Neuromuscular Synapse Formation Research Articles

Expression and localisation of dynamin and syntaxin during neural development and neuromuscular synapse formation

The expression and subcellular localisation of dynamin and syntaxin were examined during the periods of motor neuron development and neuromuscular synaptogenesis in the mouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinal cord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent ages examined. Reverse transcription and polymerase chain reaction (RT-PCR) identified low levels of all three carboxy-terminal splicing forms of dynamin I in spinal cord from as early as E10. During the period of maturation of spinal neurons, from E10 to the first postnatal day (P0), the short carboxy-terminal splicing form of dynamin I (dynamin I*b) was up-regulated, as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining became colocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hours of the commencement of synapse formation and throughout subsequent development. In contrast, dynamin, which is important for activity-dependent synaptic vesicle recycling and, thus, sustained neurotransmission, could not be detected at most newly formed synapses until several days after synapse formation. The delayed appearance of dynamin at the synapse, thus, heralds the neonatal development of robust synaptic transmission at the neuromuscular junction. J. Comp. Neurol. 410:531–540, 1999. © 1999 Wiley-Liss, Inc.

Laminin-11

Laminins are a family of glycoproteins which are ubiquitous components of basement membranes and play key structural and functional roles. Eleven isoforms have been identified to date; each is an αβγ heterotrimer assembled from a repertoire of five α, three β and two γ chains. Studies of laminin-11 (α5β2γ1) illustrate the unique expression patterns and distinct functions that can be displayed by laminin isoforms. Laminin-11 is found in the glomerular basement membrane in kidney, in the neuromuscular synaptic cleft in skeletal muscle and in other tissues such as placenta and lung. Mice lacking laminin-11 exhibit defective glomerular filtration and developmental defects in neuromuscular synapse formation, with Schwann cells invading the synaptic cleft. Consistent with these observations, both motoneurons and Schwann cells distinguish laminin-11 from other isoforms in vitro. These results suggest that laminin-11 is a structural component of the basement membrane which influences cell behavior in physiologically relevant ways. A greater understanding of laminin-11 assembly and basement membrane incorporation could provide a logical basis for therapy in merosin-deficient congenital muscular dystrophy.

Expression and localisation of dynamin and syntaxin during neural development and neuromuscular synapse formation

The expression and subcellular localisation of dynamin and syntaxin were examined during the periods of motor neuron development and neuromuscular synaptogenesis in the mouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinal cord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent ages examined. Reverse transcription and polymerase chain reaction (RT-PCR) identified low levels of all three carboxy-terminal splicing forms of dynamin I in spinal cord from as early as E10. During the period of maturation of spinal neurons, from E10 to the first postnatal day (P0), the short carboxy-terminal splicing form of dynamin I (dynamin I*b) was up-regulated, as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining became colocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hours of the commencement of synapse formation and throughout subsequent development. In contrast, dynamin, which is important for activity-dependent synaptic vesicle recycling and, thus, sustained neurotransmission, could not be detected at most newly formed synapses until several days after synapse formation. The delayed appearance of dynamin at the synapse, thus, heralds the neonatal development of robust synaptic transmission at the neuromuscular junction.

Drosophila synapse formation: regulation by transmembrane protein with Leu-rich repeats, CAPRICIOUS.

Upon reaching the target region, neuronal growth cones transiently search through potential targets and form synaptic connections with only a subset of these. The capricious (caps) gene may regulate these processes in Drosophila. caps encodes a transmembrane protein with leucine-rich repeats (LRRs). During the formation of neuromuscular synapses, caps is expressed in a small number of synaptic partners, including muscle 12 and the motorneurons that innervate it. Loss-of-function and ectopic expression of caps alter the target specificity of muscle 12 motorneurons, indicating a role for caps in selective synapse formation.

