Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.     

Innervation and acetylcholine sensitivity of skeletal muscle cells differentiated in vitro from chick embryo

Trypsin-dissociated myoblasts from leg muscle of 12-day chick embryos have been cultured in monolayers. After four days the muscle cultures have been confronted with fragments of the spinal cord of six-day chick embryos. Electrophysiological and morphological analysis demonstrate that characteristic neuromuscular transmission can develop in these cultures. Electrical stimulation of the cord fragment evokes contractions of innervated muscle fibers, from which end plate potentials and miniature end plate potentials with average frequency around one per second or more can be recorded. D-tubocurarine (1 μg/ml) suppresses reversibly these synaptic potentials. Non-innervated muscle fibers are sensitive to acetylcholine over all their surface, while innervated muscle fibers are sensitive at the regions where structures suggestive of motor end plate (“bulb-type”) are found. We can conclude that neuromuscular connections developed in vitro in our experiments are functional in respect of transmission of impulses but also in respect of neurotrophic influences for restriction of chemosensitivity.     

Multimodality in the amplitude distribution of miniature endplate potentials due to the action of spatially discrete areas of transmitter release

Miniature end plate potentials (MEPPs) were simultaneously recorded in frog sartorius muscle by two intracellular microelectrodes. Some isolated groups of points (clouds) were found on the diagram of MEPPs scatter. Several peaks each of which was composed of signals from certain clouds (or from several clouds) on the scatter diagram were found on the histograms of MEPPs amplitude distribution. It is assumed that the clouds on the scatter histogram and the peaks on the histogram of MEPPs amplitudes are formed at the cost of secretion of quantum acetylcholine from spatial separate areas of transmitter release. The data obtained do not correspond with the subquantum hypothesis of transmitter release in neuromuscular junctions.     

Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions

When the nerve to an adult frog sartorius muscle is crushed, and axons are allowed to regenerate, the level of polyneuronal innervation at reinnervated neuromuscular junctions is higher than normal. With time, much of this polyneuronal innervation is reduced by the process of synapse elimination (Werle and Herrera, 1988). Using intracellular recording, we estimated the level of polyneuronal innervation in adult frog (Rana pipiens) sartorius muscles 2 years (range: 1.7-2.4 years) after crushing the sartorius nerve. We found that 27% (S.E. = 1.4%) of the junctions in muscles 2 years after reinnervation were polyneuronally innervated, whereas only 10% (S.E. = 1.2%) of the junctions in normal frog muscles were polyneuronally innervated. Thus, the synapse elimination that occurs following reinnervation does not restore the normal level of polyneuronal innervation. Histological comparisons of junctional structure between muscles 2 years after reinnervation and normal muscles revealed substantial differences. Reinnervated junctions had a greater length of synaptic gutter apposed by nerve terminal processes, more axonal inputs, more empty synaptic gutter, more instances of single synaptic gutters innervated by more than one axon, and longer lengths of nerve terminal processes that connect synaptic gutters within a junction. On the basis of this physiological and anatomical evidence, we conclude that nerve injury causes persistent changes in the pattern of muscle innervation.     

The pattern of innervation in serially duplicated axolotl limbs: further evidence for the existence of local pathway cues?

The innervation of the biceps muscle was examined in regenerated and vitamin A-induced serially duplicated axolotl forelimbs using retrograde transport of horseradish peroxidase. The regenerated biceps muscle becomes innervated by motor neurones in the same position in the spinal cord as the normal biceps motor pool. In previous experiments in which the innervation of a second copy of a proximal limb muscle was examined in serially duplicated limbs (Stephens, Holder & Maden, 1985), the duplicate muscle was found to become innervated by motor neurones that would normally have innervated distal muscles. In the present study, the innervation of the second copy of biceps was examined under conditions designed to encourage nerve sprouting from 'correct' biceps axons. Following either partial limb denervation or denervation coupled with removal of the proximal biceps, the second copy of the muscle was still innervated by inappropriate motor neurones, which again would normally innervate distal limb muscles. These results are interpreted as evidence for the necessity for an appropriate local environment for axonal growth to allow reformation of a correct pattern of motor innervation in the regenerated limb.     

