I'm growing single isolated neurons from hippocampus of mouse pups on their own micro-islands of astrocytes for study using fluorescence microscopy of synaptic proteins. I'm only interested in excitatory neurons, which I can distinguish with a mouse model we are using encoding PSD95-eGFP to look at excitatory postsynaptic densities. But I've been told I still need to address the question of "what type of (excitatory) neuron am I looking at?". (I was hoping to somehow avoid this question for my project but nevermind!)
I've been trying to find some kind of review article on this topic but haven't been able to find anything. Can anyone recommend any good markers for distinguishing different neuronal subtypes? Or morphological characteristics that should still be conserved in culture which I can use for classification?
The hippocampus also has subclasses of inhibitory neurons. What about GAD staining to distinguish GABAergic from glutamatergic neurons to start?
Thanks for your responses. I should have explained a bit better though. So the key thing is that I'm making single isolated neurons, on their own island unable to form synapses with any other cell. They form synapses to themselves (autapses). So if its a glutamatergic cell, it will express PSD95eGFP, if it's inhibitory I won't see anything. So I've already essentially got a way to look at the excitatory ones (which are the only ones i'm interested in really).
So I suppose my real interest is being able to distinguish subtypes of excitatory hippocampal neurons.
CaMK2 is a marker for excitatory neurons. Parvalbumin and Somatostatin are markers for interneurons. Check Klausberger and Somogyi (2008), Science, 321 (5885): 53-57. This is a great review for interneurons subtypes in CA1.
As it is know the hippocampus also has subclasses of inhibitory neurons. Try to distinguish GABAergic from glutamatergic neurons just to stain with parvalbumin and also by electrophysiology approaches.
Hey Matthew,
I am not that familiar with the hippocampus but here are some genes that separates cortical excitatory cells: Fezf2, Ctip2, Otx1, Sox5, Tbr1, Couptf1, Bhlhb5, Satb2, Cux1/2, Lhx2 and Rorb. Perhaps you can have a look at Allen Brain atlas/pubmed if they are of any use for you. Have a look at Woodworth, Custo Greig et al, Cell 151, Nov 9, 2012 for a quick review.
Best of luck!
Hi Matthew, you can check "Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus" by Williams et al., Neuron 2011, for specific markers to distinguish DG, CA3 and CA1 neurons. Good luck!
You can get a list of Neuronal Lineage Markers from rnd systems website
Matthew: I am not certain your reasoning is correct for cultured neurons, here.
"So the key thing is that I'm making single isolated neurons, on their own island unable to form synapses with any other cell. They form synapses to themselves (autapses). So if its a glutamatergic cell, it will express PSD95eGFP, if it's inhibitory I won't see anything. So I've already essentially got a way to look at the excitatory ones (which are the only ones i'm interested in really)."
Couldn't you still have a GAD-positive (GABA-ergic) neuron that in culture will also release glutamate into autapses? How can you rule that out?
"So I suppose my real interest is being able to distinguish subtypes of excitatory hippocampal neurons. "
When we have generated mixed rat hippocampal cultures, we could rather easily distinguish the pyramidal phenotype from fusiform and other shapes. Is it the CA1-3 pyramidal neuron you are hoping to study? If so, there might be region specific peptides that colocalize with glutamate, e.g., opioids.
http://archives.drugabuse.gov/pdf/monographs/82.pdf
Clyde and Sophie - my understanding from a lot of reading and from speaking to people who first developed the autaptic neuron culture system is that the neurons are either inhibitory GABAergic or excitatory glutamatergic, and never both. I suppose it would be a useful control to show that PSD95 +ve autaptic neurons are not also GABAergic neurons (and who knows, I may get a surprise considering it is a weird culture system).
Sophie - I imagine there is some astro-neuron cross talk, but while I'm quite a glial cell fan its not really part of my project sadly! The astrocytes are just there in this case as a feeder layer to support the single neurons. Because I plate astrocytes first I always thought it would be a great system to study astro-neuron communication, as you could transfect astrocytes with optogenetic constructs or something, before the neurons are plated... maybe one day!
"Hippocampal Pyramidal Neurons Comprise Two Distinct Cell Types that Are Countermodulated by Metabotropic Receptors" this the title of a recent article published by Dr Nelson Spruston. I am still not sure whether these functional classifications apply to cultured neurons or not.
Hi Matthew, A marker called CTIP2 stains for CA1 and DG neurons. Whereas Prox1 stains only DG. So Neurons which are CTIP2 positive but Prox1 negative are CA1s. This is explained in Williams et al, Neuron, 2011. But I have to warn you that CTIP2 may stain some interneurons which you have to distinguish somehow.
Hi Matthew,
There is a wonderful new online metasource of rodent hippocampus-related information: http://hippocampome.org/php/index.php. There you can find tables of markers expressed in each neuron type and much more.
Enjoy!
Elena
Postnatally Prox1 expression in the CNS is restricted to the dentate gyrus of the hippocampus and cerebellum, where it continues to be expressed in adults controlling neurogenesis (PMID: 17117441). Therefore, Prox1 can be used as a marker of a subset of neuronal progenitors, as wells as a specific marker of dentate gyrus cell lineages, such as type-2 intermediate progenitors and mature granule cells.
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