The distribution of intracellular acetylcholine receptors and nuclei in developing avian fast-twitch muscle fibres during synapse elimination

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Abstract
The spatial distribution of intracellular acetylcholine receptors along the length of fibres from the avian posterior latissimus dorsi muscle has been investigated during embryonic development, when distributed synaptic sites are eliminated from the muscle fibres. Cell surface AChR were irreversibly blocked with unlabelled α-bungarotoxin (α-BGT). Muscles were then fixed and ultrasonically dissociated into fibre fragments, treated with 0.5% saponin and stained with125I-α-BGT. This revealed an intracellular pool of curare sensitive binding sites equivalent to about 10% of total cell AChR. The spatial distribution of this pool was studied by autoradiography. Large (longer than 2μ.m) AChR-clusters (AChR-C) characteristic of neuromuscular contacts were localized on the same fibres by immunofluorescence with an anti-AChR antibody. At E11, relatively high levels of intracellular AChR were observed throughout the length of fibres. Between E11 and E18 intracellular AChR declined (19 fold) in extrajunctional parts of fibres but remained high in segments of fibre corresponding to AChR-clusters. Treatment of E14 embryos with an inhibitor of protein synthesis (cycloheximide) reduced intracellular AChR to 22 ± 6% (mean ± SE) of control levels, suggesting that most of the intracellular binding represented newly-synthesized AChR. Between E11 and E18 cell nuclei were found to accumulate beneath AChR-C. The mean density of nuclei in segments of fibre corresponding to AChR-C increased 5 fold between E11 and E18, but remained unchanged in extrajunctional segments. It is suggested that the elimination of excess distributed AChR-C may be due to the preferential accumulation of nuclei at a single AChR-C on each fibre accompanied by the down regulation of AChR synthesis associated with nuclei at the remaining AChR-C.