Acetylcholine Receptor Formation in Mouse–Chick Chimera

This study investigated possible interactions between motoneurons and somitic-derived muscle cells in the formation of neuromuscular synapses in the myotome. The peculiarities of the neuromuscular synaptic pattern in chick and mouse embryos provided a model for studying the achievement of synaptogenesis between chick motoneurons and mouse muscle cells. In chick embryo, initial AChR clustering occurs well before innervation of the myotome, whereas in mouse embryo nerve axons invade the myotome extensively before the appearance of AChR clusters. Our approach was to replace somites from a chick host embryo with those derived from mouse donor embryos. We show that muscle cells from mouse myotome can differentiate in the chick embryo environment and form neuromuscular contacts with chick motor axons. Host axons invadedin ovodifferentiating mouse myotome at a time when they had not yet reached the host myotome. This particular ingrowth of motor nerves was attributable to the mouse transplant since use of a quail somite did not produce the same effect as the mouse somite, which suggests that developing mouse muscles specifically modify the time course of chick axogenesis. The synaptic areas formed between chick motor axons and mouse myotubes developed according to the mouse pattern. Both the timing of their appearance and their morphology correlated perfectly with events in mouse synaptogenesis. These results indicate the important role played by postsynaptic membrane in controlling the first steps of AChR formation.

Defective Neuromuscular Synaptogenesis in Agrin-Deficient Mutant Mice

During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here that postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation. However, some postsynaptic differentiation does occur in the mutant, suggesting the existence of a second nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.

Screening of mutants defective in neuromuscular synapse formation of Drosophila melanogaster

Screening of mutants defective in neuromuscular synapse formation of Drosophila melanogaster

ARIA, a protein that stimulates acetylcholine receptor synthesis, is a member of the neu ligand family

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human heregulin, ligands for the receptor tyrosine kinase encoded by the neu (c- erbB2, HER2) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

Development of the neuromuscular synapse

Major advances have occurred in our understanding of the signaling events involved in neuromuscular synapse formation. In particular, it has recently been shown that agrin is necessary for synapse formation, that acetylcholine receptor genes are specifically transcribed by synaptic nuclei in response to signals from the synaptic basal lamina, and that synaptic competition between motor neurons can occur by a Hebbian mechanism in cell culture.

43K Protein and Acetylcholine Receptors Colocalize during the Initial Stages of Neuromuscular Synapse Formation in Vivo

The 43K protein is a cytoplasmic peripheral membrane protein concentrated subsynaptically in skeletal muscle. Recombinant 43K has been shown to cause clustering of acetylcholine receptors (AChRs) in cultured cells. However, the role of 43K in vivo is disputed, because in some cases it appears only after AChRs have clustered. We therefore examined the expression and distribution of 43K and AChRs during synapse formation in embryonic mouse muscles. Messenger RNA for 43K was detected on Embryonic Day (E) 12, a day prior to the first AChR clusters. Immunofluorescence showed that both AChRs and 43K were colocalized in patches by E13, the stage at which intramuscular nerves were first detected. The AChR/43K patches were nerve associated, and more than 98% of AChR patches were accompanied by 43K. The precise colocalization of 43K and AChRs persisted through development. These results are consistent with 43K being involved in the nerve-induced clustering of AChRs during synapse formation.

Localization of urokinase-type plasminogen activator in human eyes: An immunocytochemical study

We investigated the presence of urokinase-type plasminogen activator (u-PA) in various structures of the human eye by using a biotin-strepavidin complex immunoperoxidase technique with a monoclonal antibody specific for human u-PA. A moderate to intense reaction product was seen in the corneal endothelium, episclera, sclera, iris muscles, lens equator and posterior capsule, peripheral vitreous, epithelium of pars plana, retinal pigment epithelium, axons, uveal fibroblasts, optic nerve fibers, and extraocular muscles. A weak reaction product was seen in the conjunctival and corneal epithelium, the trabecular meshwork, melanocytes and stroma of the iris and choroid, and the posterior layers of the retina. u-PA was not localized in the corneal stroma, conjunctival goblet cells, and certain parts of the lens. Comparison of the present results with those of our previous study (Tripathi, Geanon and Tripathi, 1987, Ophthalmology, 94, 1434-8) on the localization of tissue plasminogen activator (t-PA) in the eye indicate that the distribution of u-PA and t-PA overlaps in many tissues, but differs significantly in others, and that this finding may be related to their complementary and specific roles. As in other body systems, u-PA in ocular tissues is probably involved in processes such as fibrinolysis, wound healing, turnover of intra- and extracellular macromolecules, muscle and neuromuscular regeneration, synapse formation, and maintenance of general neuronal function.

