scholarly journals Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

1979 ◽  
Vol 83 (1) ◽  
pp. 143-158 ◽  
Author(s):  
E Frank ◽  
G D Fischbach
Keyword(s):  
Synapse Formation ◽  
Synaptic Cleft ◽  
Postsynaptic Membrane ◽  
Active Growth ◽  
Before And After ◽  
Nerve Muscle ◽  
Scanning Em ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

Barbiturates depress vagal motor pathway to ferret trachea at ganglia

1982 ◽  
Vol 53 (1) ◽  
pp. 253-257 ◽  
Author(s):  
B. E. Skoogh ◽  
M. J. Holtzman ◽  
J. R. Sheller ◽  
J. A. Nadel
Keyword(s):  
Neuromuscular Junction ◽  
Vagus Nerve ◽  
Muscle Tension ◽  
Postsynaptic Membrane ◽  
Nerve Fibers ◽  
Motor Pathway ◽  
Parasympathetic Ganglia ◽  
Before And After ◽  
Nerve Muscle

To determine which site in the vagal motor pathway to airway smooth muscle is most sensitive to depression by barbiturates, we recorded isometric muscle tension in vitro and stimulated the vagal motor pathway at four different sites before and after exposure to barbiturates. In isolated tracheal rings from ferrets, we stimulated muscarinic receptors in the neuromuscular junction by exogenous acetylcholine, postganglionic nerve fibers by electrical fluid stimulation, and the postsynaptic membrane in ganglia by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). We also developed a tracheal nerve-muscle preparation to stimulate preganglionic fibers in the vagus nerve electrically. Activation of ganglia by DMPP or by vagus nerve stimulation was depressed by barbiturates at 10-fold lower concentrations than those depressing the activation of postganglionic nerves or the neuromuscular junction. These findings suggest that the postsynaptic membrane in parasympathetic ganglia is the site in the vagal motor pathway most sensitive to depression by barbiturates.

scholarly journals Purification and characterization of a polypeptide from chick brain that promotes the accumulation of acetylcholine receptors in chick myotubes.

1986 ◽  
Vol 103 (2) ◽  
pp. 493-507 ◽  
Author(s):  
T B Usdin ◽  
G D Fischbach
Keyword(s):  
Acetylcholine Receptors ◽  
Synapse Formation ◽  
Neuromuscular Junctions ◽  
Surface Membrane ◽  
Postsynaptic Membrane ◽  
Size Exclusion ◽  
Response Curves ◽  
Sds Page ◽  
Nerve Muscle ◽  
Receptor Clusters

Acetylcholine receptors (AChRs) are packed in the postsynaptic membrane at neuromuscular junctions at a density of approximately 20,000/micron 2, whereas the density a few micrometers away is less than 20/micron 2. To understand how this remarkable distribution comes about during nerve-muscle synapse formation, we have attempted to isolate factors from neural tissue that can promote the accumulation of AChRs and/or alter their distribution. In this paper we report the purification of a polypeptide from chick brains that can increase the rate of insertion of AChR into membranes of cultured chick myotubes at a concentration of less than 0.5 ng/ml. Based on SDS PAGE and the action of neuraminidase, the acetylcholine receptor-inducing activity (ARIA) appears to be a 42,000-D glycoprotein. ARIA was extracted in a trifluoroacetic acid-containing cocktail and purified to homogeneity by reverse-phase, ion exchange, and size exclusion high pressure liquid chromatography. Dose response curves indicate that the activity has been purified 60,000-fold compared with the starting acid extract and approximately 1,500,000-fold compared with a saline extract prepared from the same batch of brains. Although the ARIA was purified on the basis of its ability to increase receptor incorporation, we found that it increased the number and size of receptor clusters as well. It is not yet clear if the two effects are independent. The 42-kD ARIA is extremely stable: it was not destroyed by exposure to intact myotubes, low pH, organic solvents, or SDS. Its action appears to be selective in that the increase in the rate of receptor insertion was not accompanied by an increase in the rate of protein synthesis. Moreover, there was no change in cellular, surface membrane, or secreted acetylcholinesterase. The effect of ARIA is apparently independent of the state of activity of the target myotubes as its effect on receptor incorporation added to that of maximal concentrations of tetrodotoxin.

scholarly journals Acetylcholine receptor aggregation parallels the deposition of a basal lamina proteoglycan during development of the neuromuscular junction.

