Calcium imaging experiments were performed with hippocampal neurons at 18-21 DIV (at least one week post-transduction with AAV2-retro-jGCaMP7f). Images were acquired by a sCMOS camera Prime 95B, 22 mm (Teledyne Photometrics, UK), mounted on a Nikon Eclipse Ti2-E (Nikon, Japan) inverted microscope with a Nikon Achro ADI 10X/0.25NA, a Nikon Pl Apo 20X/0.75NA, or a Nikon Apo LWD λ S 40X/1.15NA (water-immersion) objective. Image acquisition was performed using Micromanager (Version 1.4) at 200 Hz (5 ms exposure), which allowed for the temporal discrimination of soma depolarizations in response to single electrical pulse stimulations. Electrical stimulations were performed using the MEA2100-256 system's (MCS, Germany) internal stimulator. Per trial, five biphasic voltage pulses (-500/500 to -1000/1000 mV, 100 µs per phase) were delivered to the last microelectrode within a microchannel at or 1 Hz. To synchronize electrical stimulation, recording, and fast image acquisition, the whole setup was triggered via a transistor-transistor logic signal sent at the start of the MEA recording/stimulation protocol.
The resulting videos were median-filtered, regions of interest (i.e. somas, axons) were delineated manually and ∆F/F 0 traces were calculated in ImageJ using custom macros. ∆F/F traces were exported for analysis in MATLAB 2018a (The Mathworks, Inc., USA).
The resulting videos were median-filtered, regions of interest (i.e. somas, axons) were delineated manually and ∆F/F 0 traces were calculated in ImageJ using custom macros. ∆F/F traces were exported for analysis in MATLAB 2018a (The Mathworks, Inc., USA).