Neural influence on the postnatal changes in acetylcholine receptor distribution at nerve-muscle junctions in the mouse

The effect of denervation at different stages of development on the pattern of junctional AChR molecules has been examined in hindlimb muscles of the mouse using fluorescent α-bungarotoxin. Denervation at birth leads in a few days to a marked dispersal of the junctional AChR cluster. The mechanism of dispersal is unknown but appears to be too slow to be explained by free diffusion of individual AChR molecules. Following birth, the morphological stability of the cluster increases so that denervation at 2 weeks of age, when the mature form of the cluster begins to develop, leads to little increase in cluster size. The changes in the pattern of receptors seen when the nerve is intact are arrested by denervation.     

Acetylcholine receptor kinetics at slow fiber neuromuscular junctions

Acetylcholine receptors in slow fiber neuromuscular junctions of garter snake (sp. Thamnophis) produced synaptic responses that were more complicated than those observed from twitch fibers. Although the slow fiber miniature end plate currents decayed monoexponentially with time, both the current fluctuations spectrum and the voltage jump end plate current required two temporal components for good theoretical fits. This behavior was accurately accounted for by a generalized version of the three-state kinetic model by del Castillo and Katz. Application of the model allowed not only the rate of channel closing to be estimated, but also the rate of channel opening (from the closed state with acetylcholine bound) and the apparent rate of acetylcholine unbinding from the receptor. The results suggest that at the peak of the miniature end plate current local receptor saturation occurs.     

Acetylcholine receptors and nerve terminal distribution at the neuromuscular junction of long-term regenerated muscle fibers

Mdx mice are deficient in dystrophin and show muscle fiber regeneration. Changes in the distribution of acetylcholine receptors have been reported at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration. In this study, we examined whether the distribution of receptors was still altered in long-term, regenerated muscle fibers from C57Bl/10 mice. The left sternomastoid muscle of adult mice was injected with 60 μl of lidocaine hydrochloride to induce muscle degeneration-regeneration. In some mice, the sternomastoid muscle was denervated at the time of lidocaine injection. After 90 and 150 days, the nicotinic acetylcholine receptors were labeled with rhodamine-α-bungarotoxin for confocal microscopy. At both intervals studied, the receptors were distributed in spots. In denervated-regenerated fibers, the receptors were distributed as regular branches similar to denervated muscles without lidocaine treatment. These findings suggested that nerve-dependent mechanisms were involved in the changes in receptor distribution seen in regenerated muscle fibers after lidocaine treatment, and that a similar phenomenon could explain the changes in receptor distribution seen in dystrophic muscle fibers.     

Acetylcholine receptor clustering and nuclear movement in muscle fibers in culture

We have studied the formation of acetylcholine receptor (AChR) clusters and the behavior of myonuclei in rat and chick skeletal muscle cells grown in cell culture. These cells were treated with a factor derived from Torpedo electric extracellular matrix, which causes a large increase in their number of AChR clusters. We found that these clusters were located preferentially in membrane regions above myonuclei. This cluster-nucleus colocalization is explained by our finding that most of the nuclei near clusters remain relatively stationary, while most of those away from clusters are able to translocate throughout the myotube. In some cases, clusters clearly formed first, then nuclei migrated underneath and became immobilized. If clustered AChRs later dispersed, their associated nuclei resumed moving. These results suggest that AChR clustering initiates an extensive cytoskeletal rearrangement that causes the subcluster localization of organelles, potentially providing a stable source of newly synthesized AChRs for insertion into the cluster.     

The clustering of acetylcholine receptors and formation of neuromuscular junctions in regenerating mammalian muscle grafts

