Analysis of the mechanism for acetylcholine release at the synapse formed between rat sympathetic neurons in culture.

Superior cervical ganglion neurons (SCGNs) were isolated from 7-day-old rat SCG and cultured in MEM containing horse serum, fetal calf serum, and nerve growth factor. In this culture condition, it is well known that the SCGNs form cholinergic synapse. In 3-4 weeks cultured neurons, immunofluorescent staining for synaptophysin, a small synaptic vesicle associated protein, showed the presence of synaptophysin as small dots on the surface of the soma. Postsynaptic potentials could be recorded in 50-80% of the neurons responding to evoked action potentials elicited in neighboring neurons. Because of its relatively large cell size and the short distance to the terminal, this synapse is a useful model for studying the mechanisms of acetylcholine (ACh) release by introducing substances such as antibodies or selective inhibitors into the presynaptic neuron by means of the whole-cell clamp technique. In this model synapse we tested the possible role of myosin in ACh release. The distribution of myosin was studied by the immunofluorescent staining technique. Myosin was recognized by the anti-myosin II IgG at the same synaptic terminals that showed the presence of synaptophysin with its antibody. The functional blockade of myosin by the antibody itself, and that of myosin light chain kinase (MLCK) by a pseudosubstrate inhibitor of MLCK, SM-1, or by a selective inhibitor of MLCK, wortmannin, induced depression of synaptic transmission in a dose-dependent manner. These indicate that phosphorylation of myosin by MLCK may be necessary for ACh release mechanisms.