Functional Calcium Imaging in Hippocampal Cultures

For imaging studies of functional Ca²⁺ activity in primary hippocampal cultures, we used a Zeiss 510 NLO fluorescent confocal microscope (Carl Zeiss, Germany) with a W Plan-Apochromat 20 × /1.0 objective. This method allows visualization of the functional neural network architecture at the cellular level. Oregon Green 488 BAPTA-1 AM (OGB-1) (0.4 μM, Thermo Fisher, United States), which was used as a calcium sensor, was dissolved in DMSO (Sigma, Germany) with 4% Pluronic F-127 (Thermo Fisher, United States) and then added to the culture medium for 40 min at 37°C and 5% CO₂. OGB-1 was excited at 488 nm and recorded in the range of 500–530 nm. Time series of 512 × 512 pixel images of 420 × 420-μm fields of view were recorded at 2 Hz. A confocal pinhole of 1 airy unit was used to obtain an axial optical slice resolution of 1.6 μm. Detection and further analysis of Ca²⁺ oscillations was performed in the Astroscanner program. A more detailed description of the image analysis is provided in our previous articles (Vedunova et al., 2013 (link); Zakharov et al., 2013 (link)). The following parameters of spontaneous Ca²⁺ activity were taken into account: the percentage of functional active cells and the duration (s) and frequency (the amount of Ca²⁺ events/min) of Ca²⁺ oscillations.