LS-2-P-3405 Effects of Electrical Stimulation at Different Frequencies on the Cytoskeleton of Frog Nerve Terminals

The cytoskeleton plays a crucial role in a variety of important cellular processes including the functioning of the neuromuscular endings. Two main classes of the cytoskeletal structures: microtubules and microfilament involved in all main stages of synapse maturation and function. At the mouse model it was shown that the NMJ are normally able to switch between modes of synaptic transmission that require different biochemical pathways, depending on the frequency of stimulation [1]. At the same time response of a cytoskeleton on electrical stimulation at various frequencies remains insufficiently studied. The present study aimed to investigate changes of tubuline and actin cytoskeleton in the motor nerve terminals of frogs in response to electrical nerve stimulation at different stimulus frequencies. The experiments were done on the muscle m. cutaneus pectoris of Rana ridibunda. The nerve of the dissected muscle was stimulated with a frequency of 10 Hz or 100 Hz during 5 min or 10 min. Then the muscle was fixed by 4% PFA. Synaptic sites were identified by staining acetylcholine receptors with a-bungarotoxin conjugated with TRITC. Primary antibodies included rabbit polyclonal antibodies against actin, mouse monoclonal antibodies against beta-tubuline. Secondary antibodies were conjugated with ATTO647N and ATTO488 dyes. Visualization of cytoskeletal elements was carried out by means of laser confocal microscope Leica SP5. The obtained data processed statistically and analyzed by means of the LAS AF 4.0 and ImageJ 1.42 programs. The morphology of microtubules and microfilaments bundles and the average mean of fluorescence in the nerve terminals were estimated. We found that electric stimulation of a nerve induces the changes of the neuronal endings cytoskeleton depending on both stimulus frequency and stimulation duration. The increase dyes-labeled MTs was observed in nerve terminals after stimulation. In addition MT bundles became more branched (Fig. 1). The actin cytoskeleton density was decreased in nerve terminals depending on nerve stimulation frequency (Fig. 2). It was shown that the F-actin-based network may participate in creating a scaffold for SV clustering, and in supporting ordered vesicle mobility [2]. Ours results are consistent with a role of the cytoskeleton in the synaptic vesicle recycling pathway and the transport of cargo by molecular motors.

[1] Maeno-Hikichi Y. et al. Frequency-Dependent Modes of Synaptic Vesicle Endocytosis and Exocytosis at Adult Mouse Neuromuscular Junctions // The Journal of Neuroscience, 2011, 31(3):1093–1105.

[2] Hirokawa N. et al. The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1 // J Cell Biol, 1989, 108: 111–126.