and associated organelles and because of contraction artifacts, it is extremely difficult to locate the same fibers in serially cut longitudinal frozen sections. In order to do so, the sections have to be cut so thin that enzyme reactions visible by light microscopy are very difficult to obtain. Comparison of Figs. 1 and 2 demonstrates significant differences in the reaction for Ache activity visualized by the two cytochemical techniques. The reaction around the end plate when the i\ChE reaction alone is used is more diffuse and less intense than that observed with the combined AChE and myofibrillar ATPase procedure. The ability to observe early increases in
80
ASHMORE
ET
AL.
some nuclear and sarcolemmal reactions with the combined assay procedure which are not visible when the AChE assay is used alone also points to the value of the combined procedure. It seems possible th.at dispersion by solution when the AChE assay alone is used may in some cases provide a local concentration of enzyme which is insufficient to provide adequate reaction product to visualize cytochemically. The observation of “doughnut”-shape end plates when BuChE is used as substrate in the combined assay procedure further supports this contention. Far less reaction product is seen with this substrate when the AChE assay alone is performed compared to when the combination procedure is used. Figure 4 shows that p fibers are multiply innervated. It is clear from a previous study that all or nearly all p fibers in mixed fiber muscles of the chick are multiply innervated (5). Several studies have indicated that AChE activity in dystrophic chick muscle is not normal (12, 14, 19, 25-27). The results reported here provide additional evidence that the regulation of AChE activity in dystrophic chick muscle fibers is abnormal. The sequence of events visualized in this study by cytochemistry is that abnormally high AChE activity originates at the nucleus and subsequently appears on the sarcolemma, along fiber splits, and surrounding necrotic vacuoles. It does not appear to originate from the end-plate region because it is often visible along segments of the fiber far removed from the end plate. Conversely, the sarcolemma adjacent to the end-plate AChE was frequently devoid of enzyme. Necrotic vacuoles were usually, but not invariably, lined with AChE. Patterson and Wilson (19) found abnormally high AChE activity only around the neuromuscular junctions of G-week-old dystrophic chick muscle fibers. Using the Ache staining method alone, we were rarely able to see the intense nuclear reaction or sarcolemmal reaction that became apparent in other areas of dystrophic fibers when the combined AChE and myofibrillar ATPase procedure was used. Wake (24) recently showed that AChE granules are synthesized in the sarcoplasm and transported onto the membrane surface. Fambrough et al. ( 11) reported that acetylcholine receptor molecules are also transported from the interior of the myofiber to the sarcolemma, and it was suggested that the AChE and acetylcholine receptor molecules are transported together in granular form to the site of the myoneural junction (24). The results reported here suggest that AChE synthesis is greatly accelerated early in the dystrophic process and enzyme is moved from the nucleus and sarcoplasm to any surface available in the cell. However, at this time we do not favor the interpretation that abnormal regulation of AChE is the primary event in dystrophy of the chicken. The onset of high AChE activity appears coincidentally with many other morphologic and biochemical changes. Fibers begin to hypertrophy, and most chicks lose
AChE
AND
FIBER
TYPES
IN
DYSTROPIIIC
MUSCLE
Xl
