The single-channel basis for the slow kinetics of synaptic currents in vertebrate slow muscle fibers

The time course of synaptic currents is significantly longer in slow than in fast twitch muscle fibers. To examine the underlying basis for these slow synaptic currents, single-channel recordings were made from the synapses of slow muscle fibers. Our analysis indicates that low conductance acetylcholine receptor (AChR) channels predominate in innervated slow fibers. The high level of expression of low conductance channels is in contrast to fast twitch fibers, in which these channels are expressed in significant numbers only in embryonic or denervated muscle. Analysis of the distribution of open durations for the low conductance channel class suggests that the open time of this AChR class is the major determinant in shaping the slow time course of synaptic current decay. The predominant contribution of low conductance channel openings to synaptic currents of slow muscle fibers indicates a well-defined physiological role for this class of AChRs.     

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.     

Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses

To elucidate the nature of signals that control the level and spatial distribution of mRNAs encoding acetylcholine receptor (AChR), alpha-, beta-, gamma-, delta- and epsilon-subunits in muscle fibers chronic paralysis was induced in rat leg muscles either by surgical denervation or by different neurotoxins that cause disuse of the muscle or selectively block neuromuscular transmission pre- or postsynaptically and cause an increase of AChRs in muscle membrane. After paralysis, the levels and the spatial distributions of the different subunit-specific mRNAs change discoordinately and seem to follow one of three different patterns depending on the subunit mRNA examined. The level of epsilon-subunit mRNA and its accumulation at the end-plate are largely independent on the presence of the nerve or electrical muscle activity. In contrast, the gamma-subunit mRNA level is tightly coupled to innervation. It is undetectable or low in innervated normally active muscle and in innervated but disused muscle, whereas it is abundant along the whole fiber length in denervated muscle or in muscle in which the neuromuscular contact is intact but the release of transmitter is blocked. The alpha-, beta-, and delta-subunit mRNA levels show a different pattern. Highest amounts are always found at end-plate nuclei irrespective of whether the muscle is innervated, denervated, active, or inactive, whereas in extrasynaptic regions they are tightly controlled by innervation partially through electrical muscle activity. The changes in the levels and distribution of gamma- and epsilon-subunit-specific mRNAs in toxin-paralyzed muscle correlate well with the spatial appearance of functional fetal and adult AChR channel subtypes along the muscle fiber. The results suggest that the focal accumulation at the synaptic region of mRNAs encoding the alpha-, beta-, delta-, and epsilon-subunits, which constitute the adult type end-plate channel, is largely determined by at least two different neural factors that act on AChR subunit gene expression of subsynaptic nuclei.     

Dual motor innervation in the axial musculature of fishes

A systematic survey of motor innervation in the myotomal muscle fibres in several groups of fishes was conducted to observe the extent of dual innervation, a phenomenon found within those myotomal muscle fibres with terminal innervation. It is concluded that the dually innervated myotomal muscle fibre is a primitive vertebrate feature. Furthermore, it is suggested that this particular form of polyneuronal innervation is retained in the course of neural evolution within the homogeneous white myotomal muscle fibre region in these fish groups in order to insure synchrony of firing during sudden, rapid locomotion. The findings are examined in light of current ideas regarding the direct adrenergic innervation in addition to cholinergic innervation of striated muscle fibres.     

An acetylcholine receptor lacking both γ and ε subunits mediates transmission in zebrafish slow muscle synapses

