Muscle-specific calpain is localized in regions near motor endplates in differentiating lobster claw muscles

Calpains are Ca2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.     

Quantitative study of motor endplates in muscle fibres dissociated by a simple procedure

Large numbers of single muscle fibres can be obtained reproducibly from glutaraldehyde-fixed skeletal muscle by the method described here. With suitable modifications, one can estimate acetylcholine receptor number (alpha-bungarotoxin binding sites) and endplate area in parallel portions produced from the same muscle sample, so that small differences (e.g. with growth or between muscle types) become detectable. Microdissection further increases the precision of evaluation of junctional, perijunctional and extrajunctional binding sites. Other applications are illustrated.     

NADPH-diaphorase-positive nerve fibers associated with motor endplates in the rat esophagus: new evidence for co-innervation of striated muscle by enteric neurons

NADPH-diaphorase histochemistry was combined with demonstration of acetylcholinesterase and immunocytochemistry for calcitonin gene-related peptide to study esophageal innervation in the rat. Most of the myenteric neurons stained positively for NADPH-diaphorase, as did numerous varicose nerve fibers in the myenteric plexus, among striated muscle fibers, around arterial blood vessels, and in the muscularis mucosae. A majority of motor endplates (as demonstrated by acetylcholinesterase histochemistry or calcitonin gene-related peptide immunocytochemistry) were associated with fine varicose NADPH-diaphorase-positive nerve fibers. Analysis of brainstem nuclei, sensory vagal, spinal, and sympathetic ganglia in normal and neonatally capsaicin-treated rats, and comparison with anterogradely labeled vagal branchiomotor, preganglionic and sensory fibers led to the conclusion that NADPH-diaphorase-positive fibers on motor endplates originate in esophageal myenteric neurons. No association of NADPH-diaphorasepositive nerve fibers with motor endplates was found in other organs containing striated muscle. These results suggest extensive, presumably nitrergic, co-innervation of esophageal striated muscle fibers by enteric neurons. Thus, control of peristalsis in the esophagus of the rat may be more complex than hitherto assumed.     

The three-dimensional structure of motor endplates in different fiber types of rat intercostal muscle

Summary The three-dimensional organization of the motor end plates in the red, white and intermediate striated muscle fibers of the rat intercostal muscle was observed under a field-emission type scanning electron microscope after removal of connective tissue components by HCl hydrolysis.The motor endplate of the white fiber had terminal branches (or axon terminals), which were large, long and thin, and small but numerous nerve swellings (or terminal boutons). The motor endplate of the red fiber had terminal branches, which were small, short and thick, and had large but fewer nerve swellings. The motor endplate of the intermediate fiber was intermediate in size and structure between these two. In detached nerve-ending preparations, primary synaptic grooves with slit-like openings of the junctional folds appeared on the surface of the muscle fibers. The primary synaptic grooves were more developed in the white fiber than in the red fiber, and they were intermediate in the intermediate fiber. The numerical ratio of slit-like openings was 11.83.5 in the red, intermediate and white fiber, respectively.The Schwann cells and their processes were observed on the surface of the motor endplate, with the processes covering the upper orifices of the primary synaptic grooves and sealing the terminal branches. The number of Schwann cells was usually three in the white fiber, two in the intermediate fiber and one in the red fiber.     

Fusion between myoblasts and adult muscle fibers promotes remodeling of fibers into myotubes in vitro

Muscle satellite cells are residual embryonic myoblast precursors responsible for muscle growth and regeneration. In order to examine the role of satellite cells in the initial events of muscle regeneration, we placed individual mature rat muscle fibers in vitro along with their satellite cells. When the satellite cells were allowed to proliferate, they produced populations of myoblasts that fused together to form myotubes on the laminin substrate. These myoblasts and myotubes also fused with the adult fibers. When they did so, the fibers lost their adult morphology, and by 8 days in vitro, essentially all of them were remodeled into structures resembling embryonic myotubes. However, when proliferating satellite cells were eliminated by exposure to cytosine arabinoside (araC), the vast majority of fibers retained their adult shape. Addition of C2C12 cells (a myoblast line derived from adult mouse satellite cells) to araC-treated fiber cultures resulted in their fusion with the rat muscle fibers and restored the ability of the fibers to remodel, whereas addition of either a fibroblast cell line or a transformed, non-fusing variant of C2C12 cells, or addition of conditioned medium from C2C12 cells, failed to do so. These results imply that myoblast fusion is responsible for triggering adult fiber remodeling in vitro.     

