Dear Julieta,

Regarding expression of the GCaMP6, over-expression may (i guess) alter calcium homeostasis and I could imagine that that would be harmful for cells. If the expression is not so very strong, however, it should be fine I think. Secondly, the brightness of the GCaMP6 depends on how strongly it is expressed and on whether it is bound by calcium. What I would suggest to do is to inject a virus (of preferentially the same serotype and titer) that expresses GFP (or any other FP) and make sure that your virus injection does make it as far lateral as CA1/CA3.

How long do you wait to check your virus expression and do you check in vivo or in brain slices?

There are also some papers describing two-photon imaging of hippocampal CA1 cells (From David Tank's lab) with GCaMP, and the virus they used was aav1 or aav2.1, maybe try that serotype, maybe that will solve the expression problem. See e.g. these papers: Rickgauer JP, Deisseroth K, Tank DW. (2014) Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat Neurosci. 17: 1816-24. Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW. (2010) Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci. 13: 1433-40. PubMed

Regarding the shapes of the cells, i'm not sure through how much mirrors and glass you are imaging, but dispersion is wavelength dependent and maybe this effects your image. Since you indicate to be not sure if the cells really look different: Try imaging fluorescent beads (making a zstack of a single bead at high zoom) and you can calculate your point spread function. This will probably be more telling than looking at an actual living sample.

A colleague of mine showed me the following imageJ plugin & paper on how to to this, which I find useful: 

http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:metroloj:start

http://www.nature.com/nprot/journal/v6/n12/pdf/nprot.2011.407.pdf

Hope this helps and cheerios,

Pieter

Hi,

first of all you can find the correct coordinates in a good papers. There are a lot of  papers where you could find the coordinates for the virus injections in mouse hippocampus (P20). 

Finally, I'm not sure but the changes of shape of the cells could depend of which power laser you use to excite your cells. Check you the fluorescence emission and excitation wavelength curve. 

Best

Francesco

Hi Francesco,

Thank you very much for your useful comments. I will check the literature for the coordinates. 

Regarding the power, I didn't mention this but I am using three different power levels. But I only see differences when I change the wavelengths, no matter the power of the laser.

Thank you very much for your help!!

Kind Regards,

Julieta

Hi, 

We have good experience with AAV2 syn. GCamp6f in cortex. To begin with, make sure the titter of your virus is good, which is usually not a concern if you purchase them from Penn vector or other established facilities. Then, you might want to take into account of the toxicity of AAV expression of GCaMP6s on neurons. Especially 6s, compared to 6f, in my impression. Those whose nuclei are stained are intoxicated ones. Cytoplasmic localization of the GCaMP protein is a sign of health. You may not want to wait too long to observe them after viral infection, because most of the positive neurons may have died before you could possibly see them.

Hope this helps.

Best

Yajie

Dear Julieta,

Regarding expression of the GCaMP6, over-expression may (i guess) alter calcium homeostasis and I could imagine that that would be harmful for cells. If the expression is not so very strong, however, it should be fine I think. Secondly, the brightness of the GCaMP6 depends on how strongly it is expressed and on whether it is bound by calcium. What I would suggest to do is to inject a virus (of preferentially the same serotype and titer) that expresses GFP (or any other FP) and make sure that your virus injection does make it as far lateral as CA1/CA3.

How long do you wait to check your virus expression and do you check in vivo or in brain slices?

There are also some papers describing two-photon imaging of hippocampal CA1 cells (From David Tank's lab) with GCaMP, and the virus they used was aav1 or aav2.1, maybe try that serotype, maybe that will solve the expression problem. See e.g. these papers: Rickgauer JP, Deisseroth K, Tank DW. (2014) Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat Neurosci. 17: 1816-24. Dombeck DA, Harvey CD, Tian L, Looger LL, Tank DW. (2010) Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat Neurosci. 13: 1433-40. PubMed

Regarding the shapes of the cells, i'm not sure through how much mirrors and glass you are imaging, but dispersion is wavelength dependent and maybe this effects your image. Since you indicate to be not sure if the cells really look different: Try imaging fluorescent beads (making a zstack of a single bead at high zoom) and you can calculate your point spread function. This will probably be more telling than looking at an actual living sample.

