Quantitative ultrastructure of histochemically identified avian skeletal muscle fiber types.код для вставкиСкачать
THE ANATOMICAL RECORD 218~128-135(1987) Quantitative Ultrastructure of Histochemically Identified Avian Skeletal Muscle Fiber Types ROBERT S . HIKIDA Program i n Anatomy, Neurobiology, and Developmental Biology, Department of Zoological and Biomedical Sciences, Ohio University, Athens, OH 45701 ABSTRACT A cryostat retrieval method and combined adenosine triphosphatase (ATPase) and acetylcholinesterase (AChase) method were used to study the ultrastructure and innervation of histochemically identified skeletal muscle fibers in different pigeon muscles. The Z-line structure and volume percentage sarcotubular system were analyzed from different muscles selected for their composition by fiber type. Histochemically, three main fiber types were investigated: slow tonic fibers with a moderate ATPase activity after preincubation at acid or alkaline pH; fast-twitch fibers that had high activity after alkaline treatment and low activity after acid preincubation; and a type considered to be slow-twitch that had low activity after alkaline, and high after acid preincubation. Both the slow tonic and slow-twitch fibers had multiple, en grappe innervation, while the fast-twitch fibers had robust, single end plates. The Z-line of the fast-twitch and slow-twitch fibers had a regular square lattice pattern, in contrast to the granular, nonlattice structure of the slow tonic Z-line. The volume percentage sarcotubular system of the slow-twitch fibers was intermediate between and significantly different from that of the fasttwitch and slow tonic fibers. These correlative analyses suggest that the avian muscles contain not only the fast-twitch and slow tonic fibers previously known, but also a slow-twitch fiber that appears to be intermediate between the tonic and the mammalian slow-twitch fiber type. Based on the abundance of the sarcotubular system, this fiber type appears to be fast-contracting and -relaxing, in spite of being multiply innervated. The characterization of skeletal muscle fiber types is important for clinical, functional, and developmental studies because there may be fiber type specificity of the muscles' reactions to various experimental, ontogenetic, and pathologic changes. It is clear that a variety of skeletal muscle fiber types exist, and that the predominant fiber types in mammals differ in some respects from those of other vertebrates (reviews by Morgan and Proske, 1984; Buchthal and Schmalbruch, 1980). Most mammalian muscles consist of two major types, the fasttwitch and its subtypes, and the slow-twitch fibers (classification systems differ for both of these fiber types). Both fiber types respond to a single stimulus with a n action potential and twitch, but the slow fiber has longer twitch times than the fast fibers. Both are usually innervated by a single neuron that terminates in a single motor end plate. The internal membrane systems have been described both qualitatively and quantitatively, and the sarcotubular system is more abundant in fast fibers (Eisenberg, 1983). In contrast, some mammalian muscles and those of most vertebrates have slow tonic fibers that respond with local depolarizations rather than conducting action potentials, and have polyneuronal innervation, with small-diameter neurons terminating in grape-like clusters. The most common method for differentiating muscle fiber types in any tetrapod vertebrate muscle has been 0 1987 ALAN R. LISS, INC. to use histochemical assays for myosin adenosine triphosphatase (ATPase) activity. The fast and slow fibers have different myosins and their ATPase activities can be differentiated qualitatively by means of differing pH stabilities and labilities. Thus a fast fiber would have a high ATPase activity that is maintained after alkaline preincubation, but is much reduced after acid preincubation. In contrast, slow twitch fibers have a low activity after alkaline preincubation, and high activity after acid preincubation. The slow tonic fiber, at least in the birds, shows a moderate activity after either alkaline or acid preincubation. Several investigators have noted that some avian slow fibers have a histochemical ATPase activity that resembles mammalian slow-twitch fibers; these fibers have a low ATPase activity that reverses after acid preincubation (Barnard et al., 1982; Toutant et al., 1981). The morphological characteristics of these histochemically identified fiber types have not been investigated because of the difficulty in identifying them other than by histochemical or immunocytochemical methods. We have modified a cryostat retrieval method, developed by Eisenberg and Kuda (19771, that allows us to examine the ultrastructure of histochemically identified fibers (Staron et al., 1984).This technique is used here to analyze Received September 16, 1986; accepted January 8, 1987. Technical assistance was provided by Laurel Zirnrner. 