In vitro analysis of position- and lineage-dependent selectivity in the formation of neuromuscular synapses

The hypotheses that selective formation of nerve-muscle connections depends upon intrinsic cellular properties, endowed either by the cell's rostral-caudal position in the embryo or its lineage, were tested directly in Xenopus embryonic cell cultures. The position or the lineage of embryonic cells was traced in vitro by previous injection of fluorophore-conjugated dextran molecules into individual blastomeres. Synaptic efficacy was assayed by recording synaptic currents from neurite-contacted muscle cells in the culture, and the physical affinity of neurites for muscle cells of different positional or clonal origins was assayed by counting the frequency of association between the neurites' growth cones and the muscle cells. Both assays showed no apparent preference between nerve and muscle cells of similar rostral-caudal positions or clonal origins, suggesting that there is little position- or lineage-dependent selectivity in the initial nerve-muscle interactions.

Localization of muscle gene products in nuclear domains.

The localization of gene products is central to the development of cell polarity and pattern specification during embryogenesis. To monitor the distribution of gene products encoded by different nuclei in the same cell in tissue culture, we fused cells of different species to form multinucleated non-dividing heterokaryons. In previous fusion studies, cell-surface antigens and organelles contributed by disparate cell types intermixed within minutes. Using heterokaryons produced with differentiated muscle cells, we demonstrate here that a muscle membrane component, the Golgi apparatus mediating its transport, and a sarcomeric myosin heavy chain are localized in the vicinity of the nuclei responsible for their synthesis. These results provide direct evidence that products (organelle, membrane and structural proteins) derived from individual nuclei can remain localized in myotubes, a finding with implications both for neuromuscular synapse formation and for the carrier state of Duchenne muscular dystrophy.

Initial events in the formation of neuromuscular synapse: rapid induction of acetylcholine release from embryonic neuron.

We have studied the electrical events during the initial phase of nerve-muscle contact in embryonic Xenopus culture. Using a G omega-seal, whole-cell recording method, we monitored the membrane current of a muscle cell continuously while it was manipulated into close proximity of the growth cone of a cocultured spinal neuron. We found a rapid appearance of pulsatile inward currents at the muscle cell after the neurite-muscle contact. These currents were abolished by d-tubocurarine and alpha-bungarotoxin but were unaffected by tetrodotoxin. Both the drug sensitivity and the time course of these currents are similar to that of the spontaneous miniature end-plate currents (MEPCs) resulting from spontaneous release of pulses of acetylcholine (AcCho) from the nerve terminal. Unlike the MEPCs at the mature neuromuscular synapse, these early MEPCs varied greatly in their amplitudes, and there was a gradual increase in the frequency of the MEPCs of larger amplitudes during the first 20 min after the contact. Independent measurement of AcCho concentration near the growth cone by an excised patch of AcCho-sensitive muscle membrane showed that very little AcCho is released from the isolated growth cone, and marked release can be triggered by the contact with a muscle cell or with the excised membrane itself. The induction of release is relatively specific: contact with a neuron or the tip of a clean glass pipette was capable of inducing only a transient release, while persistent release was induced by contacts made with muscle membrane. This contact-dependent AcCho release may be responsible for an early induction of muscle activity and serve as a signal for the establishment of synaptic contact.

Molecular forms of acetylcholinesterase in Xenopus muscle

Xenopus adult muscle, whole Xenopus embryos, and cultured embryonic myocytes together contain five acetylcholinesterase forms which can be resolved by sucrose density gradient centrifugation. These are identified as the collagenase-sensitive asymmetric forms A 12 and A 8, and the globular forms G 4, G 2, and G 1. Asymmetric forms rise in whole embryos during the period of neuromuscular synapse formation, but their rise is not prevented by tricaine methanesulfonate, which abolishes motor activity. Aneural myocyte cultures synthesize primarily asymmetric acetylcholinesterase, much of which is extracellular. Prior nerve contact is not required for its expression. The proportion of asymmetric forms is neither decreased by tetrodotoxin, nor enhanced by veratridine and aconitine. We conclude that muscle activity does not modulate the expression of asymmetric acetylcholinesterase in Xenopus.