their functional ability to right themselves \~.hen placed on their backs. Glycogen phosphorylase activity begins to increase above normal chick muscle (3), cathepsins ,4 and B are increased (13)) concentrations of free taurine are more than two times higher than in normal muscle (21), and mitochondrial enzymes are also more than double those of normal muscle ( 1). It is our view that the synthesis of many (perhaps all) muscle proteins is greatly accelerated. The muscle is ultimately unable to cope with this failure of regulation and degenerative events ensue. REFERENCES 1. ASHMORE, C. R. 1970. Some aspects of oxidative metabolism in muscle of chickens with hereditary muscular dystrophy, Page 225 in M. SABOURDY, Ed., Lcs L’,lh~alc. Editions due Centre National de la Mutants Putllologiqruz Cluz Recherche Scientifique, Paris. 2. ASHMORE, C. R., AND L. DOERR. 1971. Postnatal development of fiber types in normal and dystrophic muscle of the chick. Exp. Ncwol. 30: 431-446. 3. ASHMORE, C. R., AND L. D~ERR. 1971. Phosphorylase in skeletal muscle of normal and selected lines of dystrophic chicks. Proc. Sot. Exj. Biol. Med. 137: 1066-1068. 4. ASHMORE, C. R., AND L. DOERR. 1971. Comparative aspects of muscle fiber types in different species. Exp. Ncwoi. 31 : 408-418. 5. ASHMORE, C. R., T. KIKUCHI, AND L. DOERR. Innervation patterns of different fiber types of chick muscle. Erp. Ncwol. 58: 272-284. 6. BECKETT, E. B., AND G. H. BOURNE. 1957. Cholinesterase and abnormal human muscle. J. Ncurol. Nmroswg, Psychiat. 20 : 191-197. 7. BROOKE, M. H., AND Ii. K. KAISER. 1974. The use and abuse of muscle histochemistry. AII~Z. N.Y. Acnd. Sci. 28: 121-144. 8. COERS, C., AND N. TELEMAN-TOPET. 1977. Morphological changes of motor units in Duchennes muscular dystrophy. .4urh. Nczlrol. 34 : 396-402. 9. EL-BADAWI, A., AXD E. A. SCHENK. 1967. Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholesterase and norepinephrine in cryostat sections. J. Histoclmrz. Cytochcm. 15 : 580-588. 10. ENGEL, W. K. 1974. Fiber type nomenclature of human skeletal muscle for histochemical purposes. Ncwology 31: 344-348. 11. FAMBROUGH, D., H. C. HARTZELL, J. E. ROSEI, AND A. K. RITCIIIE. 1974. Receptor molecules of developing muscle. AUG. Nrzw Yovk .Icad. Sci. 228: 47-61. 12. HARMAN, P. J., J. P. TASSONI, R. L. CURTIS, AKD M. B. HOLLINGSHEAD. 1963. Pages 407-456 in Musczrlnr Dystrophy in Marz. and Animals. Hafner, Xew York. 13. IODICE, A. A., J. CHIN, S. PERKER, AND I. M. WEINSTOCK. 1972. Cathepsins A, B, C, D, and autolysis during development of breast muscle of normal and dystrophic chickens. .4~clz. Biochcm. BiobkJls. 152 : 166-174. 14. JEDRZEJCZYR, J. J., T. WEICKOWSKI, T. RYMASZEWSKA, AND E. A. BARNARD. 1973. Dystrophic chicken muscle--altered synaptic acetylcholinesterase. Science 180 : 406-408. 15. KARNOVSKY, M. J. 1964. The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. /. Ccl1 Biol. 23 : 217-232. 16. KOELLE, G. B., AND J. J. FRIEDENWALD. 1949. A histochemical method for localizing cholinesterase activity. Proc. Sot. Exp. Biol. Med. 70 : 617-622.
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17. LAW, P. K., AND H. L. ATWOOD. 1974. Does axonal sprouting occur in dystrophic mouse muscles? Experientia 30 : 15.5-156. 18. PAGE, K. M. 1971. Histological methods for peripheral nerves. Part II. Med. Lab.
Tech~~ol.
28:
44-48.
19. PATTERSON, G. T., AND B. W. WILSON. 1976. Distribution of acetylcholinesterase activity in normal, dystrophilc and denervated muscles of the chicken. Exp. Neural. 52: 250-262. 20. PEARSE, A. G. E. 1960. Page 877 in Histochewistry, Theoretical and Applied. J. A. Churchill, London. 21. PETERSON, B. W., A. L. LILYBLADE, AND J. LYON. 1963. Serine ethamolaminephosphate, taurine, and free amino acids of muscle in hereditary muscular dystrophy of the chicken. Proc. Sot. Exp. Biol. Med. 113: 798-802. 22. TENNYSON, V. M., A. MIRANDA, AND L. T. KREMZNER. 1975. Electron-microscopic, cytochemical, and biochemical studies of acetylcholinesterase and butyrylcholinesterase activity in muscle of normal and dystrophic mice. J. Neztrol. Sci. 25 : 309-332. 23. TOOP, J. 1976. A rapid method for demonstration of skeletal muscle motor innervation in frozen sections. Stain Tccl~nol. 51 : 1-8. 24. WAKE, K. 1976. Formation of myoneural and myotendinous junctions in the chick embryo. Cell Tissue Res. 173 : 383-400. 25. WILSON, B. W., M. A. KAPLAN, W. C. MERHOFF, AND S. S. MORI. 1970. Innervation and regulation of acetylcholinesterase activity during the development of normal and dystrophic chick muscle. J. Exj. Zool. 174 : 39-54. 26. WILSON, B. W., M. A. MONTGOMERY, AND R. V. ASMUNDSON. 1968. Cholinesterase activity and inherited muscular dystrophy of the chicken. Proc. Sot. Exp. Biol. 129 : 199-206. 27. WILSON, B. W., R. G. TAYLOR, W. M. FOWLER, G. T. PATTERSON, P. A. MEBERG, S. G. LINILHARD, T. A. LINKHART, AND M. D. FRY. 1975. Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles. J. Nezwol. Sci. 26 : 133-146.