Fast and slow skeletal muscle types in larval zebrafish can be distinguished by a fivefold difference in the time course of their synaptic decay. Single-channel recordings indicate that this difference is conferred through kinetically distinct nicotinic acetylcholine receptor (AChR) isoforms. The underlying basis for this distinction was explored by cloning zebrafish muscle AChR subunit cDNAs and expressing them in Xenopus laevis oocytes. Measurements of single-channel conductance and mean open burst duration assigned α(2)βδε to fast muscle synaptic current. Contrary to expectations, receptors composed of only αβδ subunits (presumed to be α(2)βδ(2) receptors) recapitulated the kinetics and conductance of slow muscle single-channel currents. Additional evidence in support of γ/ε-less receptors as mediators of slow muscle synapses was reflected in the inward current rectification of heterologously expressed α(2)βδ(2) receptors, a property normally associated with neuronal-type nicotinic receptors. Similar rectification was reflected in both single-channel and synaptic currents in slow muscle, distinguishing them from fast muscle. The final evidence for α(2)βδ(2) receptors in slow muscle was provided by our ability to convert fast muscle synaptic currents to those of slow muscle by knocking down ε subunit expression in vivo. Thus, for the first time, muscle synaptic function can be ascribed to a receptor isoform that is composed of only three different subunits. The unique functional features offered by the α(2)βδ(2) receptor likely play a central role in mediating the persistent contractions characteristic to this muscle type.     

Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed.     

Denervation alters the size, number, and distribution of clusters of acetylcholine receptor-like molecules on frog cardiac ganglion neurons

Acetylcholine receptor (AChR)-like molecules are found in clusters on the surface of parasympathetic neurons in the frog cardiac ganglion. Electron microscopy of immunoperoxidase-stained tissue reveals that in normally innervated ganglia most of these clusters are located at synaptic sites. Denervation for 2-3 weeks results in a 64% reduction in the total surface area occupied by AChR-like clusters; this change is brought about by the combined effects of a 4-fold decrease in cluster size and a 30% increase in cluster number. Denervation also changes the distribution of AChR-like clusters: clusters, normally restricted to portions of the cell surface, are more widely distributed following denervation. Denervation of amphibian skeletal muscle for a comparable period of time has no effect on the size or the number of synaptic clusters of AChRs. These results suggest that AChRs in nerve and in muscle are regulated differently by innervation.     

Succinic dehydrogenase activity of the respiratory muscles of a fresh-water teleost, Bagarius bagarius (Ham.) (Sisoridae, Pisces).

Functional morphology including the origin, insertion, and innervation of the respiratory muscles in relation to buccal pressure pump and opercular suction pumps in a fresh-water bottom dwelling siluroid fish, Bagarius bagarius have been studied. Histochemical studies were made on the succinic dehydrogenase activity of adductor mandibulae, retractor tentaculi, levator operculi, dilatator operculi, adductor operculi, intermandibularis, interhyoideus, hyohyoideus superior and constrictor branchialis. The intensity of reaction reveals the presence of three types of muscle fibres in some of the respiratory muscles. The muscle containing red muscle fibres are mostly innervated by the branches of the VIIth cranial nerve. The retractor tentaculi consists of superficial white muscle fibres and the interior part is dominated by red muscle fibres. The muscles (adductor operculi, levator operculi, dilatator operculi, interhyoideus, hyohyoideus superior) concerned with the opercular suction pumps are of mixed type and consist of white and red muscle fibres, whereas adductor mandibulae and intermandibularis are made up entirely of white muscle fibres. The adductor muscle bundles of the constrictor branchialis, which are responsible for movement of gill filaments, are dominated by the red muscle fibres. The abductor part, however, is made up entirely of white muscle fibres.     

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.     

Expression of nicotinic acetylcholine receptors in aneural Xenopus embryos

During gastrulation in vertebrate embryos, the mesoderm moves inward and under the ectoderm and these two cell layers subsequently differentiate in close proximity to each other, providing an opportunity for the exchange of inductive signals. This study examines whether the activation of muscle nicotinic acetylcholine receptor (AChR) genes and the subsequent expression of receptors in Xenopus myotomal muscle are dependent on interaction between the ectoderm and the mesoderm, or their derivatives, after the onset of gastrulation. We eliminated such interaction by inducing total exogastrulation of Xenopus embryos. During exogastrulation, the mesoderm moves away from the ectoderm, and the nervous system fails to develop. Single channel recordings from the myotomal muscle of exogastrulated embryos revealed the presence of two major classes of AChRs, which could be distinguished on the basis of channel conductance. The current amplitudes, conductances, reversal potentials, and open times of these channels closely resembled those reported for the two major classes of AChR channels normally expressed in vivo. We conclude that interaction between ectoderm and mesoderm following the onset of gastrulation is not required for the future expression of the major classes of AChRs in myotomal muscle.     

