The effect of electric stimulation of various frequencies and durations on microtubules in frog motor axon terminals.

Abstract

The cytoskeleton carries out many functions in cells of the nervous system, including synaptic forma� tion and function. Actin microfilaments, microtu� bules (MTs), and intermediate filaments are the main components of the cytoskeleton of motor axon termi� nals. Microfilaments form a network and are often linked to the presynaptic membrane and its active regions (1). Cytoskeletal MTs are mainly located in the central part of the axon terminal (2) and carry out the transport function. Exoand endocytoses involve reorganization of cytoskeletal structure in all parts of a motor axon terminal (3) and may be considered a morphologic expression of synaptic plasticity. However, reorganization of cytoskeleton during different types of synaptic activity is poorly studied. In this study, we investigated the response of MTs in a frog peripheral axon terminal to highand lowfre� quency electrical stimulations of various durations. Experiments were carried out on neuromuscular junc� tions in an isolated m. cutaneous pectoris of a frog Rana ridibunda. The neuromuscular preparation was put into a 2 cm 3 Plexiglas chamber and perfused with standard Ringer's solution (pH 7.2-7.4) for cold� blooded animals. Experiments were carried out at 22°C. The motor nerve was stimulated by 100 μs electric stimuli with a frequency of 10 or 100 impulses per sec� ond using an absorbing electrode. The duration of stimulation was 5 or 10 min. The neuromuscular prep� arations were fixated in a 4% paraformaldehyde solu� tion immediately after the stimulation or 10 min after the stimulation (after relaxation). The cytoskeleton was visualized using a Leica SP5 TCS MP confocal microscope. MTs were labeled with specific mono� clonal antibodies against tubulin (Sigma, United States) and polyclonal secondary antibodies conju� gated with the Atto�488 (Sigma, United States) and Atto�647 (Sigma, United States) fluorophores. Post� translational tubulin modifications were investigated using monoclonal antibodies against acetylated and tyrosinated tubulins (Sigma, United States). The mean fluorescence intensity (MFI) of tubulin in axon terminals was measured. At least 50 axon terminals were studied in each experiment. The analysis and sta� tistics were performed using the ImageJ and LAS AF software. We found that the electrical nerve stimulation increased MFI of the fluorescent marker in terminals, which indicated an increase in the number of poly� merized MTs (table). MFI changed depending on the duration and frequency of stimulation. Lowfre� quency (10 impulses per second) 10�min stimulation induced a significantly greater increase in MFI than 5�min stimulation. At the same time, no differences were found between the effects of 5�min and 10�min highfrequency stimulations (100 impulses per sec� ond). In the case of lowfrequency stimulation, MFI returned to its original level 10 min after relaxation. However, 10�min relaxation did not lead to a decrease in MFI and caused its further increase in the case of highfrequency stimulation. An increase in MFI of labeled tubulin was accompanied by an increase in the complexity of MTs in terminals and formation of branched MT networks. To confirm the hypothesis that stimulation of the neurotransmitter in an axon terminal is followed by de novo MT formation, we investigated the dynamics of MT with various posttranslational tubulin modifica� tions. The cytoskeletal function is affected by the post� translational processing of cytoskeleton elements (4). Newly polymerized MTs were found to have a tyrosine