NMDA buffer toxic? - (Oct/05/2005 )
I want set up an assay to determine protection against NMDA-induced neurotoxicity. I am using E14 primary mouse neurons in Neurobasal/B27 medium. All protocols I have seen tell me you need to add NMDA in a specific high salt buffer for 10 minutes, wash the cells and culture the neurons for an additional 24 hours in the proper medium. My problem is that the NMDA buffer is killing 50% of my neurons?! Has anyone experienced similar problems?
Another related question is whether anyone knows whether the number of astrocytes present in the culture can influence NMDA-induced toxicity?
YES!! astrocytes express glutamate transporters that take up glutamate from the media and protect the neurons from over exposure to glutamate. If you do not have astrocytes in you culture or not the same amount every time you results would be different.
so: low astrocytes --> more glutamate in the media --> more NMDA toxicity
more astrocytes --> less glutamate in the media --> less toxicity
I hope I helped
Thanks! Could that also explain why my neuronal cultures are sensitive to the high salt NMDA buffer? I do not have many astrocytes in my culture (5-10%) and other protocols grow neurons on an astrocyte monolayer..?
It might, I have never done this.
you have to understand that neurons are like moviestars they get all the attention and are the stars but wiht out the costumes and directors and the other people (astrocytes in the case of brain) they can't do anything. there is a lot of unknopwn interaction between neurons and astrocytes that is noe known yet.
all the best
What a great analogy!
I was asking around and you can try to do the toxicity with high glutamate levels
Would you know whether glutamate needs a specific high salt buffer like NMDA? Or just add it to the medium?
I checked some papers and they say just to add to the medium. Personally, I tried but I have some problems to solve it !