Difficulty keeping stable recordings

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synapticstudent
synapticstudent's picture
Difficulty keeping stable recordings

Hello everyone,

I'm having difficulty keeping stable recordings from my E18 cortical cultures. I'm trying to record mEPSCs and I'm voltage clamping at -70 mV. My setup is an Zeiss axiovert 25 inverted microscope, an Axopatch 200B amp, a Axon CV203BU headstage, and a Sutter MP-225 manipulator. I'm using King Precision Glass KG-33 electrodes with filament: 1.12 ID, 1.50 OD, and pulling them at 3-5 MOhm. I can make a gigaohm seal without issue usually, but once I break in, there is so much leak and my holding current is so unstable that mEPSC events become almost completely undetectable, and I usually lose the seal entirely within <1 minute. When I make a gigaohm seal, there also seems to be drift in the Y-axis that i think may be one of the contributing factors for the instability of my seals. However, I have tried everything I can think of to get rid of the drift, but to no avail. Could this be an issue with the cells themselves? Patching cultured neurons is e a very common occurrence, so I'm not sure why I'm having such a difficulty here. 

Any suggestions will be much appreciated. Thank for you taking the time to read my question!!

Deep Forest
Deep Forest's picture
My five cents.

My five cents.
First of all, I would make sure that you're not too "rough" when you're breaking the membrane, after you form the gigaseal. I use mouth to break the membrane. Also, check the osmolarity of your internal/external solutions.
If you have a drift, after forming a gigaseal, I would suggest to re-chloride your ground electrode (I don't know if you're using the AgCl ground electrode/pellet or not) TOGETHER with your silver wire from pippette holder.