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.     

Development and survival of thoracic motoneurons and hindlimb musculature following transplantation of the thoracic neural tube to the lumbar region in the chick embryo: functional aspects

Following heterotopic transplantation of the thoracic neural tube to the lumbar region on embryonic day (E) 2, the transplanted cord differentiates normally and establishes neuroanatomical connections with the host central nervous system and hindlimb muscles. Beginning on about E12, however, the neuromuscular system begins to undergo regressive changes resulting in motoneuron degeneration and muscle atrophy (O'Brien and Oppenheim, 1990). In the present paper, we have examined the development of neuromuscular function in thoracic transplant embryos from E6 to the time of hatching on E20-21. The onset of hindlimb movements and reflexes occurred at the same time (E6-E8) in both control and thoracic transplant embryos. Further, both the nature (pattern) and frequency of these movements appeared normal in the thoracic transplants up to E10-E12, after which there was a gradual and marked reduction in the frequency, and an alteration in the pattern, of both spontaneous and reflex-evoked hindlimb movements. After E16 normal movements were virtually absent in many of the thoracic transplant cases. By contrast, movements of the head, trunk and wings were normal in these embryos throughout the observation period. Hindlimbs innervated partly by the thoracic transplant and partly by remaining host lumbar cord did not exhibit the regressive changes in function after E10 that occurred in hindlimbs innervated exclusively by the thoracic transplant. EMG recordings from specific hindlimb muscles innervated solely by thoracic motoneurons demonstrated that the activation pattern of both flexors and extensors was similar to the repetitive pattern observed in normal thoracically innervated intercostal muscles (i.e., extensor-like). Muscles did not show distinguishable EMG burst patterns with inhibitory periods as do control lumbar innervated muscles. We conclude that the development of the pattern generating circuitry in the transplanted thoracic cord was similar to normal thoracic cord and thus appeared to be uninfluenced by having contacted the foreign hindlimb muscle targets early in development. Activity blockade with curare from E6 to E14 suppressed the loss of motoneurons that occurs in the thoracic transplant after E10. Thus, the abnormal thoracic-like activation pattern of thoracically innervated hindlimbs may be a critical signal in the initiation of the neuromuscular regression that occurs after E10 in these preparations. Finally, although the innervation and formation of neuromuscular endplates in thoracic transplants appeared normal up to E12, by E14 both the intramuscular nerves and the endplates exhibited signs of degeneration and regression. Thoracic motoneurons are initially able to innervate and functionally activate hindlimb muscles in a manner similar to that of thoracically innervated intercostal muscles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Normal development of nerve-muscle synapses in mice lacking the prion protein gene.

The expression of acetylcholine receptors (AChR) at neuromuscular synapses in skeletal muscle is regulated by innervation. Recent evidence suggests that the neurotrophic factors involved in the expression of AChR subunit genes may be related to the prion protein (PrPc), a protein of unknown function expressed primarily in neurons which, in its modified form, PrPSc, is thought to have a role in the pathogenesis of transmissible spongiform encephalopathies. We have tested for an involvement of PrPc in the neurotrophic regulation of synaptic AChRs in muscle by comparing the contents of AChR epsilon- and gamma-subunit mRNAs by Northern blot analysis and by in situ hybridization in mice with normal and with deleted PrP genes. At the protein level, AChR expression was assessed electrophysiologically. No difference was found between muscles from the two types of animals, suggesting that the neural regulation of AChR subunit expression in skeletal muscle can be mediated by factors that are not derived from the PrP gene.     

