I always hold the neurons at -70mV when I break through. But after 40min of recording the cell always depolarizes (SEE THE ATTACHMENT), its resting potential increases from -70mV to -64mV (and sometimes even to -50mV!!!) Someone told me that is because I always switch between voltage clamp mode and current clamp mode.(I use voltage clamp to break through and then I switch to current clamp mode to record action potential...), but I wonder why! sometimes after I breaking through, the holding current is +200pA, then I switch to current clamp mode and then switch back to voltage clamp mode immediately, the holding current decreases to -10pA....
The neurons I patch are stellate cells in entorhinal cortex layer 2
I could be way off as I have only patched cell lines, but based on your first figure I don't think you have actually fully broken in yet. Your Ra is very high and the capacitive current looks very slow. It would help a lot to know more information of what kind of preparation and neuron type you are working with.
Over long recording times, an increase in leak current and membrane depolarization is pretty normal, mainly due to your cell getting unhealthy or your access deteriorating. Depending on your experimental configurations (age of the animal if in acute tissue, cell type patched, etc.), you want to improve conditions in order to achieve a good and stable access for the time needed for your experiment (e.g. 1 hour).
First of all, your initial Ra is not great. You can improve it by reaching a very tight seal and/or using glass pipettes with lower resistance (i.e. making a bigger hole in the membrane). However, Ra is never super-stable straight after breaking in, but it needs some time to stabilise. Thus, I would aim to obtain an Ra
Have you tried changing the electrode wire and re-chloriding the reference and recording electrodes?
Hi Wanyu,
I think that you are loosing your seal. At times I have tried to suck through again and it has helped to bring the seal back. At other times, after sucking through, I lost the cell. Also, I will re-chloride the reference electrode, try to get a better seal at the start and also try to properly break through before starting the recordings. I did this with enteric neurons and it helped. Hope it helps in your case.
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