Cells were loaded with 1 μM Fluo-4-AM (Thermo Fisher Scientific) in recording buffer, containing (in mM): CaCl2 1.2; KCl 2.67; NaCl 138; KH2PO4 1.47; Na2HPO4 8; D-glucose 5.6 (adapted from ref. 55). Cultures were incubated at 37 °C and 5% CO2 for 30 minutes and then imaged with an inverted confocal laser scanning microscope (Axiovert 100 M Carl Zeiss, combined with Zeiss LSM510 software) using a Plan-NEOFLUAR 20x objective lens (NA 0.50). 450 frames (61 frames per minute) were recorded per well, of which the first 200 frames represent baseline recordings, followed by 200 frames after acute pharmacological stimulation. Finally, 30 μM glutamate (50 frames) was added to distinguish neurons from non-neuronal cells56. Traces of non-neuronal cells, showing only a transient increase in fluorescent intensity upon glutamate addition, were discarded.
