I've been carrying out immunofluorescence staining in striatum in order to identify recorded MSNs as D1 or D2. Recorded cells are filled with Neurobiotin and then the slice is probed for the Adenosine A2A receptor (marker for D2 MSNs). I'm been having this strange problem where the recorded cell almost never stains positive for A2A. We have a few cases where the cell might be A2A positive but it's hard to tell. This is strange because the expected proportion of D2 MSNs in striatum is 50%, so out of the 15+ slices I have processed to date, I should have around 7 A2A positive MSNs. The antibody seems to work fine as I can see other clearly A2A positive cells in the vicinity of the recorded cell. I can see only two possibilities here: 1) I'm biased towards recording from D1 MSNs, which I find hard to believe, or 2) The filling of the cell with internal solution, the introduction of neurobiotin into the cytoplasm, and/or the recording procedure, is causing a downregulation of A2A receptors in the recorded cell. Has anyone heard of something like this happening with other neurons or with other markers? I'd appreciate any advice on how to proceed.
I've attached a sample image. This was taken with a confocal laser scanning microscope. You can see in the red channel (A2A) that there are many clearly labeled cells in the field of view, but the cell in the center is not labeled. This is representative of most of the cells I've imaged. The few cells which may be positive do not look like anything like the clearly positive cells in this field of view.
Why don't you try with retrobeads?
You could inject retrobeads into the SN or GP and record already identified MSNs
I would recommend you to use a Neurobiotin electroporation technique that I have developed. This method causes minimal damage to the neuron and that may allow you to double label your cells succesfully. Please See Kanjhan and Vaney 2008 (Pflugers Archive) and Kanjhan and Bellingham 2013 Book chapter. You can all the details in there. Please let me know if you need any specific advise.
cheers, Refik
Emmanuel, has your lab already been successful with using retro-beads into the NAc?
The reason I ask is that we recently tried injecting retro-beads into the LH (known projection region for the NAc), and could not find any retro-beads (Lumafluor retro-beads specifically) in the NAc. However, when we used retrograding virus at the same time, there were plenty of cell bodies lighting up in the NAc. Similarly, when we inject the same beads directly into the NAc, we get lots of cell bodies in the VTA. So for whatever reason, we have not been able to get the beads to work by retrograding back into NAc MSNs.
To the best of my knowledge, I have not been able to find a single paper that has actually used retro-beads into NAc projection regions with NAc cell body identification. Any insight would be great!
We have been doing similar post-hoc immuno... after all, who want's to cross every mouse line with a reporter strain
We've had more success with substanceP antibodies than the A2A antibodies, I suggest you try several for staining in slices that you haven't gone to all the trouble of filling cells in until you get one that really picks up ~half of the cell bodies. Also good to test these in slices from a D1 or D2 fluo reporter mouse
Asides; as to potential bias in cell targeting, it is a discussion I've had before. If you are using DIC to visually identify cells you like the look of (as most of us do) there is at least the possibility that you introduce some unexpected bias. There are slight differences in the shape and soma size of the two subtypes, which might be exaggerated in the pool of cells you 'prefer' the look of (albeit unlikely).
Also could the age at which you are recording make a difference, personally I don't look in anything younger than 4 weeks, but I know many people patch at P15-22 or less? Regardless of mRNA levels, there might be a developmental regulation for A2A protein expression?
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