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.     

Synaptic competition and the persistence of polyneuronal innervation at frog neuromuscular junctions

Mechanisms governing the elimination of polyneuronal innervation were examined by correlating the morphology and physiology of competing nerve terminals at identified dually innervated neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). Synaptic efficacy (endplate potential amplitude per unit nerve terminal length) was presumed to reflect the ability of a terminal to compete for synaptic space. The synaptic efficacies of two terminals at the same synaptic site were found to be surprisingly equal, with a median difference of 33%. Much more variation would be expected if dually innervated junctions were randomly innervated by pairs of terminals having the same range of synaptic efficacy as that found at singly innervated junctions in the same muscle. This finding supports the hypothesis that the weaker input is eliminated from dually innervated junctions when there is a large discrepancy in competitive efficacy, and that both inputs may persist if competitive efficacies are relatively equal. We also tested but failed to find support for the hypothesis that spatial proximity between competing terminals intensifies competition for synaptic space during synapse elimination.     

Temperature and neuromuscular development in the tambaqui

The development of muscle innervation pattern was investigated in larvae of the Amazonian fish, the tambaqui Colossoma macropomum. The time to hatching decreased from 28–29 h at 23.5° C to 11–12 h at 31° C. The larvae hatched after the completion of somitogenesis (38-somite stage) at 23.5° C but only at the 33-somite stage at 28–31° C. Embryos were stained for acetylcholinesterase activity and with an acetylated tubulin antibody in order to visualize neural processes. All muscle fibre types were initially innervated at their myoseptal ends. The development of motor innervation to the trunk muscle was delayed with respect to hatching at higher temperatures. At hatching, muscle fibres were innervated only to somites 16–17 at 28–31° C and somite 23–26 at 23.5–25° C (counting from the head), although the larvae swam vigorously to avoid sinking. In contrast, in newly hatched larvae myofibrils were present right along the trunk at all temperatures in both the superficial and inner muscle fibres. At hatching numerous multi-layered membrane contacts with the ultrastructural characteristics of gap junctions, were found between muscle fibres and at the inter-somite junctions, suggesting the somites were initially electrically coupled. These structures disappeared concomitant with the development of muscle endplates right down the trunk. The larvae started feeding 5 days post-hatch at 28° C. First feeding was associated with a dramatic decrease in the volume density of mitochondria and an increase in the volume density of myofibrils in the inner muscle fibres. The polyneuronal and multi-terminal pattern of innervation characteristic of adult slow-muscle fibres also developed around the time of first feeding.     

Quantitative investigation of the neuromuscular junction of rat skeletal muscle fibres after double innervation

Summary In normal (untreated) rats the mean length ratio of postsynaptic to presynaptic membrane was 2.7±0.8 for neuromuscular junctions of slow-twitch soleus muscle fibres and 4.2±1.0 for neuromuscular junctions of fast-twitch extensor digitorum longus muscle fibres; this difference was significant (P<0.001). After experimental double innervation by fast and slow muscle nerves for four months, the ratio was (1) 2.9±0.8 for the original slow-twitch fibre end-plate and 2.8±0.8 for the newly established one, both not significantly different from that of the normal slow-twitch fibres; and (2) 2.2±0.5 for the original fast-twitch fibre end-plate and 2.2±0.7 for the newly established one, both significantly smaller than that of the normal fast-twitch fibres (P<0.001). This means that the double innervated slow-twitch muscle fibres retained their original neuromuscular junction type, whereas the doubly-innervated fast-twitch muscle fibres underwent a dramatic transformation of their neuromuscular junction from the fast-muscle to the slow-muscle type. In both doubly innervated fibres, the ultrastructural characteristics of neuromuscular junctions, whether altered or not, were identical at both end-plate regions.     

An analysis of miniature synaptic events recorded from locust muscle--variations in amplitude and time courses.

