Forum - For cortex imaging

Dear Miniscope family,

I've had some success in imaging astrocytes and neurons in the visual cortex (> 8 week old mice). Here are some tips that I think are worth sharing with people who are also interested in imaging cortex.

I use AAV2/5-GfaABC1D-GCaMP6f for astrocytes, and AAV1-hSyn1-GCaMP6f for neurons. Both are available from the Penn Vector core. I didn't dilute the virus and only did one injection per animal. I've tried two different surgery protocols:

1. Virus injection --( 1 wk)--> Craniotomy + GRIN lens implantation --(> 1 wk)--> Baseplating --> Habituation before the actual recording.

2. Craniotomy + virus injection + GRIN lens implantation --( >2wk, but no more than 3 wk)--> Baseplating --> Habituation before the actual recording.

Both ways worked. I think the most critical thing, which is different from the hippocampus surgery is that I like to put the virus injection site at or at least near the center of my craniotomy. I find that it's harder for me to find good cells if the virus injection site it not at the center of the craniotomy - because it seems that virus don't spread that much in the cortex compared to hippocampus.

Regarding how deep for the GRIN lens to go, I usually get a sense of how thick the skull is when I'm doing craniotomy, and eyeball it (at my best) when lowering the lens. I recorded how much I lower the lens (usually > 200 um and < 600 um) and check the brain to see if there's an obvious dent after the animal is perfused.

I didn't remove dura because for my study, I need the brain to be as damage-free as possible.

It seems that I can see GCaMP6f activities from layer I and layer II.

Going to test if I can image through cranial window.

Please feel free to ask any question or share your experience of cortex imaging here.