NEUROLOGY
Simultaneous
60,68-82
(1978)
Cytochemical
Types
Demonstration
and Acetylcholinesterase of Dystrophic
C. R. ASHMORE, Laboratory California,
in Muscle
Fiber
Fibers
Chickens
P. VIGNERON,
L. MARGER,
of Muscle Biology, Department of Animal Davis, California 95616, and INRA, Statiorz ENSA, 34060 Montpellier-Cedex, Received
of Muscle
November
AND L. DOERR
Scieme, University of de Physiologic Anirnale,
Fraace
29,1977
A cytochemical procedure is described which allows the simultaneous observation of different types of muscle fibers and their motor end plates. The procedure utilizes an assay for myofibrillar adenosine 5’-triphosphatase (ATPase) activity followed by an assay for acetylcholinesterase (AChE) activity. This combined assay eliminates the necessity for using serial sections for observation of these two parameters. This combined assay increases the apparent AChE activity such that sites of AChE activity are revealed which are not visualized when using the AChE assay alone. In muscles from chicks with hereditary muscular dystrophy, it is shown that initially dystrophic fibers contain nuclei which react strongly for AChE activity. Subsequently many fibers exhibit an intense reaction for AChE activity over a major portion of their cell surface. AChE activity is also found along the splits of fragmenting fibers and on the periphery of necrotic vacuoles.
INTRODUCTION Classification of muscle fiber types is useful for the diagnosis of some types of neuromuscular pathologies, for observing adaptive responses to neuromuscular training, and for following developmental patterns of muscle fiber distributions. Several cytochemical techniques have been described Abbreviations : AChE-acetylcholinesterase ; ATPase-adenosine 5’-triphosphatase ; BuCh ; butyrylthiocholine. 1 This work was supported in part by grants from the Muscular Dystrophy Association of America, U.S. Public Health Science Grant AM 16716, and Delegation G&r&ale a la Recherche Scientifique et Technique Grant 75 7 1641. 68 0014-4886/78/0601-0068$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
AChE
AND
FIBER
TYPES
IN
DYSTROPHIC
MUSCLE
69
which allow the identification of different types of muscle fibers (4, 7, 10). In addition, various histological and cytochemical techniques are available for demonstration of the motor end plates of muscle fibers and the axons which imlervate these end plates (9, 15, 16, 18, 23). These techniques were also successfully applied for studying pathologies of end plates and for calculation of the terminal innervation ratio (6, 8, 22, 26). The purpose of this report is to present a cytochemical procedure for the demonstration of AChE activity in different types of muscle fibers and to examine some results from the application of this procedure to dystrophic chick muscle. In normal adult muscle fibers, AChE activity is confined to the motor end plate and its immediate vicinity and to myotendinous junctions. Several reports showed that this normal pattern of enzyme location is altered in dystrophic muscles. Beckett and Bourne (6) noted higher activity of AChE in the sarcoplasm of muscle fibers from human dystrophic patients. Wilson rt al. (27) observed high sarcoplasmic AChE activity in more than 10% of fibers from patients having a variety of abnormal neuromuscular ailments. Harman et al. (12), Law and Atwood (17), and Tennyson it nl. (22) reported the occurrence of fragmented and shrunken motor end plates in dystrophic mouse muscle. Jedrzejczyk et al. (14) reported decreased AChE activity at the motor end plates of dystrophic mouse muscle. Wilson et al. (25) followed the distribution of AChE isoenzymes in developing muscle fibers of normal and dystrophic chicks. They found that, of three AChE isoenzymes which are present in normal embryo muscle, two disappear shortly after hatching. However, all three forms are found in homogenates of fast-twitch muscles of dystrophic chicks. These embryonic forms of the enzyme are distributed diffusely in the cytoplasm of embryo muscle fibers, but they are confined to the neuromuscular junctional area of dystrophic chick muscle fibers (19). 1Vhereas AChE activity was found to be localized within 50 pm of normal fiber end plates, activity was detectable beyond 1.50pm of dystrophic fiber end plates. Using the cytochemical procedure reported here, we describe additional abnormal cellular localizations of AGE activity in dystrophic chick muscle fibers and their temporal relation to the pathogenesis of the myopathy. For classification of different types of muscle fibers, we use the term a to denote fibers which exhibit a low activity of myofibrillar ATPase and the term p to denote fibers kvhicli exhibit a high activity of myofibrillar ATI’ase activity after preincubation in an acid medium (2 j.
