One day before the experiment, D-luciferin potassium salt (Wako) was added to the culture medium (final concentration, 0.1 mM). On the day of the experiment, cultured cortical slices were placed in an incubation chamber (Tokai Hit UK-A16U, 35°C, 5% CO2) on a microscope stage. After identifying DsRed2- or EGFP-positive cells, the luciferase signals were captured by an EMCCD camera (Andor, iXon3) attached to an upright microscope (Nikon, FN-S2N) through a 20x objective lens (NA, 0.5). The signal to noise ratio was increased by 4 × 4 binning, gain 1,000, and 5- or 10- min exposure.
After confirming that the luciferase signals were stable on the microscope stage, the recording was started. The signals were recorded for 3 h before pharmacological, electrophysiological, and optogenetic stimulation, and the slices were further observed for more than 12 h. The luciferase signals were taken at 30-min intervals.
After confirming that the luciferase signals were stable on the microscope stage, the recording was started. The signals were recorded for 3 h before pharmacological, electrophysiological, and optogenetic stimulation, and the slices were further observed for more than 12 h. The luciferase signals were taken at 30-min intervals.