In vitro formation of neuromuscular junctions between adult Rana muscle fibres and embryonic Xenopus neurons

Abstract

Adult muscle fibres of the frog Rana temporaria were cultured with neurons from embryos of the frog Xenopus laevis . Electron microscopical and electro-physiological examination of the cultures showed that hetero-specific ( Xenopus- Rana ) neuromuscular junctions were formed in vitro . Nerve processes, without any Schwann cell covering, made contacts anywhere along a muscle fibre, and the junctions resembled those seen during early regeneration of neuromuscular synapses in situ . Functional contacts, as inferred by the presence of spontaneous miniature endplate potentials, or currents, were more common if the muscle fibres were denervated prior to culturing with neurons. Miniature endplate currents (m. e. p. cs) had a skewed amplitude distribution, with many small events lost in the recording noise, and their mean amplitude was much smaller than that of m. e. p. cs in the original lumbricalis muscle. The time constant of decay of m. e. p. cs in the hetero-specific junctions formed in vitro was several times longer than the decay of m. e. p. cs in the original muscle. Analysis of membrane current noise elicited by ionophoretically applied acetylcholine (ACh) suggests that the slower decay of m. e. p. cs in the junctions formed in vitro is due to a prolonged lifetime of the channels opened by ACh and to repetitive activation of ACh-receptors, which becomes possible because of a comparative lack of cholinesterase in the junctions.