Normal chicks ailtl chicks with lieretlitarx muscular dystrophy from lilies maintai~~ctl at this station \sere usc~l in this study. The snrtorius,
70
ASHMORE
ET
AL,
anterior latissimus dorsi, posterior latissimus dorsi, superficial pectoralis, and m. complexus were examined at hatching and at weekly intervals for 10 weeks. Chicks were killed by exsanguination. Muscle samples were removed, pinned to neoprene boards, and quick-frozen in dry ice and isopentane. Pieces of muscle were cut with an International cryostat, floated on a water solution of 0.1% serum albumin at 4°C picked up on coverslips, and dried at 4°C. This allowed the muscle sections to stick to the coverslips during subsequent incubations for enzyme activities. After drying, the sections were fixed 12 min at 4°C in 0.5 N formic acid adjusted to pH 4.20 with 1 M NaOH. The optimum pH may vary slightly according to species, as well as the thickness of the muscle sections. For the results described below, we found 16 pm to be the optimal thickness of the sections. After fixation, the sections were rinsed twice for 1 min in 0.1 M Tris-HCl and 18 mM CaCla, pH 7.8, and then rinsed again with distilled Hz0 for 2 min. Next the sections were incubated for myofibrillar ATPase by a method adapted from Pearse (20). The medium was 0.1 M sodium barbiturate, 0.18 M CaClz adjusted to pH 9.4 with 0.1 M NaOH, and containing 5 mM ATP. The sections were incubated 10 min at 37°C. Although the incubation time does not allow for completion of the reaction, it is sufficient for identification of the fiber types. Longer periods of incubation may make some fibers too dark for observation of the motor end plates. After incubation for ATPase activity, the sections were rinsed three times in 1% CaClz during 10 min, transferred to 2% CaC13 for 3 min, washed 10 min with distilled water, and developed 2 min in 1% ammonium sulfide. After washing 10 min in running tap water, the sections were incubated for acetylcholinesterase ( AChE) activity and stained to reveal axons by a method adapted from Toop (23). The incubation medium for AChE activity was according to Page (18). Sections were placed in 10 ml stock solution and 20 mg acetylthiocholine adjusted to pH 5.5 with 1 N HCl. (Stock solution: CuSO4 5Hs0, 0.3 g; maleic acid, 1.75 g ; glycine, 0.375 g ; MgClz*6Hz0, 1.0 g ; 1 N NaOH, 30 ml; 20-25s Na2SOI, 170 ml.) Incubation was carried out for 10 min, then the sections were rinsed three times in distilled water. Next the sections were placed 5 to 10 min in fresh 0.5% KaFe (CN)s at room temperature, rinsed three times in distilled water, and fixed 3 to 5 min, again at room temperature, in buffered formol saline, pH 7.0. After washing in several changes of distilled water (15 min), the sections were placed 30 min in fresh 20% aqueous AgNOj with 0.1% CuS04*5H20 at 37°C. Sections were subsequently rinsed in distilled water and developed 10 s in 1 g quinol and 5 g NaZSOA in 100 ml distilled
AchE
FIG. AChE
AND
FIRER
TYPES
IN
DYSTROPIIIC
1. Longitudinal section of sartorius muscle and with terminal axon staining. X 1250.
MPSXE
of a Z-week-old
chick,
reacted
water at room temperature. Sections were then rinsed in distilled fixed 2 min in 5% sodium thiosulfate, rinsed again in distilled dehydrated with alcohol, and mounted.
