Organotypic culture of embryonic electromotor system tissues from Torpedo marmorata

Abstract

An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system. The myotubes in tissue expiants, taken from the electric organ primordia of 33–38 mm body-length embryos prior to electrocyte differentiation, contract spontaneously on explantation and have electrogenic membranes. The myotubes subsequently lose these properties in vitro and can differentiate in the absence of neural tissue into immature electrocytes which have morphologically characteristic postsynaptic membranes. Isolated expiants of differentiated electric organ tissue from 60–100 mm body-length embryos can be maintained for 3 to 4 weeks in vitro but cellular outgrowth is minimal. In contrast, a rapid, dense outgrowth of cells and a subsequent regeneration of myotubes occurs when differentiated electric organ explants are co-cultured with electric lobe tissue from embryos of the same stage. Cellular outgrowth from differentiated electric-organ tissue expiants can be stimulated by spinal cord, medulla, cerebellum and heart tissues but a subsequent regeneration of myotubes has not been observed. Myotube regeneration in the presence of electric lobe tissue is maximal with tissue from 60–80 mm body-length embryos. The myotubes that regenerate from differentiated electric organ expiants have not been observed to differentiate into electrocytes. Neuritic outgrowth in vitro occurs with electric lobe tissue taken at two different embryonic stages. The first stage corresponds to a period when most of the neuroepithelial cells in the lobe anlagen are withdrawing from the mitotic cycle and projecting axons into the branchial arches. The second, later stage is when the electromotorneurones are normally generating axon collaterals that are invading the interelectrocyte space of electrocyte columns. Maximum neuritic outgrowth at this second, later stage is obtained with tissue from 60–80 mm body-length embryos. Although neuritic invasion of electrocyte column expiants can be obtained in electric organ—electric lobe co-cultures at this later stage, synapses similar to those observed during the early stages of synaptogenesis in the electric organs in vivo have not been observed in vitro.