The present investigation was undertaken to study the relationship between acetylcholine receptor (AchR) clustering and endplate formation within regenerating skeletal muscle grafts. Silver staining of nerves was combined with rhodamine-alpha-bungarotoxin labeling of AchR clusters in heterotopic grafts of the rat soleus muscle. Two major graft procedures were used: whole muscle grafts and grafts which lacked the zone of original motor endplates (MEP-less grafts). These categories were subdivided into standard grafts, where subsequent innervation was allowed, and noninnervated grafts, which were experimentally deprived of innervation. Grafting brought about the death and removal of muscle fibers, followed by regeneration of myotubes within surviving basal lamina sheaths. A transient population of small extra-junctional AchR clusters spontaneously appears shortly after myotube formation in all four muscle graft types. Early myotubes of whole muscle grafts (both innervated and standard grafts, prior to the time of innervation) also develop presumptive secondary synaptic clefts and large, organized aggregations of AchRs at original synaptic sites. At later times, nerves regenerating into standard whole muscle and MEP-less grafts lead to the formation of numerous ectopic endplates. In whole muscle grafts, endplates may also form at original synaptic sites. Functional graft innervation is achieved in whole muscle and MEP-less grafts as early as 20 days postgrafting. The results of this study support the existence of still-unknown factors associated with the original synaptic site which can direct postsynaptic differentiation independent of innervation. They also demonstrate that functional endplates may form in mammalian muscle grafts at both original synaptic sites and ectopic locations, thus indicating that the zone of original synaptic sites is not necessary for the establishment of numerous functional and morphologically well-differentiated endplates.     

Physiological properties of developing frog tadpole nerve-muscle junctions during repetitive stimulation

Physiological properties of developing neuromuscular junctions were studied in Rana catesbeiana tadpoles at different developmental stages. Developing neurons formed functional synaptic connections with a section of tail muscle implanted in place of the hind limb bud. Low frequency repetitive stimulation at these developing junctions causes a progressive reduction in the Epp amplitude. This reduction is due to a decrease in the quantal content, is reversible, and is more apparent at the most immature developmental stages, becoming less noticeable with further development. These junctions are capable of facilitation at each developmental stage studied when the quantal content is reduced by magnesium. At normal calcium and magnesium concentrations there is little or no facilitation, and often depression of the second Epp occurs.     

The distribution and arrangement of microtubules in mammalian skeletal muscle fibers

The distribution and arrangement of microtubules (MTs) in skeletal muscle fibers of the rat and mouse diaphragm were examined by thin-section electron microscopy. In the central portion of muscle fibers, most MTs ran longitudinally between myofibrils and beneath the sarcolemma, and some MTs ran transversely predominantly at the level of the I band, especially of the A-I junction, thus forming a lattice-like arrangement. At the fiber periphery, MTs were aggregated in the perinuclear region, from which they radiated to take a longitudinal course beneath the sarcolemma and to run in a transverse direction at the I-band level. In the end portion of muscle fibers, MTs were abundant and ran longitudinally into sarcoplasmic processes. MTs were often found to be spatially associated with membranous organelles. Quantitative analyses indicated that the longitudinally running MTs were remarkably more numerous in the peripheral zone of muscle fibers than in the deeper zones. The density of MTs in the central portion was almost the same in both red and white muscle fibers. The density was significantly higher at the fiber ends, though it varied considerably among different fibers. These results are discussed with special reference to the possible involvement of MTs in intracellular transport as well as structural support.     

Acetycholine receptor production and incorporation into membranes of developing muscle fibers

Using electrophysiological and quantitative autoradiographic techniques, we studied the kinetics of acetylcholine (ACh) receptor production and incorporation into membranes of muscle fibers developing in culture. These studies were performed by utilizing ¹²⁵I-labeled α-Bungarotoxin (α-BGT) which binds irreversibly to ACh receptors. α-BGT binding to ACh-sensitive muscle cells in culture correlates well with the level of ACh sensitivity. α-BGT binds to myotubes with two different apparent rates. The slow component of binding is due to the incorporation of new receptors into the membrane at a rate of 90 receptors/μm² per hour. However, the ACh receptor density increases at a rate of only 35 receptors/μm² per hour as the result of a concurrent increase in cell surface area. The α-BGT-receptor complexes turn over slowly and the rate of receptor incorporation is not affected by the presence of α-BGT. Inhibition of protein synthesis with cycloheximide depresses receptor incorporation, the percent inhibition increasing with time in cycloheximide. Overnight treatment in actinomycin D has no effect, but inhibition of ATP synthesis with dinitrophenol and iodoacetate or incubation in the cold inhibits the appearance of new ACh receptors.     

Spatial distribution of acetylcholine receptors at developing chick neuromuscular junctions

Brain Cell Biology - The development of high-density clusters of acetylcholine receptors (AChRs) and the relationship of these clusters to nerve contacts on embryonic chick wing muscle fibres has...     