for
water, water,
RESULTS Figure 1 shows motor end plates from a section of the sartorius muscle from a Z-week-old normal chick reacted for AChE activity alone by a method modified from Toop (23), and Fig. 2 shows a section from the same muscle reacted for AChE activity after preincubation for myofihrillar ATPase activity according to the procedure descril)ed above. The terminal axon is clearly visil)le in Kg. 1, -\vhereas preinculmtion for iuyofihrillar (Fig. 2). It is th(JUght that ,4TPase activity results in no as011 staining
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FIG. 2. Longitudinal section from the same muscle as in Fig. 1, but incubated for ATPase activity prior to assay for AChE and axon staining. Note the absence of terminal axon and increased intensity of AChE activity. X 1250. some aspect of the myofibrillar ATPase reaction destroys the structural integrity of the terminal axon. In many sections reacted first for ATPase activity and then for AChE activity, a thread-like structure with a light, beaded staining reaction was visible, which appeared to be the remnant of an axon. Preliminary experinlents suggest that it is the interaction with ammonium sulfide in the ATPase reaction which provokes the disappearance of the axon. A second effect of the combined reaction can be seen by comparing the end plates shown in Figs. 1 and 2. Preincubation with myofibrillar ATPase appears to intensify the area of AChE activity. Using Toop’s method for AChE activity, less than half the amount of reaction product was present at the site of end plates when butyrylthiocholine (BuCh) was substituted for AChE. The end product of BuChE
A&E
AiVD
FIBER
TYPES
Ii’G
FIG. 3. Longitudinal section from l-week-old and AChE activities, but with butyrylthiocholine Note the doughnut-shaped end plates. X 1250.
DYSTROPHIC
sartorius muscle substituted
73
MCSCLE
incubated for ACh
for ATPase as substrate.
activity is associated with the synaptic gutter, but not with the junctional folds (22). Figure 3 demonstrates the end-plate reaction when BuCh was used as substrate and the sections were reacted both for AChE activity and for myofibrillar ATPase activity. The amount of reaction product was considerably diminished when BuChE was the substrate compared to the amount of product when ACh was the substrate, and many end plates appeared to have central areas which were void of reaction product, producing a “doughnut” appearance. No reaction product was visible when both ACh and BuCh were omitted for the assay. Figures 2 and 4 demonstrate the value of using the combined ATPase and AChE reaction on the sanie section. Broth (Y ant1 /I muscle film types are clearly visil)le, along with their motor end plates. Because the sections
74
FIG. 4. Longitudinal myofibrillar ATPase bands on the multiply
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ET
AL.
section from the same muscle as in Figs. and AChE activities. Note the irregular innervated p fibers. X 800.
1 and 2, reacted for spacing of end-plate
were preincubated in an acid medium, the /3 fibers stained darkly, whereas the (Y fibers showed an absence of ATPase activity (2). In Fig. 2 it is also clearly shown that end plates on the (Y fibers are considerably larger
FIG. 5. Longitudinal section from pectoralis muscle of chick, reacted for myofibrillar ATPase and AChE activities. staining of some internally placed nuclei. X 800.
a 3-week-old dystrophic Note the intense nuclear
FIG. 6. Longitudinal section from hereditary muscular dystrophy, reacted Note the AChE reactive sites along of activity were not visible when the The motor end plates appear fragmented.
pectoralis muscle of a J-week-old chick with for myofibrillar XTPase and AChE activities. the sarcolemmae of some fibers. These sites muscle was assayed for AChE activity alone. X 875.
than those on the /3 fibers. Characteristically, there is a transverse band of end plates through each fasciculus, but p fibers have additional end plates outside the band (Fig. 4) (5). 0 n a single p fiber, there may be two or three end plates separated by only a few micrometers with another more than lOO/pm distant (Fig. 4 j (5). During the early stages of the myopathy in chicks with hereditary muscular dystrophy, only LYfibers are obviously affected ( 1). In the present study the first visible abnormality in AChE activity x-as observed at about 10 days of age. At that time a few fibers in the pectoralis muscle could be observed in longitudinal sections to have a few nuclei which exhibited intense nuclear and perinuclear staining. These were con~nonly found in a row near the center of the fiber. Sections reacted with Toop’s method alone failed to reveal these nuclear reactions. Sections cut transversely failed to show any morphologic abnormalities, with the exception of a few fibers which were beginnin g to hypertrophy. An occasional nucleus could be seen which had AChE activity. With increasing age the numbers of positive nuclei increased, as did the number of fibers containing positive nuclei (Fig. 5). By 2 to 3 weeks of age fiber hypertrophy was prevalent and AChE activity was also observed along the extrajunctional sarcolemma of several fibers, and by 4 weeks of age it was easily observed in both longitudinal and transverse sections (Figs. 6, 7). By 4 weeks of age some
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ET
AL.