1984 ◽  
Vol 99 (5) ◽  
pp. 1769-1784 ◽  
Author(s):  
M J Anderson ◽  
F G Klier ◽  
K E Tanguay
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Synapse Formation ◽  
Muscle Cells ◽  
Postsynaptic Membrane ◽  
Receptor Aggregation ◽  
Nerve Muscle ◽  
Alpha Bungarotoxin

To determine the time course of synaptic differentiation, we made successive observations on identified, nerve-contacted muscle cells developing in culture. The cultures had either been stained with fluorescent alpha-bungarotoxin, or were maintained in the presence of a fluorescent monoclonal antibody. These probes are directed at acetylcholine receptors (AChR) and a basal lamina proteoglycan, substances that show nearly congruent surface organizations at the adult neuromuscular junction. In other experiments individual muscle cells developing in culture were selected at different stages of AChR accumulation and examined in the electron microscope after serial sectioning along the entire path of nerve-muscle contact. The results indicate that the nerve-induced formation of AChR aggregates and adjacent plaques of proteoglycan is closely coupled throughout early stages of synapse formation. Developing junctional accumulations of AChR and proteoglycan appeared and grew progressively, throughout a perineural zone that extended along the muscle surface for several micrometers on either side of the nerve process. Unlike junctional AChR accumulations, which disappeared within a day of denervation, both junctional and extrajunctional proteoglycan deposits were stable in size and morphology. Junctional proteoglycan deposits appeared to correspond to discrete ultrastructural plaques of basal lamina, which were initially separated by broad expanses of lamina-free muscle surface. The extent of this basal lamina, and a corresponding thickening of the postsynaptic membrane, also increased during the accumulation of AChR and proteoglycan along the path of nerve contact. Presynaptic differentiation of synaptic vesicle clusters became detectable at the developing neuromuscular junction only after the formation of postsynaptic plaques containing both AChR and proteoglycan. It is concluded that motor nerves induce a gradual formation and growth of AChR aggregates and stable basal lamina proteoglycan deposits on the muscle surface during development of the neuromuscular junction.

scholarly journals Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields.

1985 ◽  
Vol 100 (1) ◽  
pp. 235-244 ◽  
Author(s):  
P W Luther ◽  
H B Peng
Keyword(s):  
Acetylcholine Receptor ◽  
Electric Fields ◽  
Basal Lamina ◽  
Field Exposure ◽  
Thin Sections ◽  
Fluorescence Image ◽  
Before And After ◽  
Dc Electric Field ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.

scholarly journals Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction

2008 ◽  
Vol 182 (6) ◽  
pp. 1201-1215 ◽  
Author(s):  
Hiroshi Nishimune ◽  
Gregorio Valdez ◽  
George Jarad ◽  
Casey L. Moulson ◽  
Ulrich Müller ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Synaptic Cleft ◽  
Postsynaptic Membrane ◽  
Laminin Receptor ◽  
Topological Transformation ◽  
Presynaptic Differentiation ◽  
Synaptic Maturation ◽  
Immunohistochemical Studies

A prominent feature of synaptic maturation at the neuromuscular junction (NMJ) is the topological transformation of the acetylcholine receptor (AChR)-rich postsynaptic membrane from an ovoid plaque into a complex array of branches. We show here that laminins play an autocrine role in promoting this transformation. Laminins containing the α4, α5, and β2 subunits are synthesized by muscle fibers and concentrated in the small portion of the basal lamina that passes through the synaptic cleft at the NMJ. Topological maturation of AChR clusters was delayed in targeted mutant mice lacking laminin α5 and arrested in mutants lacking both α4 and α5. Analysis of chimeric laminins in vivo and of mutant myotubes cultured aneurally demonstrated that the laminins act directly on muscle cells to promote postsynaptic maturation. Immunohistochemical studies in vivo and in vitro along with analysis of targeted mutants provide evidence that laminin-dependent aggregation of dystroglycan in the postsynaptic membrane is a key step in synaptic maturation. Another synaptically concentrated laminin receptor, Bcam, is dispensable. Together with previous studies implicating laminins as organizers of presynaptic differentiation, these results show that laminins coordinate post- with presynaptic maturation.

scholarly journals Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides.