129 AVIAN SKELETAL MUSCLE FIBER TYPES quantitatively and qualitatively the fiber types in sev- each fiber was labeled for its ATPase activity after acid eral muscles of the pigeon in order to examine this and alkaline preincubation. Fascicles that were matched difference in fiber types. The sarcotubular systems of in the resin-embedded preparation were then trimmed different fiber types were compared quantitatively. Mi- out of the block and mounted onto a blank gelatin captochondrial volume percentages were not included in sule filled with polymerized resin. The mounted prepathe analysis because both glycolytic and oxidative fibers ration was then trimmed further and a few thick (0.5make up the fast-fiber population, and variability would pm) sections were made. These sections were stained with toluidine blue and after again being matched with be too great for results to be meaningful. the drawings of the histochemical preparations, the fiMATERIALS AND METHODS bers were traced onto transparent plastic sheets a t 200 x Muscles were taken from 20 pentobarbital-anesthe- magnification so that each fiber could be matched actized adult pigeons, Columba livia, Carneau breed, ob- cording to its histochemical characteristics. Thin sections were then cut, mounted onto formvartained from the Palmetto Pigeon Plant, of Sumter, SC, or the Bowman Gray School of Medicine, Winston-Salem, coated slot grids, and initially photographed in the elecNC. The muscles examined were the tonic anterior latis- tron microscope at 150x magnification in order to again simus dorsi (ALD), the mixed posterior belly of the bi- match the fascicles with the tracings on the transparent venter cervicis (BVC), the tonic posterior head of the plastic sheets. Individual identified fibers were photo, prints enserratus superficialis metapatagialis (SSM), and the graphed at 3,000, 7,000 and 2 0 , 0 0 0 ~ and mixed extensor digitorum longus (EDL). Segments of larged 3.5 times were used for analysis. Prints at muscles were positioned for cross-sectioning on wooden intermediate magnifications were used for the sarcotutongue blades in a rnixture of tragacanth gum and Tis- bular system measurements, and the high-magnificasue Tek O.C.T. compound (Miles Laboratories, Naper- tion prints were used to analyze the Z-line structure. ville, IL), then frozen for 20 sec in a slurry of methyl The sarcotubular system volume percentage was meabutane chilled with liquid nitrogen, and warmed to sured according to the methods described by Eisenberg -20°C in a cryostat. The tongue blades were mounted et al. (1974). One to four (usually three) regions were onto the microtome chuck, and the muscles were sec- measured to give the mean value for each fiber. The tioned a t 12-pm thickness and mounted on 22-mm cover measurements were subjected to analysis by one-way glasses for histochemistry. After each 16 sections, two ANOVA and the Student-Newman-Keuls test. thicker sections, approximately 25-35 pm thick, were Innervation brushed directly into ice-cold Karnovsky’s fixative in The method of Ashmore et al. (1978)to simultaneously cacodylate buffer. This process was repeated two to four demonstrate ATPase and acetylcholinesterase (AChase) times per muscle. activities was used for muscles from five birds to deterElectron Microscopy mine the innervation patterns of the fiber types in the The thick sections were fixed for 2-4 h r by floating in muscles studied. For these preparations, the muscles cold fixative (1% glutaraldehyde and 2% paraformalde- were prepared for longitudinal cryostat sections. Sechyde in 0.1 M cacodylate buffer, pH 7.21, then rinsed tions 40 pm thick were made and mounted on 3%EDTAovernight in cacodylate-buffered sucrose. Dehydration coated slides, allowed to dry overnight, and then prethrough a n alcohol series was followed by infiltration in pared for the ATPase and AChase activity as described a propylene oxide-Epon and Araldite mixture. The sec- by Ashmore et al. (1978). tions were flat-embedded in Epon and Araldite. The Technical Considerations blocks were sectioned with diamond knives with a Reichert OMU2 