Action potentials (APs) (83 Hz) were evoked by field stimulation with a Grass Technologies S48 stimulation unit and a custom-built 24-well cap stimulator with pairs of parallel platinum wires. The microscope was an Olympus IX81 with a 10× (0.4 NA) air objective lens and EMCCD camera (Andor 897, 512 × 512 pixels, 35 frames/s), Cairn OptoLED illumination system, and GFP (Excitation: 450-490 nm; Dichroic: 495 nm long-pass; Emission: 500-550 nm) and TxRed (Excitation: 540-580 nm; Dichroic: 585 nm long-pass; Emission: 593-668 nm) filter sets. The field of view was 800 μm × 800 μm. Images were background subtracted (mean of 5% lowest pixel values). Responses were quantified for each cell as change in fluorescence divided by baseline fluorescence measured one second prior to stimulation. Signal-to-noise ratio (SNR) was quantified as peak ΔF/F0 response over the standard deviation of the signal during a one second period prior to stimulation.