1982 ◽  
Vol 92 (2) ◽  
pp. 333-342 ◽  
Author(s):  
P A St John ◽  
S C Froehner ◽  
D A Goodenough ◽  
J B Cohen
Keyword(s):  
Binding Sites ◽  
Electric Organ ◽  
Postsynaptic Membrane ◽  
Relative Molecular Mass ◽  
Alkaline Extraction ◽  
Before And After ◽  
Torpedo Electric Organ ◽  
Extracellular Side ◽  
Alpha Bungarotoxin

Experiments were conducted to examine the topographic arrangement of the polypeptides of the acetylcholine receptor (AcChR) and the nonreceptor Mr 43,000 protein in postsynaptic membranes isolated from Torpedo electric organ. When examined by electron microscopy, greater than 85% of vesicles were not permeable to ferritin or lactoperoxidase (LPO). Exposure to saponin was identified as a suitable procedure to permeabilize the vesicles to macromolecules with minimal alteration of vesicle size or ultrastructure. The sidedness of vesicles was examined morphologically and biochemically. Comparison of the distribution of intramembrane particles on freeze-fractured vesicles and the distribution found in situ indicated that greater than 85% of the vesicles were extracellular-side out. Vesicles labeled with alpha-bungarotoxin (alpha-Bgtx) were reacted with antibodies against alpha-BgTx or against purified AcChR of Torpedo. Bound antibodies were detected by the use of ferritin-conjugated goat anti-rabbit antibody and were located on the outside of greater than 99% of labeled vesicles. Similar results were obtained for normal vesicles or vesicles exposed to saponin. Quantification of the amount of [3H]-alpha-BgTx bound to vesicles before and after they were made permeable with saponin indicated that less than 5% of alpha-BgTx binding sites were cryptic in normal vesicles. It was concluded that greater than 95% of postsynaptic membranes were oriented extracellular-side out. LPO-catalyzed radioiodinations were performed on normal and saponin-treated vesicles and on vesicles from which the Mr (relative molecular mass) 43,000 protein had been removed by alkaline extraction. In normal vesicles, polypeptides of the AcChR were iodinated while the Mr 43,000 protein was not. In vesicles made permeable with saponin, the pattern of labeling of AcChR polypeptides was unchanged, but the Mr 43,000 protein was heavily iodinated. The relative iodination of AcChR polypeptides was unchanged in membranes equilibrated with agonist or with alpha-BgTx or after alkaline-extraction. It was concluded that the Mr 43,000 protein is present on the intracellular surface of the postsynaptic membrane and that AcChR polypeptides are exposed on the extracellular surface.

scholarly journals Magi-1c

2001 ◽  
Vol 153 (5) ◽  
pp. 1127-1132 ◽  
Author(s):  
Laure Strochlic ◽  
Annie Cartaud ◽  
Valérie Labas ◽  
Werner Hoch ◽  
Jean Rossier ◽  
...  
Keyword(s):  
Postsynaptic Membrane ◽  
Primary Role ◽  
Cross Link ◽  
Adult Rat ◽  
Molecular Events ◽  
The Central Nervous System ◽  
Bona Fide ◽  
Receptor Clusters ◽  
Postsynaptic Differentiation

The muscle-specific receptor tyrosine kinase (MuSK) forms part of a receptor complex, activated by nerve-derived agrin, that orchestrates the differentiation of the neuromuscular junction (NMJ). The molecular events linking MuSK activation with postsynaptic differentiation are not fully understood. In an attempt to identify partners and/or effectors of MuSK, cross-linking and immunopurification experiments were performed in purified postsynaptic membranes from the Torpedo electrocyte, a model system for the NMJ. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis was conducted on both cross-link products, and on the major peptide coimmunopurified with MuSK; this analysis identified a polypeptide corresponding to the COOH-terminal fragment of membrane-associated guanylate kinase (MAGUK) with inverted domain organization (MAGI)-1c. A bona fide MAGI-1c (150 kD) was detected by Western blotting in the postsynaptic membrane of Torpedo electrocytes, and in a high molecular mass cross-link product of MuSK. Immunofluorescence experiments showed that MAGI-1c is localized specifically at the adult rat NMJ, but is absent from agrin-induced acetylcholine receptor clusters in myotubes in vitro. In the central nervous system, MAGUKs play a primary role as scaffolding proteins that organize cytoskeletal signaling complexes at excitatory synapses. Our data suggest that a protein from the MAGUK family is involved in the MuSK signaling pathway at the vertebrate NMJ.

scholarly journals Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages.

1987 ◽  
Vol 104 (1) ◽  
pp. 97-108 ◽  
Author(s):  
D W Pumplin ◽  
R J Bloch
Keyword(s):  
Acetylcholine Receptor ◽  
Freeze Fracture ◽  
Bright Spots ◽  
Step Model ◽  
Before And After ◽  
Control Myotubes ◽  
Internalization Rate ◽  
Receptor Clusters ◽  
Alpha Bungarotoxin ◽  
Fixed Array

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.

