Calcium imaging

CA1 pyramidal neurons (13-18 DIV) in slice culture co-expressing GCaMP6 and CyRFP1 were imaged in line-scan mode (500 Hz) to assess if they were healthy and responsive using a test stimulation of a single glutamate uncaging pulse at a dendritic spine. Using a different dendritic segment than the test spine, responsive CA1 neurons were then imaged in frame-scan mode (64 pixels per line, 7.8 Hz) before and after glutamate uncaging (720 nm, 60 pulses, 8 ms duration at 6 Hz, ~7 mW at sample) adjacent to the spine head at 27°C in ACSF containing the following (in mM): 10 Ca²⁺, 0 Mg²⁺, 5 MNI-glutamate, and 0.001 TTX. Neurons with high baseline GCaMP6f and neurons that exhibited large calcium transients extending across the dendritic shaft were excluded.