FIG. 7. Cross section from the same muscle as in Fig. 6, reacted for myofibrillar ATPase and AChE activities. Note the AChE activity on the sarcolemmae of some fibers, as well as in splits of fragmenting fibers. X 1250.
fibers were observed to be splitting and AChE activity was observed to be associated with the invaginating, or newly developing, membrane (Fig. 7). some fibers were observed to have vacuoles which were In cross sections devoid of stainable material, but were surrounded with AChE activity (Fig. 8). By 6 to 8 weeks of age the majority of fibers were clearly abnormal morphologically and many exhibited necrotic “holes” surrounded by AChE activity as well as sarcolemmal AChE activity (Figs. 7, 8). In some cases the entire fiber sarcoplasm had been removed, apparently by autolysis and phayocytosis, leaving only the sarcolemmal sheath and its associated AChE activity (Fig. 9). The majority of dystrophic fibers contained some nuclei which were positively stained for AChE activity, but no fiber was observed where all nuclei were stained. Figure 10 show-s
FIG.
fibrillar on their
8. Cross section from the same muscle as in Figs. 6 and 7, reacted for myoATPase and AChE activities. Note the necrotic vacuoles with AChE activity peripheries. X 1’250.
a remarkable demonstration of a dystrophic muscle fiber cut longitudinally through a segment which apparently comtains a necrotic hole that has opened to the cell surface and is lined with AChE activity. The myofibrillar material in the interior appears to be dissolved. In the sartorius muscle of dystrophic chicks, (Y fibers exhibited abnormalities in AChE activity similar to those described above for the pectoralis muscle. However, in these muscles the onset was obviously slower, with only a small percentage of the fibers showing nuclear or sarcolemmal AChE activity by 6 weeks of age. This muscle has a high percentage of p fibers and aerobic (Y fibers, both of which are relatively resistant to the disease process (2). The posterior latissimus dorsi of dystrophic chicks exhibited similar ahnormalities in AChE activity to the pectoralis muscle.
78
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FIG. 9. Cross section from pectoralis muscle of a 6-week-old dystrophic chick, reacted for myofibrillar ATPase and AChE activities. Note the nuclear AChE reaction in some fibers and the single fiber where the sarcoplasm has been entirely removed by necrosis, leaving only sarcolemma and sarcolemmal AChE activity. X 1250.
The posterior l.atissimus dorsi is composed entirely of (Y fibers, as is the pectoralis. Although no measurements of end plates were made in this study, the only apparent abnormality was that many of the end plates on dystrophic fibers appeared to be fragmented. However, this was not seen prior to 6 weeks of age, and in longitudinal sections it was difficult to ascertain whether or not the fibers with fragmented end plates were also fibers which had undergone other substantial morphologic alterations. DISCUSSION The data presented here show that cytochemical display of myofibrillar ATPase activity and AChE activity in the same sections of muscle tissue
FIG. 10. Longitudinal section from pectoralis muscle of a 6-week-old dystrophic chick, reacted for myofibrillar ATPase and AChE activities. The fiber has been cut through the necrotic vacuole, which is lined with AChE activity. Internally, the vacuole is devoid of cross-striated material. X 1250.
