scholarly journals Degradation rates of acetylcholine receptors can be modified in the postjunctional plasma membrane of the vertebrate neuromuscular junction.

1986 ◽  
Vol 103 (4) ◽  
pp. 1399-1403 ◽  
Author(s):  
M M Salpeter ◽  
D L Cooper ◽  
T Levitt-Gilmour
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Degradation Rate ◽  
Postsynaptic Membrane ◽  
Nerve Crush ◽  
Adult Mice ◽  
Vertebrate Muscle ◽  
Degradation Rates ◽  
Alpha Bungarotoxin ◽  
Two Populations

Denervation of vertebrate muscle causes an acceleration of acetylcholine receptor turnover at the neuromuscular junction. This acceleration reflects the composite behavior of two populations of receptors: "original receptors" present at the junction at the time of denervation, and "new receptors" inserted into the denervated junction to replace the original receptors as they are degraded (Levitt, T. A., and M. M. Salpeter, 1981, Nature (Lond.), 291:239-241). The present study examined the degradation rate of original receptors to determine whether reinnervation could reverse the effect of denervation. Sternomastoid muscles in adult mice were denervated by either cutting or crushing the nerve, and the nerves either allowed to regenerate or ligated to prevent regeneration. The original receptors were labeled with 125I-alpha-bungarotoxin at the time of denervation, and their degradation rate followed by gamma counting. We found that when the nerve was not allowed to regenerate, the degradation decreased from a t1/2 of approximately 8-10 d to one of approximately 3 d (as reported earlier for denervated original receptors) and remained at that half-life throughout the experiment (approximately 36 d). If the axons were allowed to regenerate (which occurred asynchronously between day 14 and day 30 after nerve cut and between day 7 and 13 after nerve crush), the accelerated degradation rate of the original receptors reverted to a t1/2 of approximately 8 d. Our data lead us to conclude that the effect of denervation on the degradation rate of original receptors can be reversed by reinnervating. The nerve can thus slow the degradation rate of receptors previously inserted into the postsynaptic membrane.

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 Processing of ARIA and release from isolated nerve terminals

1999 ◽  
Vol 354 (1381) ◽  
pp. 411-416 ◽  
Author(s):  
Bomie Han ◽  
Gerald D. Fischbach
Keyword(s):  
Neuromuscular Junction ◽  
Action Potential ◽  
Acetylcholine Receptors ◽  
Molecular Layer ◽  
Postsynaptic Membrane ◽  
High Density ◽  
Small Contribution ◽  
Presynaptic Nerve Terminal ◽  
Voltage Gated

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage–gated Na + channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage–gated Na + channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo . ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

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.

scholarly journals Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium

1997 ◽  
Vol 138 (1) ◽  
pp. 159-165 ◽  
Author(s):  
James P. O'Malley ◽  
Charlotte T. Moore ◽  
Miriam M. Salpeter
Keyword(s):  
Neuromuscular Junction ◽  
Half Life ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Muscle Membrane ◽  
Intracellular Camp ◽  
Signaling Systems ◽  
Camp Analogue ◽  
Degradation Rates ◽  
The Stability

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ∼1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ∼3–5 d) and a stabilized form (half-life ∼10 d), depending upon the state of innervation of the muscle. Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca2+, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

scholarly journals Effect of agrin on the distribution of acetylcholine receptors and sodium channels on adult skeletal muscle fibers in culture.

1991 ◽  
Vol 115 (3) ◽  
pp. 765-778 ◽  
Author(s):  
M T Lupa ◽  
J H Caldwell
Keyword(s):  
Skeletal Muscle ◽  
Acetylcholine Receptors ◽  
Muscle Fibers ◽  
Postsynaptic Membrane ◽  
Na Channel ◽  
High Concentration ◽  
Achr Cluster ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers ◽  
Alpha Bungarotoxin

We used the loose patch voltage clamp technique and rhodamine-conjugated alpha-bungarotoxin to study the regulation of Na channel (NaCh) and acetylcholine receptor (AChR) distribution on dissociated adult skeletal muscle fibers in culture. The aggregate of AChRs and NaChs normally found in the postsynaptic membrane of these cells gradually fragmented and dispersed from the synaptic region after several days in culture. This dispersal was the result of the collagenase treatment used to dissociate the cells, suggesting that a factor associated with the extracellular matrix was responsible for maintaining the high concentration of AchRs and NaChs at the neuromuscular junction. We tested whether the basal lamina protein agrin, which has been shown to induce the aggregation of AChRs on embryonic myotubes, could similarly influence the distribution of NaChs. By following identified fibers, we found that agrin accelerated both the fragmentation of the endplate AChR cluster into smaller patches as well as the appearance of new AChR clusters away from the endplate. AChR patches which were fragments of the original endplate retained a high density of NaChs, but no new NaCh hotspots were found elsewhere on the fiber, including sites of newly formed AChR clusters. The results are consistent with the hypothesis that extracellular signals regulate the distribution of AChRs and NaChs on skeletal muscle fibers. While agrin probably serves this function for the AChR, it does not appear to play a role in the regulation of the NaCh distribution.

scholarly journals An unusual beta-spectrin associated with clustered acetylcholine receptors.