Localization of acetylcholine receptors by means of horseradish peroxidase-alpha-bungarotoxin during formation and development of the neuromuscular junction in the chick embryo

The localization of acetylcholine receptors (AChR) in the surface of developing myogenic cells of the chick embryo anterior and posterior latissimus dorsi muscles in relation to the process of innervation has been studied at the ultrastructural level utilizing a horseradish peroxidase-alpha-bungarotoxin conjugate. Localized concentrations of AChR were found in small regions 0.1-0.4 micron in width on the surface of myogenic cells of 10- to 14-d-old muscles. Surface specializations consisting of an external coating of extraneous material and an internal accumulation of dense material are associated with the plasma membrane in the regions of AChR concentration. As the muscle fibers are innervated, reactive surface patches are found at the region of contact of the growing nerve fiber and the surface of myotubes or their fusing myoblasts. After the establishment of contact, the patches of reaction product become more numerous and coextensive within the region of the neuromuscular junction and its immediate surroundings forming a dense continuous deposit on the postsynaptic sarcolemma. Activity becomes increasingly restricted to the site of the neuromuscular junction as the embryos approach hatching. At all stages, specializations external and internal to the plasmalemma are found at regions of high density of AChR, suggesting that they play a role in the maintenance of a higher concentration of receptors at these sites. These specializations also occur at the region of initial synaptic contact, indicating that they might be recognized by the nerve and represent preferred sites of innervation. Innervation appears to exert a stabilizing influence on the area of high AChR concentration in contact with the nerve and to induce a further increase in the AChR density of this site while the number of AChR in the remaining portions of the muscle surface declines.     

Absence of competitive interactions among axon terminals of regenerating motor neurons

Competition among axon terminals is usually considered to contribute to the formation of patterned synaptic connections. During axonal regeneration of motor neurons in the cockroach, leg muscles initially become innervated by appropriate and inappropriate motor neurons. All axon terminals from inappropriate neurons eventually are eliminated, resulting in the reformation of the original innervation pattern. Destruction of an identified motor neuron by the intracellular injection of pronase did not prevent the elimination of inappropriate axon terminals in the muscle normally innervated by that motor neuron. Therefore, competition does not play a role in the reinnervation of the leg muscles. This indicates a major role for specific cell-cell recognition.     

The zebrafish ennui behavioral mutation disrupts acetylcholine receptor localization and motor axon stability

The zebrafish ennui mutation was identified from a mutagenesis screen for defects in early behavior. Homozygous ennui embryos swam more slowly than wild-type siblings but normal swimming recovered during larval stages and homozygous mutants survived until adulthood. Electrophysiological recordings from motoneurons and muscles suggested that the motor output of the CNS following mechanosensory stimulation was normal in ennui, but the synaptic currents at the neuromuscular junction were significantly reduced. Analysis of acetylcholine receptors (AChRs) in ennui muscles showed a marked reduction in the size of synaptic clusters and their aberrant localization at the myotome segment borders of fast twitch muscle. Prepatterned, nerve-independent AChR clusters appeared normal in mutant embryos and dispersed upon outgrowth of motor axons onto the muscles. Genetic mosaic analysis showed that ennui is required cell autonomously in muscle fibers for normal synaptic localization of AChRs. Furthermore, exogenous agrin failed to induce AChR aggregation, suggesting that ennui is crucial for agrin function. Finally, motor axons branched more extensively in ennui fast twitch muscles especially in the region of the myotome borders. These results suggest that ennui is important for nerve-dependent AChR clustering and the stability of axon growth.     

Effects of innervation on the distribution of acetylcholine receptors in regenerating skeletal muscles of adult chickens

In order to determine the roles of nerves in the formation of clusters of acetylcholine receptors (AChRs) during synaptogenesis, we examined the distribution of AChRs in denervated, nerve-transplanted (neurotized) muscles and in regenerated skeletal muscles of adult chickens by fluorescence microscopy using curaremimetic toxins. In the denervated muscles, many extrajunctional clusters developed at the periphery of some of the muscle nuclei of a single muscle fiber and continued to be present for up to 3 months. The AChR accumulations originally present at the neuromuscular junctions disappeared within 3 weeks. In the neurotized muscles, line-shaped AChR clusters developed at 4 days after transection of the original nerve, but no change in the distribution of AChRs had occurred even at 2 months after implantation of the foreign nerve. The line-shaped AChR clusters were found to be newly formed junctional clusters as they were associated with nerve terminals of similar shape and size. Some of both the line-shaped and extrajunctional clusters were formed at least partly by the redistribution of preexisting AChRs. Finally, based on the above observations, the regenerating muscle fibers in normal muscles and in denervated muscles were examined: The extrajunctional clusters appeared in both kinds of muscles at 2 weeks after injury. Afterward, during the innervation process, the line-shaped AChR clusters developed while the extrajunctional clusters disappeared in the innervated muscles. In contrast with this, in the absence of innervation, only the extrajunctional clusters continued to be present for up to 3 months. These results demonstrate clearly that the nerve not only induces the formation of junctional clusters at the contact site, but also prevents the formation of clusters at the extrajunctional region during synaptogenesis.     