will provide valuable information which is difficult to obtain by performing these assays separately. Because of the thickness of the muscle fibers and associated organelles and because of contraction artifacts, it is extremely difficult to locate the same fibers in serially cut longitudinal frozen sections. In order to do so, the sections have to be cut so thin that enzyme reactions visible by light microscopy are very difficult to obtain. Comparison of Figs. 1 and 2 demonstrates significant differences in the reaction for Ache activity visualized by the two cytochemical techniques. The reaction around the end plate when the i\ChE reaction alone is used is more diffuse and less intense than that observed with the combined AChE and myofibrillar ATPase procedure. The ability to observe early increases in
80
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some nuclear and sarcolemmal reactions with the combined assay procedure which are not visible when the AChE assay is used alone also points to the value of the combined procedure. It seems possible th.at dispersion by solution when the AChE assay alone is used may in some cases provide a local concentration of enzyme which is insufficient to provide adequate reaction product to visualize cytochemically. The observation of “doughnut”-shape end plates when BuChE is used as substrate in the combined assay procedure further supports this contention. Far less reaction product is seen with this substrate when the AChE assay alone is performed compared to when the combination procedure is used. Figure 4 shows that p fibers are multiply innervated. It is clear from a previous study that all or nearly all p fibers in mixed fiber muscles of the chick are multiply innervated (5). Several studies have indicated that AChE activity in dystrophic chick muscle is not normal (12, 14, 19, 25-27). The results reported here provide additional evidence that the regulation of AChE activity in dystrophic chick muscle fibers is abnormal. The sequence of events visualized in this study by cytochemistry is that abnormally high AChE activity originates at the nucleus and subsequently appears on the sarcolemma, along fiber splits, and surrounding necrotic vacuoles. It does not appear to originate from the end-plate region because it is often visible along segments of the fiber far removed from the end plate. Conversely, the sarcolemma adjacent to the end-plate AChE was frequently devoid of enzyme. Necrotic vacuoles were usually, but not invariably, lined with AChE. Patterson and Wilson (19) found abnormally high AChE activity only around the neuromuscular junctions of G-week-old dystrophic chick muscle fibers. Using the Ache staining method alone, we were rarely able to see the intense nuclear reaction or sarcolemmal reaction that became apparent in other areas of dystrophic fibers when the combined AChE and myofibrillar ATPase procedure was used. Wake (24) recently showed that AChE granules are synthesized in the sarcoplasm and transported onto the membrane surface. Fambrough et al. ( 11) reported that acetylcholine receptor molecules are also transported from the interior of the myofiber to the sarcolemma, and it was suggested that the AChE and acetylcholine receptor molecules are transported together in granular form to the site of the myoneural junction (24). The results reported here suggest that AChE synthesis is greatly accelerated early in the dystrophic process and enzyme is moved from the nucleus and sarcoplasm to any surface available in the cell. However, at this time we do not favor the interpretation that abnormal regulation of AChE is the primary event in dystrophy of the chicken. The onset of high AChE activity appears coincidentally with many other morphologic and biochemical changes. Fibers begin to hypertrophy, and most chicks lose
AChE
AND
FIBER
TYPES
IN
DYSTROPIIIC
MUSCLE
Xl
