Sodium channel distribution on uninnervated and innervated embryonic skeletal myotubes

Acetylcholine receptor (AChR) and sodium (Na(+)) channel distributions within the membrane of mature vertebrate skeletal muscle fibers maximize the probability of successful neuromuscular transmission and subsequent action potential propagation. AChRs have been studied intensively as a model for understanding the development and regulation of ion channel distribution within the postsynaptic membrane. Na(+) channel distributions have received less attention, although there is evidence that the temporal accumulation of Na(+) channels at developing neuromuscular junctions (NMJs) may differ between species. Even less is known about the development of extrajunctional Na(+) channel distributions. To further our understanding of Na(+) channel distributions within junctional and extrajunctional membranes, we used a novel voltage-clamp method and fluorescent probes to map Na(+) channels on embryonic chick muscle fibers as they developed in vitro and in vivo. Na(+) current densities on uninnervated myotubes were approximately one-tenth the density found within extrajunctional regions of mature fibers, and showed several-fold variations that could not be explained by a random scattering of single channels. Regions of high current density were not correlated with cellular landmarks such as AChR clusters or myonuclei. Under coculture conditions, AChRs rapidly concentrated at developing synapses, while Na(+) channels did not show a significant increase over the 7 day coculture period. In vivo investigations supported a significant temporal separation between Na(+) channel and AChR aggregation at the developing NMJ. These data suggest that extrajunctional Na(+) channels cluster together in a neuronally independent manner and concentrate at the developing avian NMJ much later than AChRs.     

Acetylcholine sensitivity of innervated and noninnervated Xenopus muscle cells in culture

The chemosensitivity of Xenopus muscle cells grown in culture to iontophoretically applied acetylcholine (ACh) in the presence or absence of neurons was examined. Muscle cells grown without nerve cells are sensitive to ACh over their entire surface (2.4 mV/pC) with occasional spots of high chemosensitivity (“hot spots”). In cultures containing neural tube cells, the ACh sensitivity of muscle cells increased by approximately 50% regardless of the presence of nerve contacts or functional synapses. A similar increase in the ACh sensitivity was observed in muscle cells cultured in medium conditioned by neural tube cells. The ACh sensitivity of the extrajunctional region in functionally innervated muscle cells was not different from that of noninnervated cells growing in the same cultures. However, the chemosensitivity at the junctional region was about fivefold higher than that of the extrajunctional area. This increase in junctional chemosensitivity may well account for the increase in miniature endplate potential amplitude which has previously been reported to occur during nerve-induced ACh receptor accumulation.     

Effects of partial denervation on the distribution of acetylcholine receptors and other membrane properties in innervated and paralyzed fibers of rat skeletal muscle

The origin of the membrane changes induced in skeletal muscle by denervation has been investigated by examining partially denervated rat hindlimb muscles rendered inactive for 2-3 days by a chronic conduction block in the sciatic nerve. Extra-junctional sensitivity to acetylcholine and spike resistance to tetrodotoxin developed to the same extent in the denervated and the adjacent innervated but inactive fibres. On the other hand, impulse-blocked fibres of control muscles not containing denervated fibres showed, at this early time, little membrane changes. These results are interpreted as indicating that the response of muscle to denervation is due to the combined action of inactivity and products of nerve degeneration.     

Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.     

Innervation and acetylcholine sensitivity of muscle fibers of the tail of the tadpole Rana temporaria

Changes in the distribution of Ach-sensitivity in two types of muscle fibers in the tail of the tadpoles as well as the innervation pattern of these fibers have been investigated during metamorphosis. It was shown by iontophoretic application of acetylcholine that the entire muscle membrane exhibits Ach-sensitivity only during premetamorphosis. Ultrastructural studies revealed mature nerve-muscle junctions at this stage of development of the tadpoles. At later stages (prometamorphosis), Ach-sensitivity decreased and finally (climax) became restricted by the innervation region. It is suggested that special neurotrophic regulation of extra-junctional Ach-sensitivity takes place in myotomal muscles of the tadpoles.     

The distribution of acetylcholine and cholinesterase in the nervous system and in innervated organs of Octopus dofleini

• 1.1. Various parts of the nervous system and other structures of Octopus dofleini were analyzed for their acetylcholine and cholinesterase content. Acetylcholine was determined by bioassay and paper chromatography; cholinesterase determinations were made by a manometric method.
• 2.2. Both acetylcholine and cholinesterase show systematic differences in concentration. Those of acetylcholine range from 3 to 350 μg/g wet wt, while the cholinesterase quotients (mg acetylcholine hydrolyzed/g of fresh tissue homogenate/hr) range from 0·8 to 99·0. The highest amounts of both agents were found in the cerebral ganglia, notably the optic ganglia. Large amounts also occur in the ganglia of arms, viscera and mantle. There is a positive correlation between acetylcholine content and cholinesterase activity of a given tissue.
• 3.3. Acetylcholine was the only cholinester detected in the chromatographic analyses. The cholinesterase was found to behave like an acetylcholine-esterase; it caused only neglible hydrolysis of butyrylcholine and benzoylcholine and its activity was depressed by high acetylcholine concentrations.
• 4.4. There is no experimental evidence for cholinergic synaptic transmission in cephalopods, but the results make it likely that certain structures of the nervous system contain a preponderance of cholinergic neurons. The extraordinarily high concentrations of acetylcholine found in some of the tissues, together with the correlated occurrence of high cholinesterase activity, raises the question of a metabolic role of acetylcholine.