Neurite outgrowth on cryostat sections of innervated and denervated skeletal muscle

To localize factors that guide axons reinnervating skeletal muscle, we cultured ciliary ganglion neurons on cryostat sections of innervated and denervated adult muscle. Neurons extended neurites on sections of muscle (and several other tissues), generally in close apposition to sectioned cell surfaces. Average neurite length was greater on sections of denervated than on sections of innervated muscle, supporting the existence of functionally important differences between innervated and denervated muscle fiber surfaces. Furthermore, outgrowth was greater on sections of denervated muscle cut from endplate-rich regions than on sections from endplate-free regions, suggesting that a neurite outgrowth-promoting factor is concentrated near synapses. Finally, 80% of the neurites that contacted original synaptic sites (which are known to be preferentially reinnervated by regenerating axons in vivo) terminated precisely at those contacts, thereby demonstrating a specific response to components concentrated at endplates. Together, these results support the hypothesis that denervated muscles use cell surface (membrane and matrix) molecules to inform regenerating axons of their state of innervation and proximity to synaptic sites.     

Plasticity of skeletal muscle: regenerating fibers adapt more rapidly than surviving fibers

The properties of mammalian skeletal muscle demonstrate a high degree of structural and functional plasticity as evidenced by their adaptability to an atypical site after cross-transplantation and to atypical innervation after cross-innervation. We tested the hypothesis that, regardless of fiber type, skeletal muscles composed of regenerating fibers adapt more readily than muscles composed of surviving fibers when placed in an atypical site with atypical innervation. Fast muscles of rats were autografted into the site of slow muscles or vice versa with the donor muscle innervated by the motor nerve to the recipient site. Surviving fibers in donor muscles were obtained by grafting with vasculature intact (vascularized muscle graft), and regenerating fibers were obtained by grafting with vasculature severed (free muscle graft). Our hypothesis was supported because 60 days after grafting, transposed muscles with surviving fibers demonstrated only a slight change from the contractile properties and fiber typing of donor muscles, whereas transplanted muscles with regenerating fibers demonstrated almost complete change to those of the muscle formerly in the atypical site.     

Synaptic contacts between embryonic Xenopus neurons and myotubes formed from a rat skeletal muscle cell line

Acetylcholine receptors (AChRs) accumulate at the junctional region during early development. In an attempt to characterize this process of AChR accumulation, we combined embryonic Xenopus neurons with myotubes formed from a rat skeletal muscle cell line. Xenopus neurons in culture are known to induce AChR accumulation in Xenopus muscles [Anderson, M. J., Cohen, M. W., and Zorychta, E. (1977). J. (London), 268, 731–756]. Rat myotubes, however, do not exhibit AChR accumulation in culture even when they are functionally innervated by the fetal rat spinal cord explant [Kidokoro, Y. (1980) Develop. Biol., 78, 231–241]. Establishment of synaptic transmission was examined electrophysiologically by recording synaptic potentials, while the distribution of AChR clusters was visualized using fluorescent α-bungarotoxin. Our results indicate that embryonic Xenopus neurons formed functional synaptic contacts but did not cause AChR accumulation in L6-myotubes. It seems that the ability of a nerve to cause AChR accumulation is separate from that to form the functional synapse. We also found that the mean amplitude of synaptic potentials in L6-myotubes interacted with Xenopus neurons was about half of that in L6-myotubes innervated by fetal rat spinal cord explants. Possible explanations for this finding are discussed.