ultramicrotome, mounted on formvar- The sample sizes are not large for this study. Approxicoated slot grids, stained with uranyl acetate and lead mately 20% of the preparations were discarded owing to citrate, and examined with a Zeiss EM109 electron excessive ice crystal formation. Since we work with small pieces that are 25-35 pm thick, many sections were lost microscope. during processing, or became folded, or were cut Histochemistry obliquely, making it impossible to identify individual The 12-pm sections were processed for myofibrillar fibers. Finally, the freezing tended to reduce contrast ATPase activity after preincubation at pH 4.2, 4.6, or greatly, and in spite of longer staining times, thicker 10.4 (Guth and Samaha, 1969; Brooke and Kaiser, 19701, sections, and lower accelerating voltage, contrast was nicotinamide adenine dinucleotide-tetrazolium reduc- often sufficient to obtain data. For the EDL muscle, the tase (Dubowitz and Brooke, 19731, and toluidine blue for percentage of slow fibers was often quite low, so the sample size for these fibers is quite small. Because of all routine histological observations. these problems, only about 25% of the original preparaMatching the Histochemical Preparations With the tions provided data for the final analysis. Thin Sections The flat-embedded sections for electron microscopy were traced with a brightfield microscope equipped with a drawing tube; outlines of fascicles and positions of blood vessels and nerves were marked. This outline was superimposed onto the histochemical preparation until matching fascicles were identified. When regions were matched, fibers in several fascicles of the histochemical preparation were traced a t 200 x magnification, and RESULTS Histochemistry We have found in prior studies that the anterior latissimus dorsi is homogeneous in its slow tonic fiber population in about half the pigeons; in the remainder of the birds a few fast fibers (usually less than 1%)may occur in the muscle (Hikida, 1981).The tonic fibers of the ALD had a moderate ATPase activity after preincubation at 130 R.S. HIKIDA 131 AVIAN SKELETAL MUSCLE FIBER TYPES pH 4.2, 4.6, or 10.4, and the NADH activity was also uniformly moderate. The BVC and EDL had a mosaic distribution of only two fiber types at pH 4.2 preincubation (Fig. l),but a t 4.6 the fast fibers displayed either a low or moderate ATPase activity (Fig. 2). The slow fibers with high ATPase activity at pH 4.2 had a low activity a t pH 10.4 (Fig. 3), and therefore had properties similar to the mammalian slow-twitch fibers. The slow head of the SSM consisted of fibers similar to the ALD, with moderate ATPase activity after all preincubation conditions and moderate NADH activity. Thus the slow fibers of the SSM were slow tonic. The interface between the slow SSM and the adjacent fast head consisted of a small zone with a mosaic pattern of fiber types, but this portion was not used in the study. Innervation By using the method to simultaneously demonstrate the AChase and ATPase activities, the pattern of end plates on histochemically identified muscle fibers could be determined. As has been found previously, the tonic fibers of the ALD and SSM had multiple innervation (Hikida and Bock, 1974), and the pattern consisted of a group of small round terminals (en grappe) situated a t intervals along the fibers. The slow fibers of the BVC and the EDL, identified by the ATPase activity, also had multiple innervation, while the fast fibers of these muscles had only a single site of innervation (Fig. 4). only a square lattice pattern (Fig. 6). The Z-lines of the fast fibers of the EDL and BVC had the normal lattice pattern (Fig. 7). The morphometric analysis revealed that three classes of fibers could be distinguished on the basis of the amount of sarcotubular system (Table 1).The group having the least sarcotubular system was made up of the ALD and posterior SSM, the intermediate class consisted of the slow fibers from the BVC and EDL, and the group with the most abundant sarcotubular system was made up of the BVC and EDL fast fibers. There were significant differences between these groups in terms of the volume percentages of sarcotubular system. The ultrastructure of frozen sections differed slightly from fresh unfrozen sections. The contrast of the sections was markedly reduced, and some components such as the nuclei sometimes had ice crystals within them. The sarcotubular system had no perceptible swelling (Figs. 5-8), and this was verified by comparisons of the volume percentages of these avian