A study of miniature post-synaptic potentials (min. e.p.s.p.'s) in metathoracic extensor tibiae muscle fibres at neuromuscular junctions of adult locusts (Schistocerca gregaria) has been undertaken. Extracellular min. e.p.s.p.'s recorded from single junctional sites and their "marked" intracellular min. e.p.s.p. counterparts varied both in time course and amplitude and at many sites a small proportion of abnormal miniatures were observed, i.e., of large amplitude and/or of long duration. Positive correlations between the rise times and 1/2-decay times of the extracellular min. e.p.s.p.'s and between rise times and amplitudes and 1/2-decay times and amplitudes of "marked" intracellular events were found. Comparison of results obtained from differently innervated muscle fibres demonstrates that the occurrence of abnormal miniatures is independent of the type of innervation i.e., "fast" or "slow" excitatory motoneurons. Mechanisms that lead to the occurrence of abnormal miniatures are discussed in relation to the presence of large vesicles in the terminals of the excitatory motoneurons but the occurrence of these events could equally well be explained by the "post-synaptic saturation hypothesis".     

Cell types required to efficiently innervate human muscle cells in vitro

Previous studies carried out in our laboratory have shown that myofibers formed by fusion of muscle satellite cells from donors with spinal muscular atrophy (SMA) type I or II undergo a characteristic degeneration 1.5-3 weeks after innervation with rat embryonic spinal cord explants. The only cells responsible for degeneration of innervated cocultures are SMA muscle satellite cells. In order to study the kinetics of nerve and muscle cell degeneration in nerve-muscle cocultures implicating SMA muscle cells, we attempted to simplify the nervous component of the coculture and identify the nerve cell types necessary for a successful innervation. We demonstrate here that motoneurons alone were unable to innervate myotubes. However, when three cell types (motoneurons, sensory neurons, and Schwann cells) were added onto a reconstituted muscular component consisting of cloned muscle satellite cells and cloned muscular fibroblasts, myotubes contracted, indicating that functional neuromuscular junctions were formed. We concluded that the three cell types were required for a successful innervation. Moreover, we studied the effects of culture medium conditioned by different combinations of nerve cells on innervation; we observed that physical contacts among sensory neurons, motoneurons, and myotubes are required for a successful innervation; in contrast Schwann cells can be replaced by a Schwann-cell-conditioned medium, indicating that these cells produce a putative soluble "innervation-promoting factor." Obviously such a reconstituted system does not reflect the in vivo situation but it allows the formation of functional motor synapses and could therefore allow us to elucidate neuromuscular disease pathogenesis, especially that of spinal muscular atrophy.     

In vitro regulation of the innervation pattern of quail muscle fibres by quail and mouse neurons

Myoblasts from rudiments of slow and fast muscle, anterior latissimus dorsi (ALD) and posterior latissimus dorsi (PLD) respectively, of 9-day-old quail embryos were cultured in vitro for a period of up to 60 days in order to give rise to well-differentiated muscle fibres. These fibres were innervated by neurons from either quail or mouse embryo spinal cord and their innervation pattern was examined by the visualization of acetylcholine receptors (ACh-R) and of acetylcholinesterase (ACh-E) activity at the neuromuscular contacts. In the culture system used, quail neurons always innervated muscle fibres at several sites and only when a fast-type activity was imposed on these neurons did a reduction in the number of the previously established neuromuscular contacts take place. In contrast, in the muscle fibres innervated by mouse neurons, a spontaneous reduction in the number of the previously established neuromuscular contacts occurred but this spontaneous reduction depended upon the level of differentiation reached by the muscle fibres in vitro. In the cultures of muscle fibres previously innervated by mouse neurons, the addition of quail neurons did not provoke any modification in the initial innervation pattern, and no quail ACh-R cluster was observed. In contrast, in the muscle fibres previously innervated by quail neurons, the mouse neurons contacted these fibres, resulting in a decrease in the number of quail ACh-R clusters. These results emphasize the part played by neurons in the establishment of the innervation pattern when muscle fibres have reached a high level of differentiation. In vitro, the slow and fast characteristics of the muscle fibres do not influence this pattern.     

