I've been using IR-DIC in P32 mouse slice to target cells that appear larger then pyramidal cells that are not pyramidal shaped, or have a different orientation than pyramidal cells. But I've recently hit a month long dry spell where those types of phenotypes are either not present or very unhealthy. My regular spiking cell populations appear to be okay and their membranes seal okay too. Does anyone have any advise to better target FS cells? Could there be a reason that FS cell populations die when RS cell populations survive during slice preparation?
Hi Philip,
The size and shape of cortical FS interneurons can vary depending on region/layer. In particular, when I record FS interneurons in layer 5 cortex I search for small round cells with no clear apical dendrite. Since Layer 5 pyramids are very large, it is pretty easy to find the FS interneurons (assuming good morphological preservation in the slices). However, in layers 2/3 cortex the pyramids are relatively smaller and the FS interneurons are of similar or sometimes slightly larger size. They are also more irregular shaped than the small round ones in layer 5 and may have almost an inverted pyramid shape in some cases, but again no clear apical dendrite. If you can't do it by iR-DIC alone, many people use a labeled line such as G42, Pvalb-EGFP, or PV-IRES-Cre mated to tdTomato Cre reporter.
I do think FS interneurons are more sensitive than pyramids in adult slices. You may wish to also have a look at the methods I have described for improved health of adult mouse slices (www.brainslicemethods.com). This allows me to better preserve cells in many regions in adult and aging mice, including cortical FS interneurons. If you improve your IR-DIC optics (e.g. IR900nm instead of more common IR775nm) you may be able to see more healthy cells deeper in the slices.
I was recently recording from PV interneurons in both hippocampus, cerebellum, as well as cortex in mouse P14 - P30. If you have Epi on your scope you can easily target these cells. I targeted them by crossing PV-IRES-Cre homozygote mice (Jackson labs stock # 008069) with AI9 (tdTomato reporter; Jackson Labs stock #007909) homozygote mice. All PV interneurons can easily be seen in Epi. Most of the ones I saw in cortex resided in layer IV.
Thank you both. Since i dont have access to transgenic lines at the moment, i have been going off morphology under DIC. Targeting cells without clear apical dendrites appears to be working and if i dont get FS cells with that method then they are typically LTS or bursting types, which i can use as well.
Dr. Ting, i was recently referred to brain slice methods and have been considering switching from my sucrose ACSF solutions for my next experiments, thank you for putting that information out there!
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