1989 ◽  
Vol 108 (2) ◽  
pp. 481-493 ◽  
Author(s):  
R J Bloch ◽  
J S Morrow
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Room Temperature ◽  
Peptide Mapping ◽  
Postsynaptic Membrane ◽  
Human Erythrocytes ◽  
Early Event ◽  
Alpha Spectrin

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane is an early event in the formation of the neuromuscular junction. The mechanism of clustering is still unknown, but is generally believed to be mediated by the postsynaptic cytoskeleton. We have identified an unusual isoform of beta-spectrin which colocalizes with AChR in AChR clusters isolated from rat myotubes in vitro. A related antigen is present postsynaptically at the neuromuscular junction of the rat. Immunoprecipitation, peptide mapping and immunofluorescence show that the beta-spectrin in AChR clusters resembles but is distinct from the beta-spectrin of human erythrocytes. alpha-Spectrin appears to be absent from AChR clusters. Semiquantitative immunofluorescence techniques indicate that there are from two to seven beta-spectrin molecules present for every clustered AChR, the higher values being obtained from rapidly prepared clusters, the lower values from clusters that require several minutes or more for isolation. Upon incubation of isolated AChR clusters for 1 h at room temperature, beta-spectrin is slowly depleted and the AChR redistribute into microaggregates. The beta-spectrin that remains associated with the myotube membrane is concentrated at these microaggregates. beta-Spectrin is quantitatively lost from clusters upon digestion with chymotrypsin, which causes AChR to redistribute in the plane of the membrane. These results suggest that AChR in clusters is closely linked to an unusual isoform of beta-spectrin.

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

1990 ◽  
Vol 111 (2) ◽  
pp. 655-661 ◽  
Author(s):  
S Rotzler ◽  
H R Brenner
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Muscle Activity ◽  
Acetylcholine Receptors ◽  
Muscle Stimulation ◽  
Cluster Growth ◽  
Rat Skeletal Muscle ◽  
Achr Cluster ◽  
End Plate ◽  
Alpha Bungarotoxin

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.

scholarly journals The Metabolic Stability of the Nicotinic Acetylcholine Receptor at the Neuromuscular Junction

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 358
Author(s):  
Isabel Martinez-Pena y Valenzuela ◽  
Mohammed Akaaboune
Keyword(s):  
Neuromuscular Junction ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Turnover Rate ◽  
State Of The Art ◽  
Postsynaptic Membrane ◽  
Metabolic Stability ◽  
Synaptic Activity ◽  
Nicotinic Acetylcholine

The clustering and maintenance of nicotinic acetylcholine receptors (AChRs) at high density in the postsynaptic membrane is a hallmark of the mammalian neuromuscular junction (NMJ). The regulation of receptor density/turnover rate at synapses is one of the main thrusts of neurobiology because it plays an important role in synaptic development and synaptic plasticity. The state-of-the-art imaging revealed that AChRs are highly dynamic despite the overall structural stability of the NMJ over the lifetime of the animal. This review highlights the work on the metabolic stability of AChRs at developing and mature NMJs and discusses the role of synaptic activity and the regulatory signaling pathways involved in the dynamics of AChRs.

scholarly journals Subtle Neuromuscular Defects in Utrophin-deficient Mice

1997 ◽  
Vol 136 (4) ◽  
pp. 871-882 ◽  
Author(s):  
R. Mark Grady ◽  
John P. Merlie ◽  
Joshua R. Sanes
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Cultured Cells ◽  
Neuromuscular Junctions ◽  
Becker Muscular Dystrophy ◽  
Postsynaptic Membrane ◽  
Mutant Mice ◽  
Nonmuscle Cells ◽  
And Behavior

Utrophin is a large cytoskeletal protein that is homologous to dystrophin, the protein mutated in Duchenne and Becker muscular dystrophy. In skeletal muscle, dystrophin is broadly distributed along the sarcolemma whereas utrophin is concentrated at the neuromuscular junction. This differential localization, along with studies on cultured cells, led to the suggestion that utrophin is required for synaptic differentiation. In addition, utrophin is present in numerous nonmuscle cells, suggesting that it may have a more generalized role in the maintenance of cellular integrity. To test these hypotheses we generated and characterized utrophin-deficient mutant mice. These mutant mice were normal in appearance and behavior and showed no obvious defects in muscle or nonmuscle tissue. Detailed analysis, however, revealed that the density of acetylcholine receptors and the number of junctional folds were reduced at the neuromuscular junctions in utrophin-deficient skeletal muscle. Despite these subtle derangements, the overall structure of the mutant synapse was qualitatively normal, and the specialized characteristics of the dystrophin-associated protein complex were preserved at the mutant neuromuscular junction. These results point to a predominant role for other molecules in the differentiation and maintenance of the postsynaptic membrane.

Acetylcholine receptors at the rat neuromuscular junction as revealed by deep etching

1982 ◽  
Vol 215 (1199) ◽  
pp. 147-154 ◽  
Keyword(s):  
Neuromuscular Junction ◽  
Basal Lamina ◽  
Acetylcholine Receptors ◽  
Postsynaptic Membrane ◽  
Muscle Fibres ◽  
Nerve Terminals ◽  
Single Muscle ◽  
Deep Etching ◽  
Collagenase Treatment ◽  
Regular Arrangement

Collagenase treatment of rat intercostal muscles yielded single muscle fibres in which the nerve terminals and basal lamina were removed allowing an unimpeded view of the ecternal surface of the postsynaptic membrane. This was revealed by deep etching of freeze-fractured preparations and appeared as a maze of folds separated by deep troughs, showing on the crests of the folds a densely packed population of protrusions about 8⋅5 nm in diameter. These densely packed protrusions ( ca . 9000 μm -2 ) are mainly confined to the postsynaptic regions of the sarcolemma and presumably represent the acetycholine receptor molecules, which are highly concenrated in these areas. The protrusions are generally tightly packed without obvious regular arrangement, but in some areas, usually on the tops of the crests, they are arranged into irregular rows normal to the long axis of the folds.

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