Neuromuscular junction assessment
After perfusion, the TA muscle was collected and underwent cryoprotection in PB 30% sucrose solution. To label neuromuscular junctions (NMJ), the muscle was sectioned in longitudinal slices of 50 µm thickness, collected in sequential series. Following a blocking step using 5% normal donkey serum, the sections were incubated for 48 hours at 4°C with primary antibodies, chicken anti-neurofilament 200 (NF200, 1:1000; AB5539, Millipore) and rabbit anti-synaptophysin (1:500; AB32127, Abcam). After washing steps, the sections were incubated overnight with secondary antibodies Alexa Fluor 594-conjugated secondary antibody (1:200; A11042-A21207, Invitrogen) and Alexa Fluor 488-conjugated α-bungarotoxin (1:500; B13422, Life Technologies). The prepared slides were then mounted using Fluoromount-G. Images were captured under confocal microscopy (LSM 700 Axio Observer, Carl Zeiss, 20x with a numerical aperture of 0.5), and maximum projection images were generated from z projections with a thickness of 1.3 µm. To evaluate the proportion of innervated NMJs, each individual endplate was classified as either occupied (when presynaptic terminals covered the endplate) or vacant (when there was no presynaptic label in contact with the endplate). A total of at least 60 endplates from four different fields were analyzed for each mouse.