Influence of nerve on the formation and survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus muscle cells in culture

Abstract

Embryonic Xenopus muscle cells grown in culture develop discrete patches of high acetylcholine receptor (AChR) density. By following identified muscle cells after staining with fluorescent alpha-bungarotoxin, we have found that many of these AChR patches survive in a fixed position for several days. For AChR patches on the lower surface of the cell (the surface apposed to the culture dish), more than 60% of those which were followed beginning on day 2 survived for a further 4 days. The survival rate was greater when patches were followed from day 3 or later and was almost as high for AChR patches on the upper surface. New AChR patches also formed on all of the muscle cells. When muscle cells were cultured together with spinal cord cells, nerve-muscle contacts developed with a characteristic localization of AChRs along the path of contact. AChR patches did not form elsewhere on these contacted cells. Nerve-contacted muscle cells examined 2 to 3 days after adding spinal cord cells to established (2- to 5-day-old) muscle cultures also exhibited a marked reduction of AChR patches away from the site of contact. This reduction was not due to the nerve having contacted pre-existing AChR patches. Rather, the findings indicate that contact by an appropriate nerve inhibits the formation of AChR patches elsewhere on the contracted muscle cells and reduces the survival of pre-existing AChR patches. Nerve contact also inhibited the formation of cholinesterase (ChE) patches remote from the site of contact and appeared to cause some reduction in the survival of pre-existing ChE patches. Spontaneous twitching was not observed in these experiments, thereby indicating that the remote effects of nerve contact were not mediated by muscle action potentials or contraction. Such remote influences of the nerve may play a role in determining the pattern of innervation on individual muscle cells.

Distribution of synaptic specializations along isolated motor units formed in Xenopus nerve-muscle cultures

“…By contrast, muscle cells contacted by incompetent neurons often had no NARPs at all along their contacts and usually had broad AChR patches elsewhere on their surface (see Fig. lo), similar to those seen typically on noncontacted muscle cells (e.g., Moody-Corbett and Cohen, 1982). When some AChR stain was seen along the contacts made by incompetent neurons, it was either faint or appeared similar to the broad AChR patches associated with noncontacted muscle cells.…”

“…The method of preparing cultures was similar to that described previously Moody-Corbett and Cohen, 1982), with some modifications. Dorsal segments, including the spinal cord and myotomes, were removed from l-d-old X. luevis embryos (stages 22-28; Nieuwkoop and Faber, 1967) and placed in a collagenase solution (0.1-0.2 mg/ml in 67% L15) for up to 1 hr to facilitate isolation of the myotomes and spinal cord.…”

“…These studies have revealed that competent neurons, such as motor neurons and other cholinergic neurons, trigger profound changes in the distribution of AChRs on the surface of the muscle cells they innervate. Following net&e-muscle contact, AChRs accumulate in high density along the path of contact, while elsewhere on the muscle cell the survival of preexisting AChR patches is reduced and the formation of new AChR patches inhibited Frank and Fischbach, 1979;Moody-Corbett and Cohen, 1982;Kuromi and Kidokoro, 1984). As a result, AChRs become localized preferentially at sites of innervation, thereby permitting efficient synaptic transmission.…”

Acetylcholine receptors and sodium channels in denervated and botulinum‐toxin‐treated adult rat muscle.

“…Aneural embryonic muscle cells are chemosensitive to ACh and bind c-bungarotoxin (BTX) along their entire length (Devreotes & Fambrough, 1975;Kidokoro & Gruener, 1982). Extrajunctional regions of the muscle fibre membrane lose chemosensitivity following innervation (Diamond & Miledi, 1962;Gordon & Vrbova, 1975) with disappearance of extrajunctional receptors (Bevan & Steinbach, 1977) and restriction of ACh receptors to the end-plate region (Anderson & Cohen, 1977;Moody-Corbett & Cohen, 1982). Neuromuscular blockade mimicked the effect of denervation in preventing this loss of extrajunctional sensitivity (Gordon & Vrbova, 1975), indicating that the neural L. BAMBRICK AND T. GORDON effect on ACh receptor distribution in the developing muscle membrane is mediated by neuromuscular activity.…”

Potassium inward rectifier and acetylcholine receptor channels in embryonicXenopus muscle cells in culture

“…ACh receptor aggregates on the upper surface of the muscle cells, away from the site of contact with the culture dish, are more numerous in older cultures (more than 2 days after plating) ( Moody-Corbett and Cohen, 1982). To determine if inward rectifier channels were also present at these ACh receptor aggregates, we labeled the muscle cells with rhodamine-conjugated a-bungarotoxin.…”