fibers with those of various mammals, a s described below. DISCUSSION The method of matching histochemically identified fibers with adjacent frozen sections fixed and embedded for electron microscopy is tedious and produces a rather low yield because of various technical difficulties, as discussed above. We wondered whether the procedure artifactually altered the sarcotubular volume, so we compared our results with those for mammalian fiber Ultrastructure and Morphornetric Analysis types, as compiled by Eisenberg (1983), and they The Z-line structure has been used as a major morpho- matched very well (Table 2). The data for our fiber types logical characteristic for distinguishing between twitch fitted very closely with the values for the guinea pig, and tonic fiber types (Page, 1969; Hikida, 1972; Hikida rat, and mouse. If a n artifactual swelling had occurred, and Bock, 1974). The Z-line of twitch fibers has a regular the values for avian fibers would have been much zig-zag pattern in longitudinal section, and a very regu- greater. Values for the EDL and BVC slow fibers match lar square lattice pattern in cross section. The Z-line of those €or slow-twitch oxidative fibers of mammals, and the tonic fiber has a n amorphous, thick appearance in fast fibers in this study correspond to those of the guinea longitudinal sections of the myofibrils, and a dense gran- pig and mouse. ular ultrastructure containing no lattice pattern in crossThe fibers classified as slow-twitch have the following sectional appearance (Hikida and Bock, 1974). The Z- characteristics: The ATPase activity is identical to mamlines of fast-twitch and slow-twitch fibers have no such malian slow-twitch fiber activity; the innervation patdistinction (Goldstewn et al., 1982), but the Z-line of the tern is arranged in many small clusters; the sarcotubular slow-twitch fiber may be thicker than that of the fast- system volume is intermediate between that of fast and twitch in longitudinal sections. tonic fibers; the Z-line morphology is characteristic of The only fibers that had no Z-line lattice pattern were fast fibers. Until physiological studies are done, we will those of the ALD and SSM (Fig. 5). The slow fibers of assume these to be slow-twitch fibers because most of the EDL and BVC contained fibers having Z-lines with the morphological and histochemical characteristics indicate fast fibers. The existence of different avian slow skeletal muscle Figs.1-3. Biventer cervicis, serial sections, prepared for ATPase ac- fiber types has been proposed previously on the basis of tivity after preincubation at pH 4.2 (Fig. 11,4.6 (Fig. 2), and 10.4 (Fig. the histochemical ATPase activity or immunocytochem3). The same fast fibers are indicated by 0,and the slow fibers by X in istry (Barnard et al., 1982; Pierobon Bormioli et al., the adjacent sections. After incubation at pH 4.2 (Fig. 11, only two fiber types are manifested, the slow-twitch (dark) and the fast-twitch (light 1980; Toutant et al., 1981; Shafiq et al., 19841, but charfibers). After pH 4.6, the fast fibers have either a low or a moderate acterization of these slow fiber types has not yet been ATPase activity (Fig, 2). After incubation at pH 10.4 (Fig. 3), the completed. Barnard et al. (1982) recognized five major activity is the reverse of that after pH 4.2 Figures 1-3 x 130. types of fibers in muscles of chicks up to 3 months of Fig. 4. Longitudinal section of a biventer cervicis muscle prepared age; the three twitch types (including a slow twitch) and for ATPase after acid preincubation and AChase activity. The neuro- two tonic types are classified on the basis of their histomuscular junctions of the slow-twitch fibers (dark fibers), as indicated chemical ATPase activity. Although Barnard et al. (1982) by this AChase activity, consist of a group of small punctate sites (en studied the contractile and biochemical AChase characgrappe endings, indicated by arrow) that occur at intervals aIong the teristics of various muscles, they did not match the fiber. In contrast, the neuromuscular junction of the fast (light) fiber is large and single, with finger-like extensions; the end plate occupies a characteristics with specific fiber types, but only inlarge part of the fiber surface (indicated by arrowheads). x325. ferred these correlations. 132 R.S. HIKIDA Fig. 5. Electron micrograph of the histochemically identified slow tonic fiber of the anterior latissimus dorsi shows that the Z-line consists of a granular dense mass that has no regular structure. ~ 8 0 , 0 0 0 . Fig. 6. Electron micrograph of a histochemically identified slow-twitch fiber of the biventer cervicis having a regular square lattice pattern of the 2-line, and an abundant sarcotubular system. ~ 8 0 , 0 0 0 . 