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The redistribution of vesicles containing serotonin in leech neurons was studied using the fluorescent, scanning ion-conductance, and laser phase microscopy methods. During acetylcholine receptor (AChR) activation in Retzius neurons, the changes of Ca2+ desorption, cellular stiffness and the cell optical phase difference (OPD) were established. It was found that the amplitude of OPD changes in the near-membrane area (membrane and near-membrane of the cytoplasm layers) increases upon AChR activation and this is, possibly, associated with the neurons vesicle redistribution. The decrease in the cell stiffness upon AChR activation suggests the crucial role of cytoskeleton for vesicle transport and release. Ca2+ rise in the cytoplasm during AChR activation may regulate the mitochondrial recruitment to regions with high energy demand for vesicle trafficking.