Blockers of calcium permeability inhibit neurite extension and formation of neuromuscular synapses in cell culture

Growth of neurites from trypsin-dissociated retinal neurons from chick embryos is very sensitive to the extracellular calcium concentration. Blockers of calcium permeability such as Co 2+, Mn 2+, La 3+ and nitrendipine, when added to dissociated neurons, decrease the fraction of cells that extend neurites and the rate of neurite growth without influencing cell-substratum adhesion or survival capacity. Inhibition is concentration dependent, is related to the age of the donor chick embryo and can be prevented by increasing the extracellular calcium concentration. 50% inhibition in 8-day neurons is produced by 120 μM Co 2+, 250 μM Mn 2+, 50 μM La 3+ and 10 μM nitrendipine in medium containing 1.8 mM Ca 2+. Inhibition of neurite extension is accompanied by a concentration dependent inhibition of synapse formation between retinal neurons and muscle cells in culture, as determined by intracellular recording. 50% inhibition in the fraction of innervated myotubes is produced by 0.4 mM Co 2+ and 4 mM Mn 2+. These results suggest that: (1) a voltage-dependent calcium flux is a signal not only for growth cone expansion but also for neurite extension in primary dissociated neurons; and (2) that neurite extension is a prerequisite for synaptogenesis between neurons and muscle cells in culture.

Influence of neostigmine treatment on embryonic development of acetylcholine receptors and neuromuscular junctions.

The postulated role of the acetylcholine receptor in the formation of neuromuscular synapses during the course of embryonic development was investigated in the superior oblique muscle of white Peking duck embryos. The possibility that the number of receptors could be experimentally lowered by chronic injections of the anticholinesterase agent, neostigmine methylsulfate, was determined using 125I-alpha-bungarotoxin. The total number of acetylcholine receptors on incubation day 12, 2 d subsequent to the onset of treatment, was reducted 45% as compared to saline-treated controls. A similar reduction in total receptor content (49%) was also observed on day 19. Radioautographic preparations showed that clusters of acetylcholine receptors were rare and that the grain density of extrajunctional receptors was also reduced. Hence, chronic treatment with neostigimine during development was observed to exert an effect on both the number and distribution of receptors in the developing superior oblique muscle. These changes occurred in the absence of any apparent effect on muscle differentiation in general. Myoblasts and myotubes were present on day 14 and further differentiated into myofibers by day 18 in both neostigmine and saline-treated muscles. The cytology of the develop;ing muscle cells also appeared normal. This is in contradistinction to the striking morphological changes that take place in adult mammalian and avian muscle after anticholinesterase treatment. More significantly, the decreased total receptor content and sparsity of clusters had no apparent effect on the formation of developing neuromuscular junctions at the electron microscopic level. The frequency of neuromuscular junctions in neostigmine-treated muscles was similar to that of the controls. It is concluded that acetylcholine receptor clusters are not required for the events leading to the morphological formation of neuromuscular junctions during in vivo development.

Re-innervation of rat skeleton muscle in the presence of alpha-bungarotoxin.

1. The possible role of acetylcholine (ACh) receptors in controlling neuromuscular synapse formation was studied by blocking the receptors with alpha-bungarotoxin during re-innervationf the rat diaphragm. Anaesthetized and artificially respirated rats were perfused with toxin throughout the 3-day period when most re-innervation was shown to take place (from day 4 to day 6 after a nerve crush). 2. The toxin treatment blocked about 99.9% of both junctional and extrajunctional ACh receptors, thereby eliminating virtually all end-plate potentials. However, a low level of ACh sensitivity returned to the muscle after several hours of washing, and small end-plate potentials were then recorded from the majority of fibres on the side that previously had been denervated. The degree of re-innervation was nearly as great as that seen in control denervated muscles that had not been treated with toxin. 3. The presence of bungarotoxin during re-innervation did not significantly affect either the appearance of newly formed synapses in the light microscope or the amount of transmitter they released during nerve stimulation. 4. Double innervation, which was not seen in normal muscles, was found in about 10-15% of re-innervated fibres in both the untreated and toxin-treated preparations. This suggests that some of the synapses formed during re-innervation may have been made over what previously had been extrajunctional membrane. 5. It is therefore unlikely that the ACh receptor, or at least that part of the receptor to which bungarotoxin binds, plays a direct role in controlling the process of synapse formation in mammalian skeletal muscle...