Synaptic vesicles and microtubules in frog motor endplates.

Motor endplates of the cutaneous pectoris skeletal muscle of the frog have been examined by electron microscopy using a new technique. This involves pretreatment with an albumin solution, followed by fixation with 4% unbuffered tetroxide. A small proportion of the endplate axonal ramifications show microtubules clothed in synaptic vesicles and focused on the presynaptic membrane, in particular on the active zones. The microtubules run in the presynaptic cytoplasm either parallel to or across the active zones. These two sets of microtubules cross each other at the active zones, which lie opposite the dips in the post-junctional folds. The possibility that the microtubules are involved in the translocation of synaptic vesicles to the active zone is discussed.     

Scanning electron-microscopic study on the three-dimensional structure of motor endplates of the slow (tonic) muscle fibers in the frog,Rana n. nigromaculata

Summary The three-dimensional organization of the motor endplates of the slow fibers of the rectus abdominis muscle in the Japanese meadow frog (Rana nigromaculata nigromaculata Hallowell) is visualized by use of a field-emission scanning electron microscope after removal of connective tissue components by HCl hydrolysis. Clusters of shallow oval depressions 1–3 m in diameter are seen in the postsynaptic membrane at intervals of about 150 m. On the surface of these depressions, a few low bulges of postsynaptic membrane are irregularly arranged. Terminal boutons, 1–3 m in diameter, occur along the length of nerve branches and terminals and fit into the shallow oval depressions of the postsynaptic membrane. The Schwann cells covering the terminal branches exhibit a simpler organization than those in twitch fibers.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Summary Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag₁, bag₂ and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag₁ and bag₂ fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag₁, bag₂ and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

Histochemistry of rat intrafusal muscle fibers and their motor innervation.

Muscle spindles were followed in serial transverse sections of freshly frozen rat soleus muscles. Adenosine triphosphatase (ATPase) histochemical staining reaction was used to identify nuclear bag1, nuclear bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along bag1 and bag2 fibers but not along chain fibers. Bag1 fibers displayed ultrastructural heterogenity when their intra- and extracapsular regions were compared. Simple "diffuse" and more elaborate "plate" motor nerve terminals were demonstrated histochemically along the poles of bag1 and bag2 fibers by staining for cholinesterase. One motor terminal of the "plate" appearance was present on a chain fiber pole. There was no consistent spatial correlation between the intensity of regional ATPase staining along the nuclear bag fibers and the location, number and type of motor endings. Other factors, such as intrafusal fiber sensory innervation and regional differences in active and passive functional recruitment of nuclear bag fibers during muscle activity, may contribute to the ATPase staining variability along the intrafusal fibers.     

Histochemical profiles of cat intrafusal muscle fibers and their motor innervation

Muscle spindles were examined histochemically in serial transverse sections of cat tenuissimus muscles. The myofibrillar adenosine triphosphatase (ATPase) staining reaction was used to identify nuclear bag1, bag2 and nuclear chain intrafusal muscle fibers. Regional differences in ATPase staining occurred along the bag1 and bag2 fibers but not along the chain fibers. All intrafusal fiber types displayed regional variability in staining for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR). Motor nerve terminals were demonstrated along the poles of bag1, bag2 and chain fibers by staining for cholinesterase (ChE). There was no consistent spatial correlation between the intensity of regional ATPase staining along the bag fibers and location, number or type of motor endings. However, most ChE deposits occurred in intrafusal fiber regions that displayed the greatest NADH-TR variability. Some fiber poles or whole intrafusal fibers were devoid of any ChE deposits but their ATPase and NADH-TR content was comparable to that of fibers bearing ChE deposits. The observations suggested that motor nerve fibers per se may not play a major role in determining the histoenzymatic content of intrafusal fibers.     

