scholarly journals Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions.

1982 ◽  
Vol 93 (2) ◽  
pp. 417-425 ◽  
Author(s):  
M M Salpeter ◽  
S Spanton ◽  
K Holley ◽  
T R Podleski
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Marked Change ◽  
Brain Extract ◽  
Bottom Surface ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Em Autoradiography ◽  
Alpha Bungarotoxin

We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.

scholarly journals Agonist-induced myopathy at the neuromuscular junction is mediated by calcium.

1979 ◽  
Vol 82 (3) ◽  
pp. 811-819 ◽  
Author(s):  
J P Leonard ◽  
M M Salpeter
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Large Diameter ◽  
Mammalian Skeletal Muscle ◽  
Esterase Inhibitors ◽  
Bath Application ◽  
Esterase Inhibition ◽  
Muscle Necrosis ◽  
Alpha Bungarotoxin

Inactivation of cholinesterases at mammalian neuromuscular junctions (nmj) produces extensive muscle "necrosis." Fine-structurally, this myopathy begins near the nmj with an increase in large-diameter vesicles in the soleplasm, the dissolution of Z-disks, dilation of mitochondria, destruction of sarcoplasmic reticulum, and often a highly specific contracture of the muscle under the endplate. Since a Ca++-activated protease which specifically removes Z-disks is known to exist in mammalian skeletal muscle, we tested the possibility that the myopathy after esterase inactivation is due to the prolongation of acetylcholine lifetime and thus of Ca++ influx. We first produced the myopathy near endplates by inactivating esterases with diisopropylfluorophosphate (DFP) followed by nerve stimulation for 1--2 h in vitro. The myopathy was later mimicked by bath application of carbamylcholine without esterase inhibitors. This myopathy could be prevented by inactivating the acetylcholine receptors (AChR) with alpha-bungarotoxin (alpha-BGT) or by removing Ca++ from the bath with EGTA. These results favor the hypothesis that esterase inhibition leads to an agonist-induced myopathy, which is mediated by Ca++ and requires an intact AChR.

scholarly journals Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures.

1984 ◽  
Vol 32 (9) ◽  
pp. 973-981 ◽  
Author(s):  
B W Lubit
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Morphological Changes ◽  
Muscle Cells ◽  
High Concentration ◽  
Rat Muscle ◽  
Cytoplasmic Actin ◽  
High Concentrations

Previous immunocytochemical studies in which an antibody specific for mammalian cytoplasmic actin was used showed that a high concentration of cytoplasmic actin exists at neuromuscular junctions of rat muscle fibers such that the distribution of actin corresponded exactly to that of the acetylcholine receptors. Although clusters of acetylcholine receptors also are present in noninnervated rat and chick muscle cells grown in vitro, neither the mechanism for the formation and maintenance of these clusters nor the relationship of these clusters to the high density of acetylcholine receptors at the neuromuscular junction in vivo are known. In the present study, a relationship between beta-cytoplasmic actin and acetylcholine receptors in vitro has been demonstrated immunocytochemically using an antibody specific for the beta-form of cytoplasmic actin. Networks of cytoplasmic actin-containing filaments were found in discrete regions of the myotube membrane that also contained high concentrations of acetylcholine receptors; such high concentrations of acetylcholine receptors have been described in regions of membrane-substrate contact. Moreover, when primary rat myotubes were exposed to human myasthenic serum, gross morphological changes, accompanied by an apparent rearrangement of the cytoplasmic actin-containing cytoskeleton, were produced. Although whether the distribution of cytoplasmic actin-containing structures was influenced by the organization of acetylcholine receptor or vice versa cannot be determined from these studies, these findings suggest that in primary rat muscle cells grown in vitro, acetylcholine receptors and beta-cytoplasmic actin-containing structures may be somehow connected.

scholarly journals In vivo recovery of muscle contraction after alpha-bungarotoxin binding.

1975 ◽  
Vol 66 (1) ◽  
pp. 209-213 ◽  
Author(s):  
H C Fertuck ◽  
W Woodward ◽  
M M Salpeter
Keyword(s):  
Fine Structure ◽  
Neuromuscular Junction ◽  
Muscle Contraction ◽  
Acetylcholine Receptors ◽  
Actinomycin D ◽  
Recovery Period ◽  
Em Autoradiography ◽  
Alpha Bungarotoxin ◽  
In Vivo Recovery

Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.