     

Electrical properties of chick skeletal muscle fibers developing in cell culture

The membrane properties of individual skeletal muscle cells were studied with intracellular microelectrodes as the fibers developed, in vitro, from mononucleated precursor cells. Passive membrane constants were determined from analysis of transmembrane potential responses to pulses of current assuming the myotubes could be represented as sealed, finite cylinders. Resting membrane potentials increased from 10–15 mV in the shortest, youngest myotubes to ca. 60 mV in the longest, most mature fibers. The increase in membrane potential was not associated with a change in membrane resistivity. Action potentials occurred spontaneously in the most mature cells and repetitive spikes could be evoked by depolarizing current pulses. Spikes and twitches could be evoked in young myotubes provided the membrane was first hyperpolarized to 60–70 mV. Apparently the membrane potential is the rate limiting factor in the maturation of excitation-contraction mechanisms.     

Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle

In adult vertebrate skeletal muscle acetylcholine receptors are localized to the neuromuscular junction. Upon denervation, this distribution changes, with new receptors appearing in extrajunctional regions of the muscle fiber. The location of acetylcholine receptors in innervated or denervated muscle may result, in part, from the distribution of their RNAs. This was tested by assaying for receptor RNAs in junctional and extrajunctional regions of innervated and denervated rat soleus muscle using in situ hybridization and RNAase protection assays. These experiments showed alpha, beta, and delta subunit RNAs concentrated beneath the endplates of innervated muscle fibers. Following denervation, there was an unequal distribution of receptor RNAs along the muscle fiber, with highest levels occurring in extrajunctional regions near the endplate. These data are consistent with a nonuniform pattern of gene expression in adult skeletal muscle fibers.     

Variation of mitochondrial volume fraction along multiply innervated fibers in rabbit extraocular muscle

Mitochondrial volume fraction was compared among three regions along the length of six multiply innervated fibers (MIFs) in the orbital surface layer of rabbit superior rectus. These MIFs are of about 5 μm diameter toward the middle of their length, and of about 15 μm diameter toward their proximal and distal ends. The region of highest volume fraction (26%) was located toward the proximal end of their segment of minimal diameter, in apparent association with endplate-like nerve junctions. The region of lowest volume fraction (8%) was located at their distal segment of maximal diameter. The region toward the distal end of their segment of minimal diameter displayed an intermediate volume fraction (15%). These mitochondrial volume fractions were further analyzed in terms of the relative contributions of the I-band, the A-band, and the subsarcolemmal mitochondrial clusters. Comparable changes in mitochondrial content occur in both the I-band and A-band: in the fibers' distal segment of maximal diameter, however, the mitochondrial volume fraction in the A-band (5%) is lower than in the I-band (11%). These modifications of mitochondrial content along the fibers' length occur irrespective of the contributions of the subsarcolemmal mitochondrial clusters.     

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.     

Channel open time of acetylcholine receptors on Xenopus muscle cells in dissociated cell culture

The kinetics of acetylcholine (ACh) receptor channels on cultured myotomal muscle cells from Xenopus embryos were studied by analyzing focally recorded membrane currents. The mean open time for receptor channels on embryonic muscle cells grown in dissociated cell cultures showed a time-dependent decrease similar to that seen in vivo. The changes in power density spectra are consistent with the hypothesis that the decrease results from the appearance of a class of ACh receptor with a short mean channel open time (0.7 msec) and a decrease in the proportion of receptors with a long mean channel open time (3 msec). The addition of dissociated neural tube cells to muscle cell cultures resulted in an unexpected increase in mean channel open time for ACh receptors in both synaptic and nonsynaptic regions. These studies demonstrate that ACh receptor function may be altered in cultured muscle cells.     

Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers

The structure of regions with a high concentration of ACh receptors (clusters) on cultured skeletal muscle myotubes was examined by immunoperoxidase staining of bound alphaBT. The clusters did not appear to differ from the other regions except in their higher concentration of receptor.     

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.     

The effects of modified ringer's solutions on the membrane potentials of innervated and denervated frog sartorius muscle fibers

Comparison has been made between innervated and chronically denervated frog sartorius muscle fibers for resting potentials and a number of features of the action potential. Muscles were obtained from force-fed frogs maintained at room temperature for periods up to one year, and were studied with intracellular microelectrodes. Denervated muscles increased in sensitivity to acetylcholine by 100–400-fold. Studies were made in normal Ringer's solution, and in media in which concentrations of K⁺, Na⁺, Ca⁺⁺, and Cl? were altered. The only significant differences noted between the denervated and the innervated fibers were a reduction in the maximum rate of fall of the action potential (ca. 20%) and an increase in the fall time of the active membrane potential (ca. 25%). These differences were present in normal Ringer's solution and remained when the bathing medium was modified. The resting membrane potential of denervated and innervated muscles varied with log [K⁺]_o in exactly the same manner, and followed the theoretical relation proposed by Hodgkin (Proc. Roy. Soc., B, 148: 1–37, ′58), with the term representing the ratio of the sodium to potassium permeabilities assigned a value of 0.01. The results suggest that (a) the resting sodium and potassium permeabilities are reduced proportionately after denervation, since it is known that denervated frog muscle has a smaller potassium permeability, and (b) the mechanism controlling the increase in potassium conductance during the action potential is less available after denervation. Data indicate that the system controlling the sodium permeability is capable of activation to the same extent as in innervated muscles. Muslces which had been allowed to reinnervate did not show the differences presented by the denervated muscles. Innervated and denervated muscles did not show any significant changes in maximum rates of rise or fall of the action potential, nor of the active membrane potential amplitude over a 30 mV range of resting membrane potentials, indicating that the sodium and potassium permeability systems are fully available in frog muscle at membrane potentials larger than ?80 mV.