The 2-photon calcium imaging setup was identical to a previously published design15 (link). Two-photon illumination was achieved with a Ti:Sapphire laser (Chameleon Vision II, Coherent) operating at 920nm. Fluorescence was acquired using a 40× 0.8 NA objective (LUMPLFLN40X/W, Olympus) and GaAsP PMTs (H10770PA-40, Hamamatsu) after passing through a dichroic (FF670-SDi01, Semrock), an IR filter (FF01–720sp, Semrock), reflected by a second dichroic (FF562-Di03, Semrock) and passing through a final bandpass filter (FF01–520/60, Semrock). The PMT output signal was amplified (Variable High Speed Current Amplifier; #59–179, Edmund Optics) and digitized (PXIe-7961R FlexRIO, National Instrument). The microscope was controlled by ScanImage (Vidrio Technologies) software using additional analog output units (PXIe-6341, National Instruments) for the laser power control and the scanners control. Double-distilled water was used as the immersion medium for the objective. Average beam power measured at the front of the objective was 60–160 mW. The region between the objective and imaging site was shielded from external sources of light using a black rubber tube. Horizontal scans of the laser were achieved using a resonant galvanometer (Thorlabs). Typical fields of view measured approximately 500×500 μm, and data was acquired at 30Hz.