Infecting Primary Mouse Midbrain Neurons with Engineered Adeno-Associated Viruses

Begin with an embryonic mouse midbrain cell culture on a coverslip.

Treat with engineered adeno-associated viruses encoding a fluorescent calcium indicator complex under a neuron-specific promoter.

Incubate for viral attachment and internalization.

Remove the medium to eliminate unbound viruses, then add a culture medium and incubate.

Internalized viruses release their genome in the nucleus, where the neuron-specific promoter drives the production of calcium indicator complexes exclusively in primary neurons.

These complexes include a fluorescent protein tagged with a calcium-binding protein and an M13 peptide.

Transfer the coverslip to a dish with a recording buffer rich in calcium ions, then place it into a recording chamber for live confocal imaging.

Introduce a glutamate recording buffer, causing glutamate to bind with ion channels, leading to the entry of calcium ions.

These ions bind to the complex, causing a conformational change and fluorescence emission.

In live imaging, the bright neurons indicate the viral infection.

Begin by preparing 1 milliliter of serum-free DMEM medium with 1 microliter of hSyn-GCaMP6f AAV per dish. Aspirate the cell culture medium from each dish, and replace it with 1 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 3 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 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 35-millimeter 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 bright-field 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 photobleaching 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 0. 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 5 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.