Caracterização ultraestrutural de junções neuromusculares de diafragma de camundongos com disfunção colinérgica

Abstract

One of the most critical processes of cholinergic transmission involves the event of storage of acetylcholine (ACh) in synaptic vesicles (SVs) through the vesicular acetylcholine transporter (VAChT). To assess the functional importance of the VAChT in the cholinergic transmission, Prado and colleagues (2006) generated a strain of mice knockdown for the VAChT gene and these animals have an important deficit of muscle strength and motor performance. However, the structural characteristics of neuromuscular synapses of these animals have not been described. The aim of this study is to evaluate the morphofunctional changes of synaptic elements of neuromuscular junctions (NMJ) of diaphragm from adult mice with cholinergic dysfunction (knockdown for the vesicular acetylcholine transporter (VAChT) gene) under different stimuli paradigm. Using hypertonic stimulation paradigm (500 mM sucrose solution) we recorded miniature endplates potentials (MEPPs) in NMJ of diaphragm from VAChT WT and VAChT KDHOM mice. We observed that the MEPPs frequency was lower in KDHOM compared to WT. For optical analysis, NMJ were stimulated with hypertonic sucrose in the presence the dye FM1-43fx to stain synaptic vesicles recycling from readily releasable pool (RRP). We observed that nerve terminals of KDHOM diaphragm presented less fluorescence. For ultrastructural studies, the NMJ were stimulated with hypertonic sucrose, fixed in modified Karnovsky and processed for transmission electron microscopy (TEM). We observed a reduction in the total number of SVs/m2 of terminal and an altered distribution of SVs in nerve terminals of KDHOM mice. Using electrical stimulation paradigm (20 Hz/5 min) the NMJ were stimulated, fixed and processed for TEM. We observed an altered distribution of SVs in nerve terminals of KDHOM mice. Additionally, we conducted an ultrastructural study in non-stimulated NMJ and also observed altered distribution of SVs in nerve terminals of KDHOM mice. Finally, using TEM we compared the circumference, shape and distribution of SVs of electrically stimulated nerve terminals from VAChT WT and VAChT KDHOM mice untreated and WT treated with (±)-vesamicol (a VAChT blocker). We observed that the nerve terminals of VAChT KDHOM untreated and WT treated with vesamicol exhibited flattened and smaller SVs in relation to VAChT WT untreated. However, treatment with vesamicol did not alter the distribution of VSs. In conclusion, our results suggest that expression of VAChT is important for the vesicle recycling from RRP and distribution of VSs, while vesicular ACh content is important for maintaining the morphology of VSs from diaphragm nerve terminals.