Can removal of Mg2+ ion from external buffers help in this regard? How much endogenous glutamate influences NMDA activation. My holding potential is -80mV.
You want AP5. No removing Magnesium allows NMDA receptors to open at any potential. I suggest a quick read of Principles of Neural Science.
Mark is right! MK-801 is extremely potent and specific. Moreover it is usually well tolerated (dependent on the cell type). Your question suggests, that you want to be well above the IC50 to yield full inhibition. In an acute setting you may use a final concentration of 1um or even 10um. In more chronic experimental settings (over hour) I would tend to approach the nanomolar range.
Thanks William, I am relatively naive and trying to read. My aim to get transporter currents and I don't want background of receptors. Hence wanted to know how can that be achieved while dealing with single cells in culture and not slices. Thanks for teh insight though. I will try basics.
mark and Just genius: Thanks for inputs.
MK-801 is a use dependent NMDAR blocker, so you have to activate the receptors first before you can achieve a complete block of NMDAR currents. I too would suggest D-APV as the more direct way to fully block NMDARs in cultured cells independent of NMDAR channel opening. If you are trying to isolate transporter currents you will also need to consider blocking additional receptor classes that might confound your measurements.
Another possibility—MK801 and APV can be used internally.
You should be careful in your assumption that you holding potential of –80 mV is the same potential your synapses are being held at, especially if you are working branched structures with dendrites and spines. Space-clamp errors are often ignored but they are significant. See: Williams, S. R., & Mitchell, S. J. (2008). Direct measurement of somatic voltage clamp errors in central neurons, 11(7), 790–798. doi:10.1038/nn.2137
The issue of spines being un-clampable because of the high neck resistance is covered here: Harnett, M. T., Makara, J. K., Spruston, N., Kath, W. L., & Magee, J. C. (2012). Synaptic amplification by dendritic spines enhances input cooperativity. Nature, 491(7425), 599–602. doi:10.1038/nature11554
Hi Raymond.
I am using HEK and glioma cell line and the suggested holding for them through literature as well as guidelines from the Nanion guys is -80mV. Although your points are surely valid but since I am not working on slices or neurons, it might not be applicable here?
Hello Kalpita,
You are correct—cells without dendrites are the perfect (and only) system for accurate voltage clamp recordings. No space clamp issues here. Good luck.
If you have Mg in your internal pipette solution and are holding at -80mV, you shouldn't need to block NMDA receptors at all, since they will already be blocked/inactive due to the Mg block. But if you want to be completely sure, as others said, dAP5 and MK801 work well.
Thanks Aoron. I do have Mg is internal but what conc is sufficient to block possibly such currents?
hi Kalpita. I completely agree with Aaron. If you are holding at -80mV and have Mg++ in your solution then you shouldn't see any NMDA currents due to the channel blockage. If you gradually depolarize your cell you will see the gradual increase of your NMDA currents due to the removal of Mg++ blockage.
I normally use 1.2mM in my ACSF solution for acute brain slices. As Aaron also said, if you want to add blockers for extra caution, either of them should be fine. My experience with dopaminergic cells in acute slices tells me that APV at 100uM will completely block NMDA currents.
Good luck :)
- Badges
- Science topic
- Similar topics
- Methodology
- Research Methodology
- Research Papers