Hi all,
I am doing some voltage clamp experiment to look at AMPAR mEPSC in cultured hippocampal neurons. And at the moment, I think I nailed down a good pair of internal (CsGlu) and external (Hibernate E) to keep the access stable and the baseline quite flat. My biggest concern is the drop in the holding current. This drop could be as big as 100 pA within 30 min or even worse in some cases. From my quick search in the literature, it looks like it might stem from the Cs ions! Although there could be other less controllable factors like membrane integrity. Please check the paper below. Did you experience this when recording from cultured neurons? Or any other neurons? How much was the drop? And in your opinion, how much is acceptable (I meant, publishable)?
Thank you!!!
http://personal.us.es/pnunez/pdfs/9-J.Neurophy.2000b.pdf
100pA over 30min is not bad! It is normal for the integrity of the seal to break down over time, so I think you would see this in a non-Cs internal as well. It's fine, as long as you control for it.
What are you testing during the experiment? Are you applying drugs? If so, I always do a wash-out period as well as a pre-drug control. That way if the cell is changing during your recording, you can normalize to the average of the pre- and post-drug values. If the drop is about linear, and the variable you are measuring (amplitude? decay rate?) is not different between pre- and post- drug, then I wouldn't worry too much about the holding current.
If you can't wash out your drug or intervention, then you can record a separate set of cells for the same length of time, to control for time-dependent changes in the membrane.
Aside from that, if you want to improve the length of your recording, getting better gigaseals is likely to help. Heat-polishing the tips of my pipettes generally takes me from 500megohm to 3+gigohm, which makes the recordings much more stable.
Good luck!
Hi Christine, thank you for a very detail suggestion! My advisor also told me that it is not bad, but I just want to hear opinions from more experts. :)
Yes, I am trying to do time series experiment. Which is recording baseline from a cell, then applying drug, then washing the drug out. That is why I am recording for quite a long period of time. I do care about both rise time, amplitude and frequency. The rise time show no trend of increase or decrease. But the amplitude and frequency do reduce over 40 min. (from 11 to 8 pA and from 0.7 to 0.48 Hz). I definitely should record a few more cells and see if they all behave like this.
Sounds really cool about polishing the tips! What is the resistance of your pipettes before and after polishing? Are you also doing whole cell recording in hippocampal neurons? I did some single channel recording last summer and it was definitely a must to get good sealing. :) Just wasn't sure what is the parameter for whole cell recording.
Thanks again Christine!!!
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