Hey everyone,
I know the question seems paradoxical, but I am currently trying to measure sEPSC in the medial Hippocampus CA1 cells in adult mice voltage clamped at -70mV, and I find that there are these huge (1500~4000 pA) currents that pop up every minute or so (image attached). The internal solution is CsMeSO4 based and contains 5mM QX-314 (alomone labs). From my understanding, the voltage clamp and QX-314 should both stop the clamped cell from firing, but it does. Frustrated that I was, using the same int sol I changed to current clamp and found that there are depolarizations that go up to 10mV, which is why I am saying that the cells are "firing." Has anyone experienced this? As a last resort I borrowed some internal solution with the same composition from another lab, but got the same results. The alomone lab website, by the way, says that the particular chemical, at 5mM the Na current is reduced by ~20%, and complete blockage of sodium currents require 50+mM concentrations. But from what I know the conventional knowledge is that 5mM is enough to block them, so I am at a complete loss as to what to do. Any help would be appreciated. Thank you all in advance!
Dear Haram,
The neurons you are trying clamp are very large and therefore there is a problem with space-clamp issue, meaning that you are not actually fully clamping the cell at the holding voltage outside the immediate location of the electrode tip. Action potentials are coming from the axon hilux are sometimes difficult to clamp, especially if you are using small tip (high resistance) glass micropipettes. What is your access resistance readings? My recommendation would be to use larger tip electrodes 2-3 megaohm (~1 micron) in the bath solution. The larger the electrode-membrane interface the lower the access resistance will be, meaning you will be able pass larger amounts of current that will give you better voltage-clamp. The access resistance ideally should be less than 10 megaohm, especially in neurons with larger dendritic trees. This should also help passage of QX-314 to the intracellular compartments easier. For inspiration see my following publications.
Best wishes, Refik
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Hi Haram,
I see that you hold a cell nicely and that the amplitudes of currents are reasonable. I am pretty sure that your internal is not working. I'd make a new batch of internal with new QX-314. Best regards, Ewa
Dear Refik and Ewa,
Thank you so much for your feedbacks! It delights me that I can find answers here that I would be hard pressed to find on my own :)
I tried the experiment again with lower resistance pipette and the effect was instant. I am in the process of collecting more data but there is definitely a correlation between the pipette resistance and the presence of "firing" currents! It does seem like the space clamp issue was the problem. When the problematic currents were present my pipette resistance was between 3-4 MOhms. Now I am doing it again ~2MOhm.
Thank you so much once again!
Dear Haram,
Thank you for the feedback and I am delighted to hear that you are collecting more reliable data now. Best wishes,
Refik
Its important to remember also that your sEPSCs come from near and far synapses. The ones from far away will be filtered and will have a slow kinetics. There is not much you can do about that... only the nearby EPSCs will be well voltage-clamped.
I think the answers above are great - but have you tried the obvious of adding TTX in the external solution to see if the pulse-inward currents go away?
Wow, that guys got some juice in em. I agree with the external TTX idea. The MAJOR issue I would look into is synaptic inputs. It may look like the cell's firing, but glutamate channels are one hell of a sodium source. Block them.
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