For imaging, cultured RGCs were plated as a monolayer on 12-mm glass coverslips coated with poly-D-lysine (Sigma). Astrocytes were incubated with 5 µM Fura-2 AM plus 1 µM pluronic acid (Molecular Probes) for 30 min at room temperature. External solution contained 150 mM NaCl, 10 mM HEPES, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 22 mM sucrose, and 10 mM glucose (pH adjusted to 7.4 and osmolarity to 325 mOsm). Intensity images of 510 nm wavelength were taken at 340 nm and 380 nm excitation wavelengths using iXon EMCCD (DV887 DCS-BV, ANDOR technology, UK). Imaging Workbench version 6.0 (Indec System, CA, USA) was used for acquisition of intensity images and conversion to ratios as previously described [37 (link)].