133 AVIAN SKELETAL MUSCLE FIBER TYPES munofluorescence methods indicated that slow fibers, as revealed by the ATPase reaction, showed two levels of positive response, indicating that a n ALD-type and another slow-type muscle fiber existed in the leg muscles (Shafiq et al., 1984). These studies agree with an earlier study by Pierobon Bormioli et al. (19801, who used antibodies against the ALD and guinea pig soleus. In that study, fibers of mixed muscles from various vertebrates, including birds, designated as slow by myosin ATPase activity reacted with anti-ALD antibodies or with antisoleus, but not with both. This suggests that both a slowtwitch (antisoleus) and slow tonic (anti-ALD) population exists in many vertebrate muscles. These different studies point to the existence of at least two slow fiber types in the chicken, but defining the characteristics of these other than by histochemistry or immunocytochemistry has been difficult. Our study shows that the slow fibers, a s identified by ATPase activity, can be characterized ultrastructurally. This correlative analysis reveals that a muscle such as the BVC has a slow fiber type that differs from the ALD slow fiber. The difference exists not only in Z-line morphology, but the amount of sarcotubular system present. Toutant et al. (1981)have shown that the chick's BVC has two slow fiber types, both of which they classify as slow tonic. Barnard et al. (1982) would call one type slow-twitch, based on the histochemistry. Our study indicates that in the pigeon, this is a slow-twitch fiber type, but it also suggests that slow tonic fibers may not occur in the BVC of the pigeon at all. The histochemistry of the ATPase activity shows that slow fibers with a moderate activity TABLE 1. Sarcotubular system volume percentages in after both acid and alkaline preincubation are not preshistochemically identified fibers ent in the pigeon's BVC. That the chicken and pigeon Muscle' Fiber type Mean f SE (Fibers measured) should differ in this manner is not too surprising, since these two species differ in ALD fiber composition (HikSSM Slow tonic 1.04 + 0.45 (38) ALD Slow tonic 1.67 I 0.35 (15) ida and Wang, 1981)and response to tenotomy (Hikida, BVC Slow-twitch 3.21 k 0.83 (32) ns, o,05 1981). EDL Slow-twitch 3.51 f 0.98 (11) The existence of several slow muscle fiber types, espeBVC Fast-twitch 5.00 + 0.90 (38) 1 cially ones having multiple innervation, is significant EDL Fast-twitch 5.22 1.13 (9)' ( ns, P < 0.05 because it would influence specificity of innervation and There were significant differences between each set of values except interpretation of past studies concerned with neuromusfor those bracketed. cular interactions and regeneration of specific muscle 'Abbreviations: SSM, serratus superficialis metapatagialis; ALD, anterior latissimus dorsi; BVC, biventer cervicis; EDL, extensor fiber types. It raises several questions, such as what controls the manifestation of a slow-twitch versus a slow digitorum longus. Toutant et al. (1981) correlated the innervation pattern with the ATPase activity and distinguished two tonic fiber types, one having a high activity a t preincubation pH 4.2, 4.35, and 10.4, while the other had a n intermediate activit,y at pH 4.2 or 4.35, and low activity at 10.4. Both of these tonic fiber types were multiply innervated. The latter tonic type of Toutant et al. (1981) would be considered twitch by some of the other investigators. Suzuki et al. (1982, 1985) have subdivided fibers of young chicks into sjx types, and these appear to encompass all fiber types characterized by other investigators. Their type Isw is variously indicated a s tonic (Toutant et al., 1981; Suzuki et al., 19821, or twitch (Barnard et al., 1982; Phillips and Bennett, 1984). Barnard et al. (1982) and Toutant et al. (1981) indicate that this type is multiply innervated. The results of the present study demonstrate that both slow tonic and slow-twitch fibers occur in the pigeon muscles. Slow fibers of the BVC and EDL have a n acidstable and alkaline-labile ATPase, multiple innervation, a Z-line with a lattice substructure, and a sarcotubular system volume that is intermediate between fast-twitch and slow tonic fibers. The mammaIian slow-twitch fiber has a Z-line with a square lattice structure, similar to other fast-twitch fiber types (Goldstein et al., 