Demonstration of multiple innervation of skeletal muscle fibers in the rat following localized repetitive freezing of the sciatic nerve]

The medial head of the gastrocnemius muscle of the rat was denervated by three successive freezings of the sciatic nerve at the same focal localization. After the third denervation, the compound potentials obtained during a transient period of time indicated that some muscle fibres are innervated by two or several axonal terminals. It can be concluded from the electrophysiological, histological and cytoenzymological results that this multiple innervation is polyneural and located within a single end-plate.     

Molecular forms of the cholinesterases inside and outside muscle endplates

Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic9 were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.     

Changes in muscle proteins following different neural lesions

The influence of various nerve lesions on the relative concentrations of proteins in the extensor digitorum longus (EDL), plantaris (PLN), and soleus (SOL) muscles from adult female rats was examined by electrophoresis on polyacrylamide gels. After cordotomy, levels of myosin light chains Lcs1 and -2 decreased with concomitant increases in Lcf1, -2, and -3 in SOL but not in EDL and PLN. The relative levels of several other proteins in all three muscle types were changed also. Following section of the sciatic nerve, corticospinal tract and removal of the motor cortex, the relative levels of distinct sets of protein bands in all three muscle types were altered. Only a 14,000-dalton protein band appeared to be affected by all four types of lesions. This experimental evidence supports the view that neural influences upon muscle properties are exceedingly complex. Some of these influences may be exerted via separate, central pathways to the motor neurons.     

Ultrastructural demonstration of acetylcholinesterase activity of motor endplates via osmiophilic diazothioethers

Summary The ultrastructural chemical localization of acetylcholinesterase of motor endplates of rat intercostal muscle has been studied with three new esterase substrates. These substrates, although not specific for acetylcholinesterase, have differential affinities for various types of esterases; two of them (NTA and TAB) are hydrolyzed preferentially by acetyl esterase enzymes, and the third (TPB) is a propionic acid ester and is hydrolyzed preferentially by pseudocholinesterase and other esterases. The end-product of the enzymatic reaction is converted to a diazothioether (droplet form) and upon osmication this is converted to a coordination polymer of osmium which has ideal properties for electron microscopy.Although this study supports previous observations that enzymatic activity can be found primarily on the post-synaptic membranes of the motor endplate, no enzymatic activity was noted on the pre-synaptic membrane, within the synaptic cleft, or on the basement membrane unless incubation was prolonged, resulting in overstaining. Neither was enzyme activity seen on membrane-free ribosomes and the ribosome-studded sarcoplasmic reticulum. Axonal vesicles also failed to exhibit enzymatic activity which had been noted with the method using thiolacetic acid and lead. A correlation of esterase activity with ultrastructural localization, using the substrate TPB, suggests that a buffer zone of nonspecific esterase activity is present beneath the subneural apparatus which limits the aberrant, accidental, or abnormal distribution of acetylcholine within a clearly defined area of sarcoplasm in the vicinity of the motor endplate of the muscle fiber.This investigation was supported by research grants from the National Cancer Institute (CA-2078 and CA-02478) and National Institute of Neurological Diseases and Blindness (NB 04096). Acknowledgement for technical assistence is due Miss Julia Silhan.This investigation was carried out during the tenure of a Public Health Service research career program award NB 5820 from the N.I.N.D.B.     

Roles for ephrins in positionally selective synaptogenesis between motor neurons and muscle fibers

Motor axons form topographic maps on muscles: rostral motor pools innervate rostral muscles, and rostral portions of motor pools innervate rostral fibers within their targets. Here, we implicate A subfamily ephrins in this topographic mapping. First, developing muscles express all five of the ephrin-A genes. Second, rostrally and caudally derived motor axons differ in sensitivity to outgrowth inhibition by ephrin-A5. Third, the topographic map of motor axons on the gluteus muscle is degraded in transgenic mice that overexpress ephrin-A5 in muscles. Fourth, topographic mapping is impaired in muscles of mutant mice lacking ephrin-A2 plus ephrin-A5. Thus, ephrins mediate or modulate positionally selective synapse formation. In addition, the rostrocaudal position of at least one motor pool is altered in ephrin-A5 mutant mice, indicating that ephrins affect nerve-muscle matching by intraspinal as well as intramuscular mechanisms.