To simultaneously measure action potential and wave propagation, we differentiated neurons as described above for an additional 4–8 weeks, loaded them with the intracellular calcium-sensitive dye Fluo-4AM, (10 mM, Molecular Probes, Eugene, OR, USA). After 30 min, non-bound dye was removed and cells were examined in a Nikon A1R laser scanning confocal microscope system (Nikon Corporation, Chiyoda, Tokyo, Japan) to identify spontaneous activity. To evoke action potentials, 50 mM KCl was added to the dish and signaling was recorded.47 To determine whether lithium pretreatment would alter signaling, sets of neurons (3 BP and 3 C) were cultured in the presence of LiCl (1 mM) for 24 h before loading and image acquisition. For image analysis, regions of interest were defined as cell bodies or axons, and wave amplitude and propagation were measured from 5 to 10 regions per sample.