1982). The presence of different types of slow fibersin avian muscles has also been demonstrated by reactions of monoclonal antibodies to the chicken ALD myosin. Im- 1 , TABLE 2. Sarcotubular svstem volume Dercentaees of various vertebrates Animal Muscle' Fiber tvwe Cat Ciastroc Fast Slow Slow FG 1.282 0.712 0.622 4.86 Schmalbruch, 1979 FOG 3.54 3.29 5.90 3.12 1.04 1.67 3.21 3.51 5.09 5.22 Eisenberg and Kuda, 1976 Eisenberg et al., 1974 Luff and Atwood, 1971 Guinea Pig Mouse Pigeon Soleus Vastus Soleus EDL Soleus SSM ALD HVC EDL RVC EDL so Fast Slow ST ST so so Fast Fast Volume wercentaee Reference Eisenberg and Kuda, 1975 This study 'Abbreviations: FG, Fast glycolytic; SO, slow twitch oxidative; FOG, fast oxidative glycolytic; ST,slow tonic; EDL, extensor digitorum longus; SSM, serratus superficialis metapatagialis, slow head; ALD, anterior latissimus dorsi; BVC, biventer cervicis; Gastroc, gastrocnemius. 'Sarcoplasmic reticulum only. 134 R.S. HIKIDA AVIAN SKELETAL MUSCLE FIBER TYPES tonic muscle fiber type. It is known that the slow-twitch fiber is maintained and regulated by its neural impulse activity (Pette, 1984; Pette and Vrbova, 1985). Experiments with avian muscles have suggested that the slow tonic fiber type can also be developed under the influence of a tonic neural activity (Renaud et al., 1978; 1983). If both slow tonic and slow-twitch fibers are formed because of a constant tonic activity pattern, then what determines the type to be formed when genes are present for both types, as in the pigeon, and probably other species, including man? The tonic fiber has a multiple innervation, and is also polyneuronally innervated (KuMer and Vaughan Williams, 1953). It responds to indirect stimulation with a local depolarization that increases with longer stimulation, and does not normally propagate a n action potential. The adult mammalian slow-twitch fiber is usually innervated by a single neuron that terminates in a n end plate, and indirect stimulation results in a n action potential and twitch. The innervation of the avian slowtwitch fiber is similar to the tonic fiber with its multiple innervation, and multiple innervation suggests that the avian slow-twitch fiber is also activated by local depolarization. This contradicts its morphology, which suggests it is designed for a rapid activation and relaxation. Therefore this study shows that by using a correlative technique such as this cryostat retrieval method, whereby histochemistry and electron microscopy can be done on the same identified muscle fibers, it is possible to gain much more information about the nature of the muscle fibers and their relationships between various species. This study also indicates that mammals may not be as unique in their muscle fiber type composition a s previous studies might have suggested, as both mammalian and avian muscles have fast-twitch, slow-twitch, and tonic fiber types; however, this avian slow-twitch fiber is intermediate between the slow tonic and the mammalian slow-twitch fiber. 135 Dubowitz, V., and M.H. Brooke (1973) Muscle Biopsy: A Modern Approach. W.B. Saunders, London. Eisenberg, B.R. (1983)Quantitative ultrastructure of mammalian skeletal muscle. In: Handbook of Physiology, Section 10: Skeletal Muscle. L.D. Peachey, ed. Williams and Wilkins, Baltimore, pp. 73112. Eisenberg, B.R., and A.M. Kuda (1975)Stereological analysis of mammalian skeletal muscle. 11. White vastus muscle of the adult guinea pig. J. Ultrastruct. Res., 51t176-187. Eisenberg, B.R., and A.M. Kuda (1976) Discrimination between fiber populations in mammalian skeletal muscle by using ultrastructural parameters. J. Ultrastruct. Res., 54t76-88. Eisenberg, B.R., and A.M. Kuda (1977) Retrieval of cryostat sections for comparison of histochemistry and quantitative electron microscopy in a muscle fiber. J. Histcchem. Cytochem., 251169-1177. Eisenberg, B.R., A.M. Kuda, and J.B. Peter (1974)Stereological analysis of mammalian skeletal muscle. I. Soleus muscle of the adult guinea pig. J. Cell Biol., 60:732-754. Goldstein, M.A., J.P. Schroeter, and R.L. Sass (1982)The Z-band lattice in a slow skeletal muscle. J. Muscle Res. Cell Motil., 3.333-348. Guth, L., and F.J. Samaha (1969) Qualitative differences between actomyosin ATPase of slow and fast mammalian muscle. Exp. Neurol., 25:138-152. Hikida, R.S. (1972)Morphological transformation of slow to fast muscle fibers after tenotomy. Exp. Neurol., 35t265-273. Hikida. R.S. (1981) Tenotomv of the avian anterior latissimus dorsi muscle. 