Functional characteristics of Rana temporaria arteries and veins with different densities and neuromediator properties of vasomotor innervation

Pharmacological studies have been made of frog's arteries and veins with different density and transmitter nature of their vasomotor innervation. It was found that the difference in vasomotor innervation of the blood vessels is related to the level of their maximal contractile response. The higher the adrenergic innervation, the higher the response to exogeneous catecholamines. The higher the cholinergic innervation, the higher the response to acetylcholine. Vasomotor innervation affects the sensitivity of the blood vessels, however catecholamine sensitivity is also observed in non-innervated vascular smooth muscle cells. alpha-Adrenoreceptors were found both in the innervated and non-innervated vessels, whereas beta-adrenoreceptors are present only in the innervated ones.     

Changes in the efficacy of transmission at synapses of the dorsal horn of the cat spinal cord observed during repetitive activation of cutaneous afferents

Inhibitory patterns in monosynaptic components of field potentials and potentials in the dorsal spinal cord surface were investigated during acute experiments on cats involving low-frequency stimulation of cutaneous peripheral nerves. Approximation of experimental data obtained from a theoretical plot was performed adopting a standard model of transmitter storage and release. Parameters of fractional release and replenishment of transmitter depleted from synaptic junctions were determined. Processes of replenishing supplies of transmitter for release were seen to intensify under rhythmic stimulation. A comparison between these experimental findings on the character of synapses of the monosynaptic reflex arc and others found in the literature indicated a similarity between the parameters of the mechanism underlying transmitter mobilization and release at different synaptic junctions formed by primary afferent fibers.Dnepropetrovsk State University Commemorating the 300th Aniversary of Ukraine-Russian Reunion, Dnepropetrovsk. Translated from Neirofiziologiya, Vol. 19, No. 4, pp. 491–497, July–August, 1987.     

Critical period for the androgenic block of neuromuscular synapse elimination

Juvenile androgen treatment during developmental synapse elimination changes the pattern of innervation in the adult levator ani (LA), an androgen-sensitive muscle (Jordan, Letinsky, and Arnold, 1989b). Most notably, such adult muscles contain an unusually high number of muscle fibers that are innervated by two or more axons indicating that these fibers are multiply innervated. Juvenile androgen treatment also increases the adult level of preterminal branching, the number of junctional sites per adult fiber, and the size of adult LA muscle fibers and motoneurons in the spinal nucleus of the bulbocavernosus (SNB). The present study was designed to determine when in development androgen treatment is most effective in maintaining multiple innervation in adulthood and whether there are different critical periods for the different effects of juvenile androgen treatment. Male rats were castrated on 7, 21, or 34 days after birth (roughly corresponding to the beginning, middle, and end of synapse elimination in the LA muscle) and treated daily with testosterone propionate for the next 2 weeks. All rats were sacrificed at 9 weeks and their spinal cords and LA muscles were stained and analyzed. Only during the first treatment period (7-20) did androgen treatment result in increased levels of multiple innervation at 9 weeks. During this period, androgen also increased the number of junctional sites per fiber and the size of SNB somata but did not influence the adult level of preterminal branching or the diameter of adult LA muscle fibers. Androgen treatment during the two later periods increased the level of preterminal branching and the size of LA muscle fibers without influencing the level of multiple innervation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuromuscular transmission in cultures of adult human and rodent skeletal muscle after innervation in vitro by fetal rodent spinal cord