Distribution and properties of myosin isozymes in developing avian and mammalian skeletal muscle fibers

Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.     

Characterization of acetylcholine receptor subunits in developing and in denervated mammalian muscle

We have used subunit-specific antibodies to identify and to characterize partially the alpha, beta, gamma, and delta subunits of rat skeletal muscle acetylcholine receptor (AChR) on immunoblots. The alpha subunit of rat muscle is a single band of 42 kDa, whereas the beta subunit has an apparent molecular mass of 48 kDa. Both alpha and beta subunits are glycosylated and contain one or more N-linked oligosaccharide chains that are sensitive to endoglycosidase H digestion. The gamma and delta subunits, on the other hand, each appear as doublets on immunoblots, with apparent molecular masses of 52 kDa (gamma), 48 kDa (gamma') and 58 kDa (delta), 53 kDa (delta'), respectively. In each case, the two bands are structurally related and the lower band is probably the partial degradation product of the corresponding upper band. Each of the four gamma and delta polypeptides is N-glycosylated and contains both endoglycosidase H-sensitive and endoglycosidase H-resistant oligosaccharides. When the AChRs purified from embryonic, neonatal, adult, and denervated adult rat muscles were compared, no differences in the mobilities of alpha, beta, or delta subunits on sodium dodecyl sulfate gels were detected among them, either with or without endoglycosidase treatment. The gamma subunits, which were present in AChRs purified from neonatal, embryonic, or denervated rat muscles, were also identical; no gamma subunit was detected, however, in AChRs of normal adult rat muscle.     

Acetylcholinesterase is functional in embryonic rat muscle before its accumulation at the sites of nerve-muscle contact

We have examined the expression, the location, and the physiological activity of acetylcholinesterase (AChE) in developing intercostal muscles in the rat. Although focal accumulations of AChE at developing end plates do not appear until Embryonic Day (ED) 16-17, 16 S AChE is present at ED 14. Experiments with permeable and impermeable inhibitors established that prior to focal accumulation most of the 16 S enzyme is on the surface of muscle fibers, where it constitutes the major species. Intracellular recording from developing muscle fibers showed that as early as ED 14, AChE inhibitors prolonged evoked end-plate potentials. We conclude that prior to its focal accumulation, AChE is present on the surface of muscle fibers and is physiologically active. Histochemical staining of the focally accumulated enzyme demonstrated that the enzyme is concentrated both intracellularly and extracellularly at the sites of developing nerve-muscle contacts.     

Formation of acetylcholine receptor clusters in mammalian sternohyoid muscle regenerating in the absence of nerves

When the sternohyoid muscle from the rat is grafted, the original muscle fibers, including the membranes at the neuromuscular junction, degenerate irreversibly. New muscle fibers regenerate inside of the basal laminae remaining from the original muscle fibers. In this study rhodamine-alpha-bungarotoxin and electron microscopy have been used to demonstrate that acetylcholine receptor (AchR) clusters and synaptic folds are restored to the regenerating myotubes even when innervation to the grafts is prevented. The AchR clusters and synaptic folds colocalized with acetylcholinesterase that persisted at the original synaptic basal lamina. The AchR clusters were not restored if the original innervation band was removed from the muscle at the time of grafting. Lengths of the AchR clusters were measured in animals ranging in weight from 50 to 700 g. The lengths of clusters in the grafts were proportional to the lengths of those in the preoperative controls, suggesting that quantitative morphogenetic information persists through the period of degeneration and regeneration. However, the distribution of the AchRs within the clusters differed slightly from controls. Extrajunctional AchR clusters were present initially, but later disappeared. The sizes of these clusters were unrelated to the sizes of the junctional AchR clusters. This study demonstrates that morphogenetic cues persist within the region of the original motor and plate, possibly associated with the synaptic basal lamina.     

