Phillips_1987_J.Neurocytol_16_785

Changes in the distribution of large acetylcholine receptor clusters (AChR-Cs) on developing fast-twitch fibres of the chicken posterior latissimus dorsi (PLD) muscle have been studied during the period of loss of polyneuronal innervation using fluorescein-conjugated alpha-bungarotoxin. Embryonic muscles were ultrasonically dissociated into single fibre fragments and presumptive fast-twitch fibres were distinguished from the minority of slow-type fibres in the PLD by immunofluorescence using an antibody against slow-type myosin. Whereas mature PLD muscle fibres are focally innervated, at embryonic day 11 (E11) many fibre fragments from the PLD displayed two or more large (longer than 2 micron) AChR-Cs. Double labelling with anti-neurofilament antibody suggested that most of these AChR-Cs (82 +/- 2%) were associated with neuromuscular contacts. There was a progressive decline in the number of large (synaptic) AChR-Cs per 1000 micron of fibre, from 3.2 +/- 0.5 at E11 to 0.4 +/- 0.1 at E18. No further decline occurred between E18 and one week post-hatch. Primary generation muscle cells identified at E11 and E16 by tritiated thymidine labelling showed a decline in the number of large AChR-Cs per 1000 micron proportional to that seen in the fibre population as a whole, suggesting that distributed synaptic AChR-Cs are eliminated from individual fibres as they mature. When embryos were treated with d-tubocurarine starting at E6 the loss of distributed AChR-Cs from fast-type PLD fibres between E11 and E14 did not occur, suggesting that neuromuscular activity may play an important role in establishing the focal synaptic site AChR-C.