Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

Take a coverslip with a culture of ventral midbrain neurons derived from mouse embryos.

The cells are infected with a viral vector to express a calcium indicator.

Transfer the coverslip to the recording chamber of a confocal microscope perfused with a recording buffer.

Using visible light, identify a region containing multiple neurons, then switch to fluorescence imaging.

At low intracellular calcium levels, the indicator remains unbound, exhibiting low fluorescence intensity.

Intrinsic properties of the neurons generate spontaneous action potentials, triggering voltage-gated calcium channels to open and allow calcium influx.

The excess calcium binds to the indicator, inducing a conformational change that increases fluorescence intensity.

Record the fluorescence intensity over time to track the spontaneous calcium activity.

Introduce a neurotransmitter at a high concentration, which binds to receptors on the neurons, generating continuous action potentials.  

The sustained voltage-gated calcium channel activation prolongs calcium influx, causing a sustained increase in fluorescence intensity.

Prepare 1 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 millimetre Petri dish with 3 milliliters of the recording buffer.

Take the Petri dish with the infected cultures from the incubator, then carefully grab the edge of one coverslip with fine tip forceps, and transfer it into the dish with the recording buffer. Place the remaining coverslip 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 2 milliliters per minute. Transfer the infected coverslip 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 Alexa Flour 488 in the dyes list window. In order to prevent overexposure and photobleaching of the fluorophores, start with low HV and laser power settings. For the Alexa Fluor 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 sub saturation 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 cultured neurons have been exposed to glutamate for a total of 10 minutes.

Repeat this process for each coverslip to be imaged. It is possible to view calcium traces in 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 coverslip from the bath with fine forceps, and place it back into the Petri dish with the recording buffer until all imaging is completed.