Expression and distribution of acetylcholinesterase among the cellular components of the neuromuscular junction formed in human myotube in vitro.

The results of our recent investigations on the expression and distribution of acetylcholinesterase (EC. 3.1.1.7, AChE) in the experimental model of the in vitro innervated human muscle are summarized and discussed here. This is the only model allowing studies on AChE expression at all stages of the neuromuscular junction (NMJ) formation in the human muscle. Since it consists not only of the motor neurons and myotubes but also of glial cells, which are essential for the normal development of the motor neurons, NMJs become functional and differentiated in this system. We followed AChE expression at various stages of the NMJ formation and in the context of other events characteristic for this process. Neuronal and muscular part were analysed at both, mRNA and mature enzyme level. AChE is expressed in motor neurons and skeletal muscle at the earliest stages of their development, long before NMJ starts to form and AChE begins to act as a cholinergic component. Temporal pattern of AChE mRNA expression in motor neurons is similar to the pattern of mRNA encoding synaptogenetic variant of agrin. There are no AChE accummulations at the NMJ at the early stage of its formation, when immature clusters of nicotinic receptors are formed at the neuromuscular contacts and when occasional NMJ-mediated contractions are already observed. The transformation from immature, bouton-like neuromuscular contacts into differentiated NMJs with mature, compact receptor clusters, myonuclear accumulations and dense AChE patches begins at the time when basal lamina starts to form in the synaptic cleft. Our observations support the concept that basal lamina formation is the essential event in the transformation of immature neuromuscular contact into differentiated NMJ, with the accumulation of not only muscular but also neuronal AChE in the synaptic cleft.

Nonradioactive northern blotting for the determination of acetylcholinesterase mRNA. Comparison to the radioactive technique.

A sensitive nonradioactive northern blotting for the detection of acetylcholinesterase mRNA in mammalian tissues is described and compared to its radioactive version. Best results were obtained if digoxigenin labeled RNA probe was used for hybridization and CDP-Star™, a chemiluminescent alkaline phosphatase substrate, for detection. The described nonradioactive technique for acetylcholinesterase mRNA determination is as sensitive as the radioactive one, but requires no protection against radiation and is less time consuming. Because of higher stability of the labeled probe, nonradioactive technique is also more convenient from the standpoint of experimental planning.