ACCUMULATION OF ACETYLCHOLINE-RECEPTORS IS A NECESSARY CONDITION FOR NORMAL ACCUMULATION OF ACETYLCHOLINESTERASE DURING IN-VITRO NEUROMUSCULAR SYNAPTOGENESIS

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acety lcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes culture d in the presence of spinal cord cells from embryonic rat, numerous AC hE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters lar gely exceeds that of AChE clusters. Very few AChE clusters formed when such cc-cultures were treated with monoclonal antibodies (mAbs) again st the main immunogenic region (MIR) of the AChR, which provoke intern alization and degradation of the AChRs of the muscular membrane. The t otal levels of AChE and proportions of molecular forms were unaffected . We also used non-innervated myotubes in which addition of agrin, a p rotein normally synthesized by motoneurons, transported to nerve termi nals and inserted into the synaptic basal lamina, induces the formatio n of small clusters of AChE. When added to rat myotubes devoid of memb rane AChR, agrin-induced AChE clusters did not form. Finally, we analy sed the capacity of the variant of the C2 mouse muscle cell fine defic ient in AChR (1R-) to form clusters of AChE in cc-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prev ented clustering of AChR (anti-AChR antibodies, deficiency of the vari ant C2 cell line) also suppressed AChE clustering, We concluded that c lustering of AChR is a prerequisite for clustering of AChE, so that NM J formation implies the sequential accumulation of these two component s.