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 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 Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion.

1987 ◽  
Vol 105 (4) ◽  
pp. 1847-1854 ◽  
Author(s):  
M H Jacob ◽  
D K Berg
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Ciliary Ganglion ◽  
Ciliary Ganglion Neurons ◽  
Membrane Components ◽  
Ganglion Neurons ◽  
Alpha Bungarotoxin ◽  
Binding Component

The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs.

scholarly journals Regulation of the Rapsyn Promoter by Kaiso and δ-Catenin

2004 ◽  
Vol 24 (16) ◽  
pp. 7188-7196 ◽  
Author(s):  
Marianna Rodova ◽  
Kevin F. Kelly ◽  
Michael VanSaun ◽  
Juliet M. Daniel ◽  
Michael J. Werle
Keyword(s):  
Transcription Factor ◽  
Neuromuscular Junction ◽  
Acetylcholine Receptors ◽  
Consensus Sequence ◽  
Specific Protein ◽  
C2c12 Cells ◽  
Congenital Myasthenic Syndrome ◽  
P120 Catenin ◽  
Myasthenic Syndrome

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.

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 Activity-Dependent Regulation of the Binomial Parameters p and n at the Mouse Neuromuscular Junction In Vivo

2010 ◽  
Vol 104 (5) ◽  
pp. 2352-2358 ◽  
Author(s):  
Xueyong Wang ◽  
Qingbo Wang ◽  
Kathrin L. Engisch ◽  
Mark M. Rich
Keyword(s):  
Neuromuscular Junction ◽  
Muscle Activity ◽  
Acetylcholine Receptors ◽  
Quantal Content ◽  
Spontaneous Release ◽  
Postsynaptic Activity ◽  
Activity Dependent

Block of neurotransmission at the mammalian neuromuscular junction triggers an increase in the number of vesicles released (quantal content). The increase occurs whether nerve and muscle activity are both blocked by placement of a tetrodotoxin (TTX) containing cuff on the nerve or whether muscle activity is selectively blocked by injection of α-bungarotoxin (BTX). We used ANOVA to examine whether the mechanism underlying the increase in quantal content differed between the two types of activity blockade. We found that TTX-induced blockade increased the probability of release ( p), whereas BTX-induced blockade increased the number of releasable vesicles ( n). The lack of increase in p when postsynaptic activity was blocked with BTX suggests that block of presynaptic activity triggers the increase. To determine whether n is regulated by mismatch of pre- and postsynaptic activity introduced by BTX injection we combined BTX and TTX and still found an increase in n. We conclude that block of acetylcholine binding to acetylcholine receptors during spontaneous release triggers the increase in n.

scholarly journals The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo

Development ◽  
2006 ◽  
Vol 133 (22) ◽  
pp. 4485-4493 ◽  
Author(s):  
E. G. Bruneau ◽  
M. Akaaboune
Keyword(s):  
Neuromuscular Junction ◽  
Acetylcholine Receptors

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 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-aggregating activity of agrin isoforms and mapping of the active site.

1995 ◽  
Vol 128 (4) ◽  
pp. 625-636 ◽  
Author(s):  
M Gesemann ◽  
A J Denzer ◽  
M A Ruegg
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Neuromuscular Junction ◽  
Motor Neurons ◽  
Active Site ◽  
Acetylcholine Receptors ◽  
Muscle Cells ◽  
Effective Concentration ◽  
Achr Clustering

Agrin is a basal lamina protein that induces aggregation of acetylcholine receptors (AChRs) and other molecules at the developing neuromuscular junction. Alternative splicing of chick agrin mRNA at two sites, A and B, gives rise to eight possible isoforms of which five are expressed in vivo. Motor neurons express high levels of isoforms with inserts at sites A and B, muscle cells synthesize isoforms that lack amino acids at the B-site. To obtain further insights into the mechanism of agrin-induced AChR aggregation, we have determined the EC50 (effective concentration to induce half-maximal AChR clustering) of each agrin isoform and of truncation mutants. On chick myotubes, EC50 of the COOH-terminal, 95-kD fragment of agrinA4B8 was approximately 35 pM, of agrinA4B19 approximately 110 pM and of agrinA4B11 approximately 5 nM. While some AChR clusters were observed with 64 nM of agrinA4B0, no activity was detected for agrinA0B0. Recombinant full-length chick agrin and a 100-kD fragment of ray agrin showed similar EC50 values. A 45-kD, COOH-terminal fragment of agrinA4B8 retained high activity (EC50 approximately equal to 130 pM) and a 21-kD fragment was still active, but required higher concentrations (EC50 approximately equal to 13 nM). Unlike the 45-kD fragment, the 21-kD fragment neither bound to heparin nor did heparin inhibit its capability to induce AChR aggregation. These data show quantitatively that agrinA4B8 and agrinA4B19, expressed in motor neurons, are most active, while no activity is detected in agrinA0B0, the dominant isoform synthesized by muscle cells. Furthermore, our results show that a fragment comprising site B8 and the most COOH-terminal G-like domain is sufficient for this activity, and that agrin domains required for binding to heparin and those for AChR aggregation are distinct from each other.

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