Calcium imaging

Primary hippocampal neurons were seeded onto poly-L-lysine coated 22 × 22 mm glass coverslips and cultured for 14 days. The cells (DIV14) were loaded with 2 μM Fura-2/AM (Invitrogen) in HBSS buffer (100 mM NaCl, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, 1 mM NaH₂PO₄, 4.2 mM NaHCO₃, 12.5 mM HEPES, and 10 mM glucose at pH 7.4) and incubated at 37 °C for 45 min. The excess Fura-2/AM was washed out 3 times with HBSS and incubated at 37 °C in HBSS for at least 30 min prior to imaging. The images were acquired using a Zeiss Axiovert 200 inverted fluorescence microscope equipped with an open flow chamber (filled with 1 ml HBSS buffer), heated stage, Xenon lamp, and Photomatrics Cool Snap HQ CCD (Photometrics, Tucson, AZ, USA). Fluorescence ratio of calcium images was analyzed using MetaFlour (Molecular Devices, Downington, PA, USA). The stabilization period of the resting cells was recorded for 5 min. Ca²⁺ bound- and Ca²⁺ free- Fura-2 images were acquired every 2 sec after exciting the cells at 340 nm and 380 nm, respectively. The emission wavelength was 510 nm. In general, 5–10 cells were presented and recorded per field. The intracellular concentration of Ca²⁺ was calculated from the 340/380 nm fluorescence ratios at each time point based on a previously described equation⁵⁸.