An improved method for culturing myotubes on laminins for the robust clustering of postsynaptic machinery

2019 ◽  
Author(s):  
Marcin Pęziński ◽  
Patrycja Daszczuk ◽  
Bhola Shankar Pradhan ◽  
Hanns Lochmüller ◽  
Tomasz J. Prószyński
Keyword(s):  
Motor Neurons ◽  
High Throughput Screening ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Neuromuscular Disorders ◽  
Cluster Assembly ◽  
Achr Cluster ◽  
Robust Clustering ◽  
Coated Surfaces

AbstractMotor neurons form specialized synapses with skeletal muscle fibers, called neuromuscular junctions (NMJs). Cultured myotubes are used as a simplified in vitro system to study the postsynaptic specialization of muscles. The stimulation of myotubes with the glycoprotein agrin or laminin-111 induces the clustering of postsynaptic machinery that contains acetylcholine receptors (AChRs). When myotubes are grown on laminin-coated surfaces, AChR clusters undergo developmental remodeling to form topologically complex structures that resemble mature NMJs. Needing further exploration are the molecular processes that govern AChR cluster assembly and its developmental maturation. Here, we describe an improved protocol for culturing muscle cells to promote the formation of complex AChR clusters. We screened various laminin isoforms and showed that laminin-221 was the most potent for inducing AChR clusters, whereas laminin-121, laminin-211, and laminin-221 afforded the highest percentages of topologically complex assemblies. Human primary myotubes that were formed by myoblasts obtained from patient biopsies also assembled AChR clusters that underwent remodeling in vitro. Collectively, these results demonstrate an advancement of culturing myotubes that can facilitate high-throughput screening for potential therapeutic targets for neuromuscular disorders.

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

1982 ◽  
Vol 94 (3) ◽  
pp. 540-548 ◽  
Author(s):  
G S Sohal ◽  
W R Boydston
Keyword(s):  
Embryonic Development ◽  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Electron Microscopic Level ◽  
Oblique Muscle ◽  
Apparent Effect ◽  
Alpha Bungarotoxin ◽  
Superior Oblique Muscle ◽  
Superior Oblique

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.

A Simple In Vitro Model of Mechanical Injury of Confluent Cultured Endothelial Cells to Study Quantitatively the Repair Process

1986 ◽  
Vol 56 (02) ◽  
pp. 232-235 ◽  
Author(s):  
Claude Klein-Soyer ◽  
Alain Beretz ◽  
Régine Millon-Collard ◽  
Joseph Abecassis ◽  
Jean-Pierre Cazenave
Keyword(s):  
Endothelial Cells ◽  
In Vitro Model ◽  
Repair Process ◽  
Mechanical Injury ◽  
Brain Extract ◽  
Type I ◽  
Culture Dish ◽  
Vitro Model ◽  
Adhesive Proteins

SummaryA model of in vitro mechanical injury of confluent human endothelial cells (EC) in culture was developed. Human EC were obtained from umbilical veins and grown to confluence. Application on the EC monolayer of a calibrated disk of cellulose poly acetate paper resulted in removal of the EC, leaving a continuous subendothelial extracellular matrix (ECM) on the culture dish. The regeneration time depended on the original size of the lesion. Regeneration was similar with EC grown on different substrates such as human fibronectin, human subendothelial ECM, bovine collagen type I or surfaces coated with Transglutine®, a surgical glue containing adhesive proteins. A human brain extract containing growth factor activity accelerated significantly the repair of the lesion, especially at low serum concentration. This simple in vitro model of mechanical injury allows the quantitative study of the effects of matrices, growth factors and pharmacological agents on the repair process.

scholarly journals Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes.

1984 ◽  
Vol 98 (2) ◽  
pp. 498-506 ◽  
Author(s):  
S Bursztajn ◽  
G D Fischbach
Keyword(s):  
Acetylcholine Receptors ◽  
Prolonged Exposure ◽  
Addition Reaction ◽  
Surface Membrane ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Saline Extract ◽  
Permeabilized Cells ◽  
Alpha Bungarotoxin

Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.

scholarly journals Mesenchymal Cell Growth and Differentiation on a New Biocomposite Material: A Promising Model for Regeneration Therapy

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 458 ◽  
Author(s):  
Leslie Pomeraniec ◽  
Dafna Benayahu
Keyword(s):  
Fluorescein Isothiocyanate ◽  
Mesenchymal Cell ◽  
Biological Properties ◽  
Mesenchymal Cells ◽  
Nano Particles ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Tissue Organization ◽  
Proliferation And Differentiation

