NMJ analyses

For endplate band quantification, fluorescent images of α‐BTX‐stained right hemidiaphragms were captured with a Nikon Eclipse 80i fluorescent microscope, and AChR cluster distribution was analyzed. The endplate bandwidth of 20–40 bins per animal, obtained every 134 μm at the central region of each hemidiaphragm was measured with ImageJ. To analyze postsynaptic morphologies, z‐stack pictures were imaged at 1‐μm intervals with a Zeiss LSM 780 confocal microscope at the CMA Bio‐Bio facility, University of Concepcion, Chile. Maximal intensity projections images were reconstructed in 3D using ImageJ software and analyzed to determine the proportion of the different NMJ morphologies, grouped into “pretzels”, normal shapes with continuous stripes, “fragmented”, where pretzels are separated into > 4 fragments, “O” or “C” shaped, less complex postsynaptic densities with no internal stripes, and “dismantled”, structures with a shrinked shape. The morphology of > 200 NMJs was manually determined and expressed as the percentage of the total. The area and perimeter of > 60 AChR densities per animal were automatically determined in a 60 μm² ROI for each postsynaptic structure using ImageJ software. Representative 3D videos where acquired with Imaris Scene Viewer 7.6.4 software (Bitplane, Zurich, Switzerland).