We used a two-photon excitation scanning microscope controlled by Slidebook (ver. 6, 3i) with a Ti:sapphire laser (Chameleon Vision, Coherent) at 920 nm and a 40× objective (0.8 numerical aperture, NIR Apo, Nikon). For each brain area imaged, we aimed to capture the full extent of the volume of labeled neurons, using a maximum step-size of 4 μm between imaging planes, and maintained a volume-rate of at least 1 Hz. Image resolution varied depending on the number of planes captured but was not less than 100 pixels in the longest dimension. We recorded frame capture markers and stimulus events on a DAQ (6259, NI) sampling at 10 kHz.

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