Basically, I need to record the mEPSC(in TTX and AMPAR blocker) of pyramidal neurons in culture; but I am not sure whether it is pyramidal or not, partly because I can find very few cells with typical triangular soma. The situation seems to get no better even by testing the firing pattern before extracting the mEPSCs as there are so much varieties of them.
I also have similar problem before and I used simple clue to distinguish them.
First check the resting membrane potential, interneurons tend to have a slightly depolarized resting membrane potential. Then check the action potential by injecting currents into the cell, interneurons have action potential with deep after hyperpolarization.
Second add biocytin or rhodamin in your patch pipette for post hoc analysis. After finish recording, check them with markers for GABA or glutamatergic neurons. You can do this after collecting several recordings.
I hope this would help you
Biocytin in a pipette with either more work after, or to collaborate with a local histologist. Knowing precisely what kind of cell was recorded makes data interpretations much stronger.
Hi,
I'm a morphologist and by eye I can tell you that you can not discriminate pyramidal cell from interneurons in culture. You can do immunolabelling with GAD67 antibody (Millipore/Chemicon AB1511) which labels the GABA biosynthesis enzyme in the soma. You can also use GAD65 antibody (Millipore/Chemicon MAB351) that will stain the GABA biosynthesis enzyme isoform that is mainly in presynaptic boutons but which is perhaps less usefull in your case. If you wan't to discriminate different types of interneurons, you can then use double labeling with calcium binding protein (calbindin, calretinin, somatostatin, NPY,...). Keep in mind that you have more than 14 types of interneurons in hippocampus (Danglot et al, Hippocampus 2006). Hope it can help.
Lydia.
Agree with Lydia, maybe, (just maybe) enrichment in CamKII is an easier identifier. If anyone knows that some interneurons could have lots of CamKII please post.
As stated by Viktor, CamKII would be easier to identify directly pyramidals cells of CA1 without labeling inhibitory interneurons. But I do not know if expression is preserved in culture. Viktor, did you already tried a good antibody ? I would be interested. Actually a very good marker to identify glutamate neurons is also VGLUT.
unfortunately, not in culture: http://www.ncbi.nlm.nih.gov/pubmed/22509321
it requred HIER and worked well only with it. HIER on culture will be tricky...
In hippocampus like CA1/CA3 interneurons tend to spike without strong accommodation also spike heights may vary they may be shorter than pyramidal. also interneurons may lack a slow AHP. some times if you have a nice patch pyramidal neurons will have a hump when doing accommodation (current injection) near the first 100-200ms of current injection.
In sharp microelectrode method, we can catch interneuron. If you have the speaker attached to the electrophysiological set, We can easily hear characteristic sound of interneuron at the time of impalement which is different than the pyramidal cell. Later on you can study the intrinsic property as DIna suggested !
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