Quantifying Spontaneous Ca²⁺ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

This protocol quantifies calcium fluxes in dopaminergic neurons, which are lost in Parkinson's disease. This is useful for understanding how abnormal calcium causes dopaminergic neuron loss in Parkinson's disease. For the first time, we demonstrate spontaneous calcium fluxes in cultured primary midbrain neurons.

This method can be used to dissect specific receptor contributions involved in calcium-mediated apoptosis. Our method provides an avenue for high content drug screens to discover specific and effective neuroprotective compounds for Parkinson's disease. These neuroprotective compounds would prevent calcium-mediated apoptosis in dopaminergic neuron.

Begin by preparing one milliliter of serum-free DMEM medium with one microliter of hSyn-GCaMP6f AAV per dish. Aspirate the cell culture medium from each dish and replace it with one milliliter of the prepared DMEM with hSyn-GCaMP6f. Place the dishes back into the 37 degrees Celsius incubator for one hour.

After the incubation, aspirate the medium with AAVs and replace it with three milliliters of cell culture medium. Incubate the dishes at 37 degrees Celsius for five to seven days. Changing the medium every two to three days throughout this period of viral infection.

Prepare one liter of the HEPES recording buffer, 200 milliliters of 20 micromolar glutamate recording buffer, and 200 milliliters of 10 micromolar NBQX recording buffer according to manuscript directions. Fill a sterile 35 millimeter Petri dish with three milliliters of the recording buffer. Take the Petri dish with the infected cultures from the incubator, then carefully grab the edge of one cover slip with fine tip forceps and transfer it into the dish with the recording buffer.

Place the remaining cover slip in medium back into the 37 degrees Celsius incubator and transport the dish with recording buffer to the confocal microscope. Start the imaging software. While it is initializing, start the peristaltic pump and place the line into the recording buffer.

Then calibrate the flow to two milliliters per minute. Transfer the infected cover slip from the Petri dish to the recording bath with fine forceps. Using the 10X water immersion objective and brightfield light, find the plane of focus and look for a region with a high density of neuron cell bodies, then switch to the 40X objective and refocus the sample.

Select and apply AlexaFluor 488 in the Dyes list window. In order to prevent overexposure and photo bleaching of the fluorophores, start with low HV and laser power settings. For the AlexaFluor 488 channel, set the HV to 500, the gain to 1x, and the offset to zero.

Set the power to 5%for the 488 laser line. Increase the pinhole size to 300 micrometers and use the focus x2 scanning option to optimally adjust emission signals to subsaturation levels. Settings can then be adjusted for optimal visibility of each channel.

Once the microscope settings are optimized, move the stage to locate a region with multiple cells displaying spontaneous changes in GCaMP6f fluorescence and focus on the desired plane for imaging. Use the Clip rect tool to clip the imaging frame to a size that can achieve a frame interval of just under one second. Set the interval window to a value of 1.0 and the Num window to 600.

To capture a t-series movie, select the Time option, then use the Xyt scanning option to begin imaging. Watch the imaging progress bar and move the line from the HEPES recording buffer into the 20 micromolar glutamate recording buffer at the appropriate time point. When imaging is complete, select the series done button and save the finished t-series movie.

Continue to perfuse the glutamate for an additional five minutes so that the culture of neurons have been exposed to glutamate for a total of 10 minutes. Repeat this process for each cover slip to be imaged. It is possible to view calcium traces and neuronal cell bodies immediately following the experiment.

Use the Ellipse tool to draw the desired number of ROIs around neuronal soma. And use the Series Analysis button to visualize the traces. After the additional five minute exposure to glutamate, remove the cover slip from the bath with fine forceps and place it back into the Petri dish with the recording buffer until all imaging is completed.

On the day of imaging, the VM cultures were treated with either glutamate or a combination of glutamate and NBQX. In both conditions, heterogeneous and spontaneous changes in GCaMP6f florescence were observed indicating spontaneous calcium fluxes. Application of glutamate generated a robust and sustained calcium response in both spontaneously active and quiescent neurons.

Application of NBQX reduced spontaneous activity, and partially blocked the glutamate response. The extent to which glutamate application stimulated a calcium response in each condition was quantified using the area under the curve, peak amplitude, and latency to respond. The latency to response increased under the NBQX and glutamate condition.

To measure glutamate-mediated apoptosis, the cells were fixed and stained with an anti-Caspase-3 antibody. Mean caspase-3 intensity was significantly higher in both treatment conditions compared to untreated controls. A more thorough analysis of excited toxic cell death can be done by counting the number of immunostain tyrosine hydroxylase positive dopaminergic neurons in control and glutamate treated condition.

This technique has paved the way for understanding the relative contributions of different receptors and iron channels involved in calcium-mediated apoptosis in dopaminergic neuron.