Mesenchymal stem cells serve as the body’s reservoir for healing and tissue regeneration. In cases of severe tissue trauma where there is also a need for tissue organization, a scaffold may be of use to support the cells in the damaged tissue. Such a scaffold should be composed of a material that can biomimic the mechanical and biological properties of the target tissues in order to support autologous cell-adhesion, their proliferation, and differentiation. In this study, we developed and assayed a new biocomposite made of unique collagen fibers and alginate hydrogel that was assessed for the ability to support mesenchymal cell-proliferation and differentiation. Analysis over 11 weeks in vitro demonstrated that the scaffold was biocompatible and supports the cells viability and differentiation to produce tissue-like structures or become adipocyte under differentiation medium. When the biocomposite was enriched with nano particles (NPs), mesenchymal cells grew well after uptake of fluorescein isothiocyanate (FITC) labeled NPs, maintained their viability, migrated through the biocomposite, reached, and adhered to the tissue culture dish. These promising findings revealed that the scaffold supports the growth and differentiation of mesenchymal cells that demonstrate their full physiological function with no sign of material toxicity. The cells’ functionality performance indicates and suggests that the scaffold is suitable to be developed as a new medical device that has the potential to support regeneration and the production of functional tissue.

scholarly journals Natural Formulations Provide Antioxidant Complement to Hyaluronic Acid-Based Topical Applications Used in Wound Healing

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1847
Author(s):  
Pooyan Makvandi ◽  
Caterina Caccavale ◽  
Francesca Della Sala ◽  
Stefania Zeppetelli ◽  
Rosanna Veneziano ◽  
...  
Keyword(s):  
Wound Healing ◽  
Hyaluronic Acid ◽  
Vitamin E ◽  
Culture Dish ◽  
Rheological Characterization ◽  
Tissue Culture Dish ◽  
Biological Studies ◽  
A Cell ◽  
Topical Applications

Hyaluronic acid (HA) promotes wound healing, and, accordingly, formulations based on HA have been widely used in regenerative medicine. In addition, naturally derived compounds, e.g., plant-based extracts and vitamin E, have exhibited antioxidant activity. In this study, a formulation containing hyaluronic acid, vitamin E, raspberry extract, and green tea was developed for potential topical applications, targeting wound healing. Rheological analysis was performed along with antioxidant and biological studies. The rheological characterization showed that the HA-based formulation is a thixotropic platform and possesses higher mechanical properties than the control formulation. To evaluate the wound healing potential of the formulation, an in vitro “wound healing” assay was carried out using human derived fibroblasts (HDF) with a cell-free gap on the tissue culture dish. The formulation showed better wound healing ability than the control formulation.

scholarly journals Constructing and Tuning Excitatory Cholinergic Synapses: The Multifaceted Functions of Nicotinic Acetylcholine Receptors in Drosophila Neural Development and Physiology

2021 ◽  
Vol 15 ◽  
Author(s):  
Justin S. Rosenthal ◽  
Quan Yuan
Keyword(s):  
Nervous System ◽  
Nicotinic Acetylcholine Receptors ◽  
Neural Development ◽  
Acetylcholine Receptors ◽  
Animal Studies ◽  
Neuromuscular Junctions ◽  
Nicotinic Acetylcholine ◽  
Molecular Features ◽  
Cholinergic Synapses

Nicotinic acetylcholine receptors (nAchRs) are widely distributed within the nervous system across most animal species. Besides their well-established roles in mammalian neuromuscular junctions, studies using invertebrate models have also proven fruitful in revealing the function of nAchRs in the central nervous system. During the earlier years, both in vitro and animal studies had helped clarify the basic molecular features of the members of the Drosophila nAchR gene family and illustrated their utility as targets for insecticides. Later, increasingly sophisticated techniques have illuminated how nAchRs mediate excitatory neurotransmission in the Drosophila brain and play an integral part in neural development and synaptic plasticity, as well as cognitive processes such as learning and memory. This review is intended to provide an updated survey of Drosophila nAchR subunits, focusing on their molecular diversity and unique contributions to physiology and plasticity of the fly neural circuitry. We will also highlight promising new avenues for nAchR research that will likely contribute to better understanding of central cholinergic neurotransmission in both Drosophila and other organisms.

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