Neurite Degeneration and Calcium Signaling

Neurite degeneration was defined by beading (swellings along neurite) and fragmentation as previously described [60 (link), 68 (link), 69 (link)]. Briefly, cultured neurons were incubated by ammonia with or without pre-incubation of the calpain inhibitor (ALLM, 10 μM, Sigma), the pan-caspase inhibitor (ZVAD, 20 μM, Sigma) and the GSK-3 inhibitor (AR-A014418, 10 μM, Sigma) for 30 min, then neuronal morphology was evaluated. For calcium release indication, calcein-AM and Fluo-4 (all purchased from Thermo Fisher Scientific, Carlsbad, CA, USA) was applied to cultured neurons. Briefly, neurons grown on coverslips were incubated with calcium indicator 2 μM calcein-AM for 30 min at 37°C and viewed through using a fluorescent microscopy.