In Vitro Calcium Imaging in Short-Term and Long-Term Rat Hippocampal Cultures

Take a short-term rat hippocampal neuronal culture representing young neurons and a long-term culture representing aged neurons.

Aged neurons exhibit a higher density of L-type voltage-gated calcium channels, or L-VGCCs, which remain open for a longer duration upon membrane depolarization.

Incubate with a fluorescent calcium indicator that diffuses into the cells, then wash to remove any excess dye.

Place the cultures in a perfusion chamber under a fluorescence microscope.

Introduce a solution containing neurotransmitters that bind to receptors on the neurons, inducing the influx of sodium ions and triggering membrane depolarization.

The depolarization activates the L-VGCCs, enabling calcium influx into the cytoplasm.

In the long-term culture, the higher density and prolonged opening of L-VGCCs leads to calcium overloading, a hallmark of neuronal aging.

Calcium binding to the indicator generates fluorescence, with aged neurons exhibiting higher fluorescence intensity, reflecting increased intracellular calcium levels upon stimulation.

In this procedure, incubate the cells with Fura2-AM for 60 minutes at room temperature in the dark, then transfer the coverslips to the perfusion chamber. A