Cortical imaging through cranial windows with V4

[Amit Koren]

Hi all,

Following many attempts, we have still not succeeded with cortical imaging through cranial windows through the V4 Miniscope (with viral injections or transgenic mice). Has anyone had luck with this protocol or knows of a paper that shows its feasibility?

Thank you very much,

Amit Koren

PhD Candidate at Prof. Pablo Blinder's lab

[Roman Doronin]

What are the problems that you are having in particular?

[Amit Koren]

Hi,

I have been trying to image cortical activity through 3-4mm cranial windows (Retrosplenial/Somatosensory cortex) with transgenic Thy1-GCaMP6s mice or viral injections of GCaMP 6f/7f/8m. I know this should be feasible, yet I have not been successful in seeing single-neuron resolution. I see a blood vessels and cells very well.

I have done many controls such as validating activity in every mouse in the 2p and going back to exact same area of neurons,
successful imaging of GFP-labeled neurons on a slide, imaging of deep blood vessels with FITC injections, different kinds of cranial windows, etc.

I do see light waves in the tissue of what seems like neuronal activity in response to stimulation, but no single neuron resolution no matter how close I am to the window,
or where the EWL is set.

Thanks in advance for any thoughts or details about the troubleshooting journey you went through in order to receive good imaging data.

[cke...@gmail.com]

If you have a 2P head-fixed setup, have you tried imaging with a benchtop widefield fluorescence microscope? In general, you can expect a 1P signal through ~100 um of brain tissue, but a 2P signal through ~300 um. So it's quite possible that scattering is generally limiting what you see.

[Roman Doronin]

Uploading the video could be helpful with troubleshooting. Under which promoter and with which titer did you inject the construct? In general we found that with high titers and with the promoter which intensely labels layer 5 cells it is hard to see any somatic activity (our guess is because local dendrites and axons of L5 occlude the fluorescence from layer 2/3), so switching to ef1a promoter from hsyn and using titers around 10^13 helped

[Amit Koren]

Thanks for the helpful and interesting pointers!

I tried several constructs - all had titers around 10^13, part were under a hSYN promotor (AAV1-syn-gcamp6s and AAV5-hsyn-6xHis-soma-gcamp8m) and one was AAV2/php.eb.-Ef1a-gcamp6f. What AAV serotype did you use? I'm thinking that we might need to try AAV9 for cortical neurons. 

Here are a few of my videos from the different attempts:

https://drive.google.com/drive/folders/1qhq_SUFqQuFLVj0PkvJ8WF9LRZuGiMia?usp=share_link

Thank you,

Amit

[Ana Carolina Bottura de Barros]

Hi Amit,

We are using AAV1 Syn-GCaMP7s in retrosplenial cortex. Before I used to have similar videos as you do. It got better when diluting the virus about 10x. Initial titer was around 10^13. Let me know if you would like more details on injection procedure.

Best wishes,

Ana

-- 
You received this message because you are subscribed to the Google Groups "Miniscope" group.
To unsubscribe from this group and stop receiving emails from it, send an email to miniscope+...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/miniscope/08d5188d-b226-4ccc-8613-c6506e15cacen%40googlegroups.com.

[Amit Koren]

Hi Anna,

Thank you so much!! It would be great to hear more details if possible, I’ll send you an email. 

[Георгий Буков]

Hi all. 

I'm trying calcium imaging of the mouse somatosensory cortex through cranial window with V4 Miniscope with viral injections and it's still not successful. 

Can anyone give some advises or protocol for cortical imaging? I think we should try retrosplenial cortex with dura mater removing - does it makes sense?

Thank you for answers.  

суббота, 1 апреля 2023 г. в 22:25:32 UTC+3, ami...@mail.tau.ac.il:

[Antoine Bergel]

Hi everyone,

We too are going to try to image through cranial windows over the retrosplenial cortex in rats using V4 miniscopes (and miniscope LFOV when we receive it !).

I have had encouraging results (clear window, some cells) with a 1mm relay GRIN lens placed right above the cortex surface, and also by stacking 2 1.8 mm GRIN lenses.

Removing the dura seems necessary to me. Also, getting the miniscope objective as close as possible to brain tissue during baseplating.

What kind of glass coverslip are you typically using ? Do you add agarose ? Has anyone tried imaging through flexible film like PMP or PDMS ? Do you observe tissue motion and how bad is it ?

For what it's worth here a useful reference: https://doi.org/10.1016%2Fj.jneumeth.2021.109100

Best,

Antoine