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. After that, place the perfusion chamber with the coverslip in the holder on the inverted microscope. Select the microscopic field and perfuse the cells continuously with pre-warmed HBS.

Subsequently, epi-illuminate the cells at 340 and 380 nanometers ultimately, then record the light emitted at 520 nanometers every 5 to 10 seconds with a fluorescence camera, which is filtered by a Fura-2 dichroic mirror. To analyze the recorded fluorescent images. First, open the experiment file.

Using the AquaCosmos software, click on Ratio and select the desired ratio range. Calculate the pixel by pixel ratio in the resulting images in order to obtain a sequence of ratio images. Then subtract the background by adjusting the background illumination button. After that, press Start Calculation. next, press the All Time Sequence button and erase the ancient ROIs.

For quantitative analysis of individual cells, establish new ROIs or the ROIs corresponding to the individual neurons. To graph the individual recordings, export the ratio fluorescence values corresponding to each ROI to the origin lab program by clicking Graph. Then click Calculate and save the TXT file. After that, make the corresponding calculation for estimating the size of the rise in the fluorescence ratio in response to each stimulation.