Formation de novo and development of neuromuscular junctions in vitro.

The purpose of this study has been to examine, by means of isolation in vitro, the conditions under which neuromyal junctions develop. More than a hundred years ago Doyere (1840) observed in the water-bear Milnesium tardigradum that the nerve fibers terminate in characteristic eminences of muscle fibers. This observation, made on an arthropod, stimulated research into the connexion between muscle and nerve fiber, and resulted in abandonment of the older concept that motor nerves after having formed loops around the muscles return to the central nervous system as sensory pathways. Although investigations during the intervening century have identified and characterized morphologically the 'motor end plate' (as it was designated by Krause, 1863), and have revealed microscopic and fine-structural differences between types of motor endings, there is as yet no general agreement on the respective roles played by nerve, muscle and ambient influences in the development of the neuromuscular junction. The comprehensive light-microscope description of the motor end plate by Couteaux (1941, 1947) has been confirmed and extended at the electron microscopic level by Reger (1955) and subsequently by others (reviewed by Couteaux, 1960). A correlation of the structural and functional characteristics of the subneural apparatus has been proposed in a monograph by Csillik (1965). Experimental approaches in vivo to the nerve-muscle relationship using denervation have been reviewed by Gutman & Zelena (1962) and by Zelena (1962). In tissue culture studies it has been shown conclusively that skeletal muscle tissue can proceed to an advanced stage of morphological differentiation, coupled with function, in the absence of nervous tissue (cf. Murray, 1960, 1965). Crucial evidence to support these points has been accumulated from experiments utilizing explants of varying ages, primordial to adult, which commenced with the early work of the Lewises (1917) and has continued to the present day. Van Weel (1948) demonstrated that mesenchymal areas of the embryonic chick