162. FRIEND OR FOE? THE ROLES OF PAF AND p53 DURING EMBRYONIC DIAPAUSE

2010 ◽  
Vol 22 (9) ◽  
pp. 80
Author(s):  
J. C. Fenelon ◽  
C. O'Neill ◽  
G. Shaw ◽  
M. B. Renfree
Keyword(s):  
Tammar Wallaby ◽  
Early Embryo ◽  
Human Reproduction ◽  
Embryonic Diapause ◽  
Embryonic Cells ◽  
Active Growth ◽  
Molecular Control ◽  
Before And After ◽  
P53 Mrna

In the tammar wallaby, Macropus eugenii, the blastocyst normally remains in embryonic diapause for 11 months without cell division or apoptosis occurring. Progesterone regulates reactivation by inducing active secretion from the endometrium, but the molecular cross-talk between the endometrium and blastocyst is unknown. This process may involve the phospholipid paf. Paf is an embryotrophin that acts as a trophic/survival factor in the early embryo, partly by inactivating (via the PI3K/Akt pathway) the expression of p53, a cell cycle arrest factor (1,2). In vitro, paf production from the tammar endometrium increases after diapause (3). This study examined the expression of the paf receptor (pafr) and p53 in the tammar endometrium and embryo at entry into, during and reactivation from diapause. Both pafr and p53 mRNA were expressed in the endometrium at all stages. However there was no quantitative change in pafr expression. In the endometrium, pafr protein is present on the membrane of the glandular epithelium at all stages examined, but p53 was not expressed in the endometrial nuclei at any stage and hence does not appear to be active. Both pafr and p53 mRNA were also expressed in the embryo from the early cleavage stages, during diapause and in the reactivated blastocyst. Pafr protein was present in the embryo both before and after diapause, but levels were greatly reduced during diapause, indicating it may be necessary for active growth. Unexpectedly, the expression of p53 in the embryo does not appear to depend on the presence or absence of pafr. p53 was expressed in the nuclei of the cleavage stage embryonic cells before diapause, but not during or after diapause. These results suggest that paf and pafr may participate in the molecular control of embryonic diapause in the tammar independent of p53. (1) Jin XL et al. (2009) Biology of Reproduction 80: 286–294.(2) O’Neill C (2005) Human Reproduction Update 11(3): 215–228.(3) Kojima T et al. (1993) Reproduction Fertility Development 5: 15–25.

scholarly journals Neurotrophin signaling among neurons and glia during formation of tripartite synapses

2004 ◽  
Vol 1 (4) ◽  
pp. 339-349 ◽  
Author(s):  
SARINA B. ELMARIAH ◽  
ETHAN G. HUGHES ◽  
EUN JOO OH ◽  
RITA J. BALICE-GORDON
Keyword(s):  
Gabaa Receptor ◽  
Molecular Mechanisms ◽  
Synapse Formation ◽  
Synaptic Modulators ◽  
Central Synapses ◽  
Receptor Clusters ◽  
And Function ◽  
Neurotrophin Signaling

Synapse formation in the CNS is a complex process that involves the dynamic interplay of numerous signals exchanged between pre- and postsynaptic neurons as well as perisynaptic glia. Members of the neurotrophin family, which are widely expressed in the developing and mature CNS and are well-known for their roles in promoting neuronal survival and differentiation, have emerged as key synaptic modulators. However, the mechanisms by which neurotrophins modulate synapse formation and function are poorly understood. Here, we summarize our work on the role of neurotrophins in synaptogenesis in the CNS, in particular the role of these signaling molecules and their receptors, the Trks, in the development of excitatory and inhibitory hippocampal synapses. We discuss our results that demonstrate that postsynaptic TrkB signaling plays an important role in modulating the formation and maintenance of NMDA and GABAA receptor clusters at central synapses, and suggest that neurotrophin signaling coordinately modulates these receptors as part of mechanism that promotes the balance between excitation and inhibition in developing circuits. We also discuss our results that demonstrate that astrocytes promote the formation of GABAergic synapses in vitro by differentially regulating the development of inhibitory presynaptic terminals and postsynaptic GABAA receptor clusters, and suggest that glial modulation of inhibitory synaptogenesis is mediated by neurotrophin-dependent and -independent signaling. Together, these findings extend our understanding of how neuron–glia communication modulates synapse formation, maintenance and function, and set the stage for defining the cellular and molecular mechanisms by which neurotrophins and other cell–cell signals direct synaptogenesis in the developing brain.

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