Formation and survival of a postsynaptic specialization in cultures of embryonic Xenopus nerve and muscle cells

Abstract

The formation and survival of nerve-induced clusters of acetylcholine receptors (AChRs) was monitored over a synaptogenic period of several days in cultures of myotomal muscle cells and spinal cord neurons derived from embryos of Xenopus laevis. AChRs were labeled with fluorescent α-bungarotoxin so that neurite-associated receptor patches (NARPs) could be viewed at daily intervals throughout the neuritic arbor of selected neurons. To avoid bleaching the NARPs and damaging the neurons, the intensity of the fluorescence excitation was reduced to 3%. Images were digitized and NARPs were measured with a computer-based image analysis system. Virtually all newly formed NARPs (>90%) were detected at the same time as neurite-muscle contact and in the same proximal-distal sequence as neuritic growth. Those which formed in 6- to 13-day-old cocultures had similar distributions with respect to length, area, intensity, and area × intensity to those which formed in 1- to 2-day-old cocultures. NARPs exhibited variable daily changes in these parameters but on average they grew and reached close to their ultimate values within 1–2 days. Almost all (>95%) survived as long as their contacts. In cases where NARP formation occurred on the same muscle on 2 or more different days, the ones which formed first were the most extensive. Spontaneous neurite withdrawal occurred mainly from young NARPs and resulted in their rapid disappearance. It is suggested that during the period when neurons grow and make new contacts with muscle cells there is no substantial change in their capacity to trigger the formation of new synaptic sites and maintain preexisting ones, and that the first-forming synapses on a muscle cell tend to be the largest because muscle cells have a limited capacity to generate postsynaptic membrane. Additional implications of the findings for synapse formation and elimination are discussed.