Electrophysiologic analyses have been carried out on in vitro-coupled explants of fetal rodent spinal cord and adult skeletal muscle of human as well as rodent origin. The studies demonstrate that characteristic neuromuscular transmission can develop and be maintained in these unusual tissue combinations during long-term culture. After coupling periods of 2–7 weeks in vitro, selective stimulation of spinal cord evokes widespread coordinated contractions in the muscle tissue. Simultaneous microelectrode recordings of cord and muscle responses to local cord, or ventral root, stimuli show that muscle action potentials (and contractions) generally occur with latencies of several msec after onset of cord discharges. Similar temporal relations are often seen during spontaneous rhythmic discharges of the coupled cord and muscle tissues. Long series of repetitive discharges, at 2–5 sec intervals, may occur synchronously between these cord and muscle explants, in response to single cord (or dorsal-root ganglion) stimuli, and they may also appear spontaneously. d-Tubocurarine (1–10 μg/ml) selectively and reversibly blocks neuromuscular transmission in these cultures. Eserine accelerates recovery of normal function. Spontaneous repetitive fibrillations of many of the cultured muscle fibers are observed sporadically, and these contractions often continue unabated after block of neuromusclar transmission by d-tubocurarine. Many of the fibers which show asynchronous fibrillations are probably not innervated (as in denervated muscle in situ). In some cases, however, extracellular as well as intracellular recordings indicate that similar fibrillations may also occur in fibers which are clearly innervated. Repetitive cord and muscle discharges are greatly augmented after introduction of strychnine. Complex rhythmic oscillatory (ca. 10/sec) afterdischarges generated in strychninized cord explants lead to similarly patterned muscle discharges (and contractions), which may also occur, at, times, in normal medium.     

The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture

This paper describes the physiological and pharmacological parameters of the response of mature muscle fibers that develop from myoblasts in vitro to iontophoretically applied acetylcholine (ACh) and the distribution of ACh sensitivity over fibers innervated in vitro by spinal cord cells and uninnervated (control) fibers. Peaks of sensitivity were detected near nerve terminals on functionally innervated fibers, but the “extrasynaptic” chemosensitivity remained high. The distribution of chemosensitivity over uninnervated fibers is not uniform: peaks or “hot spots” were detected over most fibers. Autoradiography of cultures exposed to ¹²⁵I-α-bungarotoxin is consistent with the uneven distribution detected by iontophoresis. Sensitivity peaks were usually located in the immediate vicinity of obvious muscle nuclei and conversely the membrane near most nuclei was more sensitive than that over other regions along the same cell. The relation between innervation and distribution of ACh sensitivity is discussed.     

Penis-retractor muscle of Aplysia: excitatory motor neurons

Cobalt axonal iontophoresis and intracellular recordings were used to identify a cluster of several motor neurons innervating the penis-retractor muscle of Aplysia. Intracellularly recorded motor neuron action potentials elicited direct, one-for-one, constant latency excitatory junctional potentials (ejps) in individual muscle fibers. The axons of motor neurons could be recorded extracellularly in the penis-retractor nerve and stimulation of the nerve backfired the motor neurons. Perfusion of the ganglion, the muscle, or both with solutions of either increased Mg++/decreased Ca++ or increased Ca++ sea water indicated that the presumed motor neuron impaled was not a sensory cell and that interneurons were not intercalated in the pathway. Innervation of muscle fibers was found to be functionally polyneuronal and diffuse. The ejps were found to undergo marked facilitation with repetitive motor-neuron stimulation. The motor neurons were isolated in a distinct cluster in the right pedal ganglion. Their electrical activity was characterized by spontaneous irregular action potentials and a moderate input of postsynaptic potentials.     

The adult fast isozyme of myosin is present in a nerve-muscle tissue culture system

Abstract. Organotypic nerve-muscle cultures were prepared from foetal mouse spinal cord and adult mouse muscle fibres. In this system, the adult fibres degenerate and new myotubes form. The regenerated muscle fibres become innervated, develop cross-striations, and survive for several months. We have investigated the isozymes of myosin present in these muscle fibres using histochemistry and immunocytochemistry with antibodies to rat embryonic, neonatal, and adult fast myosins. We demonstrate that some of the regenerated fibres contain adult fast but not embryonic or neonatal myosin. This is the first demonstration of the production of adult myosin heavy chain in tissue culture. This system therefore offers the possibility for further study of the development of adult myosin isoforms in vitro.     

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.