Three-week-old neurons derived from BD and normal iPSCs were previously infected with a synapsin promoter-driven lentiviral vector expressing DsRed (Syn::DsRed). Cell cultures were washed twice with sterile Krebs HEPES Buffer and incubated with 3 μm Fluo 4-AM (Molecular Probes) in Krebs HEPES Buffer for 40 min at room temperature. Excess dye was removed by washing twice with Krebs HEPES Buffer, and cells were incubated for an additional 20 min to equilibrate the intracellular dye concentration and allow de-esterification. Time-lapse image sequences (×100 magnification) of 3,000 frames were acquired at 28 Hz with a region of 336 pixels × 256 pixels using a Hamamatsu ORCA-ER digital camera (Hamamatsu Photonics) with a 488 nm (FITC (fluorescein isothiocyanate)) filter on an Olympus IX81 inverted fluorescence confocal microscope (Olympus Optical). To assess changes in calcium signalling in response to perturbation of neuronal activity, tetrodotoxin (1 μm) was applied by bath application. Images were acquired with MetaMorph 7.7 software (MDS Analytical Technologies). Images were subsequently processed using ImageJ software and custom written routines in Matlab 7.2 software (Mathworks).