A colleague of mine showed me the following imageJ plugin & paper on how to to this, which I find useful: 

http://imagejdocu.tudor.lu/doku.php?id=plugin:analysis:metroloj:start

http://www.nature.com/nprot/journal/v6/n12/pdf/nprot.2011.407.pdf

Hope this helps and cheerios,

Pieter

Hi Yajie,

Thank you very much for your help. Yes, I am buying the virus from Penn Vector. I do see lots of neurons with dark nuclei, and some completely bright, but I guess it is normal to have some dead cells in a slice. But, again, the nucleus is quite dark in most of the cells, so I guess I should be happy about that. 

It is true that the few times I left them three weeks after the injection I did see more dead neurons, so two weeks sounds like a good trade-off. 

Thank you very much for your help.

I am new at this, so any suggestion or observation is more than helpful.

Kind Regards,

Julieta

Dear Pieter,

Thank you very much for your useful suggestions.

I don't think I'm having a toxicity problem with the GCaMP, because although it's true that it can chelate the calcium and affect the homeostasis of the cells, I guess in the case I was having a toxic effect, I should see very bright cells, as Yajie mentioned, and this is not the case, I don't see anything fluorescent in CA3 and CA1, only in the DG. And though I see some cells that are too bright, most of them have a dark nucleus, which indicates they are alive (and I see activity as well, after stimulating with an electrode).

In most of the cases I wait two weeks after the injection, but in a few cases I waited three, to see if there was any difference. I use slices, I haven't started in vivo yet.

I've read the papers you mentioned here, thanks. They are indeed very helpful.

The fluorescent bead suggestion sounds good, I might try that. It's going to be easier to see it than with living tissue. And I will also take a look at the paper you mention.

Once again, thank you so much for your time and suggestions.

Kind Regards,

Julieta

Dear Julieta,

I see, in that case, it sounds indeed more like too low expression, or that the cells are not infected by the virus at all.

I just remembered a paper from Simon Rumpels group where they tested different aav's and according to them, aav9 should work just fine in the hippocampus. Paper: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076310

In figure 1B, one can see the labeled hippocampus (CA1) and in addition see the injection tract going through the cortex (with cells labeled by the backflow of the virus), showing the 'sort of exact' location of the injection. You could try a similar approach for checking your injection location (By deliberately keeping on injecting while retracting the pipette).

In any case, once you solve the problem, i'd be interested to learn what it was.

Cheers, Pieter

Dear Pieter,

That paper is the reason why I chose to buy AAV9. I also have AAV5, which I haven't tried yet, but according to that paper it should be more or less the same.

I might try what they did to see the injection site. Thanks for the suggestion.

I will let you know how it goes in a couple of weeks!!

All the best,

Julieta

Hi Julieta, how did you resolve your problems with getting expression in CA3 and CA1? 

This is too late to help you, most likely, but in case someone else finds this discussion: the standard Paxinos & Watson mouse brain atlas is perfectly fine for developing injection coordinates for use at P20. It seems at that age the mouse brain and skull has reached its approximate adult size and shape (although I believe the skull itself does still become a bit thicker with age).

Regarding wavelength: there are multiple optical components in the excitation path that have wavelength-dependent behavior. Someone above mentioned dispersion already. Besides that, chromatic aberration could cause what you describe.

Furthermore, you mention that you try 3 different power levels, but that you only see differences when changing the wavelength, "no matter the power". How are you measuring the power at each wavelength? With most 2p lasers, the power output changes substantially as you change the wavelength - thus, even if you use all the same settings in the remaining components, the power at the specimen may be quite a bit less at e.g. 950 nm than at 920 nm. Even if you account for that, your Pockels cell may perform differently at different wavelengths, and so on. So you would need to use a power meter to verify/calibrate the true power at specimen for each wavelength that you use, if you want to be sure that the effects you are seeing don't reflect differences in excitation intensity.