their functional ability to right themselves \~.hen placed on their backs. Glycogen phosphorylase activity begins to increase above normal chick muscle (3), cathepsins ,4 and B are increased (13)) concentrations of free taurine are more than two times higher than in normal muscle (21), and mitochondrial enzymes are also more than double those of normal muscle ( 1). It is our view that the synthesis of many (perhaps all) muscle proteins is greatly accelerated. The muscle is ultimately unable to cope with this failure of regulation and degenerative events ensue. REFERENCES 1. ASHMORE, C. R. 1970. Some aspects of oxidative metabolism in muscle of chickens with hereditary muscular dystrophy, Page 225 in M. SABOURDY, Ed., Lcs L’,lh~alc. Editions due Centre National de la Mutants Putllologiqruz Cluz Recherche Scientifique, Paris. 2. ASHMORE, C. R., AND L. DOERR. 1971. Postnatal development of fiber types in normal and dystrophic muscle of the chick. Exp. Ncwol. 30: 431-446. 3. ASHMORE, C. R., AND L. D~ERR. 1971. Phosphorylase in skeletal muscle of normal and selected lines of dystrophic chicks. Proc. Sot. Exj. Biol. Med. 137: 1066-1068. 4. ASHMORE, C. R., AND L. DOERR. 1971. Comparative aspects of muscle fiber types in different species. Exp. Ncwoi. 31 : 408-418. 5. ASHMORE, C. R., T. KIKUCHI, AND L. DOERR. Innervation patterns of different fiber types of chick muscle. Erp. Ncwol. 58: 272-284. 6. BECKETT, E. B., AND G. H. BOURNE. 1957. Cholinesterase and abnormal human muscle. J. Ncurol. Nmroswg, Psychiat. 20 : 191-197. 7. BROOKE, M. H., AND Ii. K. KAISER. 1974. The use and abuse of muscle histochemistry. AII~Z. N.Y. Acnd. Sci. 28: 121-144. 8. COERS, C., AND N. TELEMAN-TOPET. 1977. Morphological changes of motor units in Duchennes muscular dystrophy. .4urh. Nczlrol. 34 : 396-402. 9. EL-BADAWI, A., AXD E. A. SCHENK. 1967. Histochemical methods for separate, consecutive and simultaneous demonstration of acetylcholesterase and norepinephrine in cryostat sections. J. Histoclmrz. Cytochcm. 15 : 580-588. 10. ENGEL, W. K. 1974. Fiber type nomenclature of human skeletal muscle for histochemical purposes. Ncwology 31: 344-348. 11. FAMBROUGH, D., H. C. HARTZELL, J. E. ROSEI, AND A. K. RITCIIIE. 1974. Receptor molecules of developing muscle. AUG. Nrzw Yovk .Icad. Sci. 228: 47-61. 12. HARMAN, P. J., J. P. TASSONI, R. L. CURTIS, AKD M. B. HOLLINGSHEAD. 1963. Pages 407-456 in Musczrlnr Dystrophy in Marz. and Animals. Hafner, Xew York. 13. IODICE, A. A., J. CHIN, S. PERKER, AND I. M. WEINSTOCK. 1972. Cathepsins A, B, C, D, and autolysis during development of breast muscle of normal and dystrophic chickens. .4~clz. Biochcm. BiobkJls. 152 : 166-174. 14. JEDRZEJCZYR, J. J., T. WEICKOWSKI, T. RYMASZEWSKA, AND E. A. BARNARD. 1973. Dystrophic chicken muscle--altered synaptic acetylcholinesterase. Science 180 : 406-408. 15. KARNOVSKY, M. J. 1964. The localization of cholinesterase activity in rat cardiac muscle by electron microscopy. /. Ccl1 Biol. 23 : 217-232. 16. KOELLE, G. B., AND J. J. FRIEDENWALD. 1949. A histochemical method for localizing cholinesterase activity. Proc. Sot. Exp. Biol. Med. 70 : 617-622.
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17. LAW, P. K., AND H. L. ATWOOD. 1974. Does axonal sprouting occur in dystrophic mouse muscles? Experientia 30 : 15.5-156. 18. PAGE, K. M. 1971. Histological methods for peripheral nerves. Part II. Med. Lab.
Tech~~ol.
28:
44-48.
19. PATTERSON, G. T., AND B. W. WILSON. 1976. Distribution of acetylcholinesterase activity in normal, dystrophilc and denervated muscles of the chicken. Exp. Neural. 52: 250-262. 20. PEARSE, A. G. E. 1960. Page 877 in Histochewistry, Theoretical and Applied. J. A. Churchill, London. 21. PETERSON, B. W., A. L. LILYBLADE, AND J. LYON. 1963. Serine ethamolaminephosphate, taurine, and free amino acids of muscle in hereditary muscular dystrophy of the chicken. Proc. Sot. Exp. Biol. Med. 113: 798-802. 22. TENNYSON, V. M., A. MIRANDA, AND L. T. KREMZNER. 1975. Electron-microscopic, cytochemical, and biochemical studies of acetylcholinesterase and butyrylcholinesterase activity in muscle of normal and dystrophic mice. J. Neztrol. Sci. 25 : 309-332. 23. TOOP, J. 1976. A rapid method for demonstration of skeletal muscle motor innervation in frozen sections. Stain Tccl~nol. 51 : 1-8. 24. WAKE, K. 1976. Formation of myoneural and myotendinous junctions in the chick embryo. Cell Tissue Res. 173 : 383-400. 25. WILSON, B. W., M. A. KAPLAN, W. C. MERHOFF, AND S. S. MORI. 1970. Innervation and regulation of acetylcholinesterase activity during the development of normal and dystrophic chick muscle. J. Exj. Zool. 174 : 39-54. 26. WILSON, B. W., M. A. MONTGOMERY, AND R. V. ASMUNDSON. 1968. Cholinesterase activity and inherited muscular dystrophy of the chicken. Proc. Sot. Exp. Biol. 129 : 199-206. 27. WILSON, B. W., R. G. TAYLOR, W. M. FOWLER, G. T. PATTERSON, P. A. MEBERG, S. G. LINILHARD, T. A. LINKHART, AND M. D. FRY. 1975. Incidence of acetylcholinesterase in the sarcoplasm of human and chicken muscles. J. Nezwol. Sci. 26 : 133-146.