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The molecular mechanisms underlying synaptic formation are one of the central themes of modern neuroscience. The best-established role of acetylcholinesterase (AChE) is the termination of signal transmission in the neuromuscular junction (NMJ) and other cholinergic synapses by catalyzing hydrolysis of acetylcholine. Most of the AChE in the NMJ is bound to the specialized basal lamina located between the presynaptic membrane contributed by the nerve ending and the postsynaptic membrane belonging to skeletal muscle fiber. Both cells can therefore be a source of synaptic AChE. Current evidence strongly supports a muscular origin for synaptic AChE; however, the possibility of neural origin has never been eliminated and is supported by another line of evidence.

The objective of this work was to study the origin of synaptic AChE at the earlier stages of the formation of mammalian NMJ under in vitro conditions.

At least part of the synaptic AChE found in the NMJ at the earlier stages of its formation is contributed by the motor neuron.

In our study, an in vitro model was employed in which human muscle was co-cultured with the explants isolated from embryonic rat spinal cord. In these co-cultures the motor neurons originate from the rat spinal cord explants and form differentiated and long-lived NMJs with human muscle fibers. By using species-specific antibodies against human and rat AChE, respectively, we were able to distinguish between AChE of muscle (human) and neural (rat) origin. Species specificity of anti-AChE antibodies was tested by Western blotting. Phase-contrast microscopy and fluorescent identification of acetylcholine receptors were used for visualization of the NMJs.

A strong signal was observed after staining with anti-human AChE antibodies, which indicated a relatively strong muscular AChE contribution in the NMJ. The signal was also observed at the NMJ after the staining of rat AChE, suggesting neural origin of a part of synaptic AChE. The lower intensity of this signal suggests that the neural contribution was smaller than the muscular one. Quantitative analysis based on the intensities of the signals revealed that 62% of the examined NMJ were human (muscle) AChE-positive and 32% of them were rat (nerve) AChE-positive.

The above results support our hypothesis. The majority of AChE at the earlier stages of NMJ formation is of muscular origin; however, a small part is also contributed by the motor neuron.