11. Can regeneration from the stump occur in the pigeon? Am. J. Anat., 16Ot409-418. Hikida, R.S., and W.J. Bock (1974) Analysis of fiber types in the pigeon’s metapatagialis muscle. I. Histochemistry, end-plates and ultrastructure. Tissue Cell, 6t411-430. Hikida, R.S., and R.J. Wang (1981) Tenotomy of the avian anterior latissimus dorsi muscle. I. Effect of age on the fiber type transformation and regeneration from the stump in chicks. Am. J. Anat., 160t395-408. Kuffler, S.W., and E.M. Vaughan Williams (1953) Properties of the “slow” skeletal muscle fibres of the frog. J. Physiol. (Lond.), 12lr318-340. Luff, A.R., and H.L. Atwood (1971) Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal muscles of the mouse during postnatal development. J. Cell Biol., 51r369-383. Morgan, D.L., and U. Proske (1984) Vertebrate slow muscle: Its structure, pattern of innervation, and mechanical properties. Physiol. Rev., 64t103-169. Page, S.G. (1969) Structure and some contractile properties of fast and slow muscles of the chicken. J. Physiol. (Lond.),205t131-145. Pette, D. (1984)Activity-inducedfast to slow transitions in mammalian muscle. Med. Sci. Sports Exerc., 16~517-528. ACKNOWLEDGMENTS Pette, D., and G. Vrbova (1985)Neural control of phenotype expression This study was supported in part by grants from the in mammalian muscle fibers. Muscle Nerve, 8t676-689. National Institutes of Health (AM 26992) and the Baker Pierobon Bormioli, S., S. Sartore, M. Vitadello, and S. Schiaffino (1980) “Slow” myosins in vertebrate skeletal muscle. An immunofluoresFund Committee of Ohio University. I wish to thank the cence study. J. Cell Biol., 85t672-681. following persons fix their very capable assistance in PhilIips, W.D., and M.R. Bennett (1984) Differentiation of fiber types in wing muscles during embryonic development: Effect of neural this study: Ms. Natalie Veres, Steve Hikida, and Bruce tube removal. Dev. Biol., 106t457-468. Hather. Renaud, D., G.H. LeDouarin, and A. Khaskiye (1978) Spinal cord stimulation in chick embryos: Effects on development of the posteLITERATURE CITED rior latissimus dorsi muscle and neuromuscular junctions. Exp. Neurol., 6Ot189-200. Ashmore, C.R., P. Vigneron, L. Marger, and L. Doerr (1978) Simultaneous cytochemical demonstration of muscle fiber types and acetyl- Renaud, D., M.-F. Gardahaut, T. Rouaud, and G.H. LeDouarin (1983) cholinesterase in muscle fibers of dystrophic chickens. Exp. Neurol., Influence of chronic spinal cord stimulation upon differentiation of 60t68-82. p muscle fibers in a fast muscle (posterior latissimus dorsi) of the Barnard, E.A., J.M. Lyle::, and J.A. Pizzey (1982)Fibre types in chicken chick embryo. Exp. Neurol., 8Ot157-166. skeletal muscles and their changes in muscular dystrophy. J. Phys Schmalbruch, H. (1979)The membrane systems in different fibre types iol. (Lond.),331t333-:354. of the triceps surae muscle of the cat. Cell Tissue Res., 204t187Brooke, M.H., and K.K. Kaiser (1970) Three “myosin ATPase” sys200. tems: The nature of their pH lability and sulfydryl independence. Shafiq, S.A., T. Shimizu, and D.A. Fischman (1984) Heterogeneity of J. Histochem. Cytochem., 18:670-672. type 1 skeletal muscle fibers revealed by monoclonal antibody to Buchthal, F., and H. Schmalbruch (1980) Motor unit of mammalian slow myosin. Muscle Nerve, 7t380-387. muscle. Physiol. Rev., 6Or90-142. Staron, R.S., R.S. Hikida, F.C. Hagerman, G.A. Dudley, and T.F. Murray (1984) Human skeletal muscle fiber type adaptability to various workloads. J. Histochem. Cytochem., 32:146-152. Fig. 7. A histochemically identified fast-twitch fiber from the biven- Suzuki, A., T. Tsuchiya, and H. Tamate (1982) Histochemical properter cervicis has the Z-line with a square lattice pattern in this electron ties of myofiber types in thigh muscles of the chicken. Acta Histomicrograph. ~ 8 0 , 0 0 0 . chem. Cytochem., 15:362-371. Suzuki, A., T. Tsuchiya, S. Ohwada, and H. Tamate (1985) Distribution Fig. 8. An electron micrograph of a histochemically identified slowof myofiber types in thigh muscles of chickens. J. Morphol., 185:145twitch fiber from the bibenter cervicis shows little damage due to the 154. freezing and preparat0r.y procedures used for this cryostat retrieval Toutant, J.P., T. Rouaud, and G.H. LeDouarin (1981) Histochemical properties of the biventer cervicis muscle of the chick: A relationmethod. The sarcotubular system (in this case the sarcoplasmic reticship between multiple innervation and slow-tonic fibre types. Hisulum) shows little evidence of dilation, and the filament arrangement tochem. J.,13:481-493. is normal. x68,400.