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.

Heparin blocks functional innervation of cultured human muscle by rat motor nerve.

In vitro innervated human muscle is the only experimental model to study synaptogenesis of the neuromuscular junction in humans. Cultured human muscle never contracts spontaneously but will if innervated and therefore is a suitable model to study the effects of specific neural factors on the formation of functional neuromuscular contacts. Here, we tested the hypothesis that nerve derived factor agrin is essential for the formation of functional synapses between human myotubes and motoneurons growing from the explant of embryonic rat spinal cord. Agrin actions were blocked by heparin and the formation of functional neuromuscular contacts was quantitated. At a heparin concentration of 25 µg/ml, the number of functional contacts was significantly reduced. At higher concentrations, formation of such contacts was blocked completely. Except at the highest heparin concentrations (150 µg/ml) neuronal outgrowth was normal indicating that blockade of neuromuscular junction formation was not due to neuronal dysfunction. Our results are in accord with the concept that binding of neural agrin to the synaptic basal lamina is essential for the formation of functional neuromuscular junctions in the human muscle.