Hi, I am trying to do mouse hippocampal CA1 neuron in vivo 2P calcium imaging with Thy1-GCaMP6f mice. I followed some protocols, remove part of the cortex above the hippcampus with aspiration until I saw the axon of the external capsule. The problem is under the 2P microscope, I can only see very bright axons but beneath them, where I should see the soma of CA1 neurons, there was completely black. Does anyone have any idea? Did I damage the CA1 neurons during the aspiration? Or did the blood cover the green signal? Thanks!
Is GCaMP6f membrane bound? We see a similar distribution of expression in CA1 of Thy1-ChR2-EYFP mice. There is much more expression in the dendrites than in the soma (presumably because of the difference in surface area of the membrane). The expression in the soma is there, it just appears dark in comparison to the surrounding dendrites. I would suggest you check expression in fixed slices to determine if this is true of your mice as well. You can compare your results to the images in this paper: Article Expression of Channelrhodopsin-2 Localized within the Deep C...
Thanks David, we have used the mice from the same strain to do slice imaging and we could see strong expression in the soma.
In that case I am not sure what might be causing your issue. Perhaps someone else might have the solution?
In our experience, there are a lot of things that can go wrong that aren't obvious in the publication record. Even if you damaged CA1 by going too far (something I've done before) you should see green signal in some deep cell bodies, it'll just look wrong (cells will be filled in instead of doughnut-like, and transients will be longer) unless you've gone REALLY far, which is pretty hard to do.
Assuming you're using the Dombeck/Tank cannula strategy, if the metabond sealing the cannula to the skull/headplate is too high above the lip of the metal cannula, the objective can get stuck, and moving the Z direction won't actually go deeper. Being careful to keep the metabond thin can help, and if it's too thick you can shave it down (carefully) with a dental drill. If the power out of the objective of your 2p is good (at least ~20-40mW) and you can see signal in slices, that's my best guess for what went wrong. If you're using quikseal between the cannula glass and brain, bubbles can sometimes obscure the field as well. Going a little less far (stopping aspiration right as you hit fibers) and not putting in any quikseal seems to work fine for us, and prevents that issue.
There's also the possibility that the particular mouse you used was incorrectly genotyped, if you didn't slice that specific animal and looked at its expression. We generally find that injecting CAMKII-GCaMP6f into CA1 is the fastest/most reliable way to get a lot of animals for imaging, if you don't need expression for over 3 weeks in any given animal.
Thanks Heather for your great suggestions!
I have figured out the issue and found out the depth of how much cortex need to be removed is the key. Now we can get similar images as shown on your lab's website.
Regards!
Glad to hear it! Depth of cortex removal is for sure important to not damage hippocampal tissue.
Jin-jing Yao, may I ask you what you landed on in terms of depth? I see some protocols refer to stopping the aspiration when hitting the layer of fibers that moves in the AP direction, while others claim to stop when they see the diagonal fibers. Yet again, if I understood Heather Macomber correctly, some simply stop as soon as they see any type of white fiber altogether. If as you say, depth is key, I would love to hear which depth gave you success and not.
Any answer is much appreciated!
Best,
Emilie
Hi Emilie, sorry for the late reply. Usually, after taking out the cortical region, I will see 3 layers of fibers, the top one looks vertical, the middle one looks horizontal, the third one looks vertical again. I will try to take out the first two layers, try not to touch the third layer. Also, If you are imaging with aged mice (9-11 month old) there might be an extra very thin diagonal layer above the third layer, tried to remove it also, but be very careful. Hope it helps.
Good luck!
Jinjing
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