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, a