Discrepancies between spontaneous and evoked synaptic potentials at normal, regenerating and botulinum toxin poisoned mammalian neuromuscular junctions

Amplitudes and times to peak of spontaneous miniature endplate potentials (m.e.p.ps) and evoked quantal endplate potentials (e.p.ps) were compared at normal, regenerating and botulinum toxin poisoned neuromuscular junctions of the extensor digitorum longus muscle of the rat. At normal junctions the mean time to peak of m.e.p.ps was longer and more variable than that of similar-sized e.p.ps. At endplates where nerve regeneration was induced by mechanical crushing of the motor nerve the frequency of m.e.p.ps was reduced and their amplitude distribution was broader than normal. The distribution of times to peak of m.e.p.ps was considerably broader than that of quantal e.p.ps recorded at the same endplates. At neuromuscular junctions poisoned with botulinum toxin type A, spontaneous and evoked transmitter release were greatly reduced. The amplitude distribution of m.e.p.ps was wider than that of e.p.ps and the time to peak of e.p.ps was about twice as fast as and less variable than that of m.e.p.ps. To explain the observed differences in time to peak among m.e.p.ps and between m.e.p.ps and quantal e.p.ps we suggest that some m.e.p.ps, but not e.p.ps, originate from transmitter quanta released from sites at a greater distance from postsynaptic receptors or that the release or diffusion process for acetylcholine is more prolonged when producing some of the m.e.p.ps. Such mechanisms produce at normal junctions a small population of m.e.p.ps with prolonged times to peak, at regenerating junctions a greater proportion of such m.e.p.ps and in botulinum toxin poisoning a majority.     

Sodium and potassium channels in regenerating and developing mammalian myelinated nerves

A study has been made of how the normal complementary distribution of sodium and potassium channels in mammalian myelinated nerve fibres (all the sodium channels being in the node with all the potassium channels in the internode) is altered in regenerating and in developing rabbit sciatic nerves. In regenerating nerve fibres, where a marked increase in the number of nodes per unit length occurs, there is a corresponding increase in the sodium channel content (determined from the maximum saturable binding of labelled saxitoxin), consistent with the idea that the number of sodium channels per node remains roughly constant. The use of 4-aminopyridine, which by blocking potassium channels prolongs the action potential, has shown that both in regenerating nerve fibres and in developing nerve fibres potassium currents contribute to the mammalian action potential. In both cases, with the passage of time, the sensitivity to 4-aminopyridine progressively decreases.     

Differential distribution of myosin isoforms among the myofibrils of individual developing muscle fibers

Myosin was localized in situ in the posthatch chicken pectoralis using isoform-specific mAbs. The distribution among myofibrils was demonstrated by immunofluorescence and by immunogold EM. Fluorescein- or rhodamine-labeled antibody (12C5) specific for the head region (S1) of myosin was used as a marker to identify "embryonic" myosin. In longitudinal semithin frozen sections, a minority population of myofibrils stained intensely with 12C5. All other myofibrils in the same cell stained only weakly. Similarly, in Lowicryl-embedded ultrathin sections prepared for EM, a minority population reacted preferentially with gold-labeled 12C5. An antibody (5B4) specific for the rod portion of "neonatal" myosin reacted strongly with nearly all myofibrils, and this was evident by light and electron microscopy. A few of the fibrils that reacted strongly with 12C5 reacted weakly with 5B4. These observations demonstrate that an epitope reacting with 12C5 is more abundant in some myofibrils than in others within the same cell. Three categories of myofibrils can be identified by their relative proportions of embryonic and neonatal forms of myosin: in nearly all fibrils, a neonatal isoform predominates; in a minority population, embryonic and neonatal isoforms are both abundant; and in a few fibrils, an embryonic isoform predominates. It is concluded that there are distinct populations of myofibrils in which specific isoforms are segregated within an individual cell.     

Regulation of myosin expression in developing and regenerating extrafusal and intrafusal muscle fibers with special emphasis on the role of thyroid hormones

Expression of the muscle phenotype is the result of interaction between intrinsic and extrinsic factors, the latter including innervation, mechanical influences and hormonal signals. This minireview summarizes some of the current knowledge regarding the regulation of myosin heavy chain (MHC) isoform transitions during muscle development and regeneration. It describes the role of genetic factors, neural and mechanical influences and it focuses on the contribution of thyroid hormones to the differentiation of muscle fiber phenotypes as shown by the regulation of the expression of MHC isoforms and development of myofibrillar ATPase activity. Finally, it shortly summarizes results regarding the differentiation of MHC isoforms in regenerated muscle fibers of the graft after heterochronous isotransplantation in rats with different thyroid status.