Technical electrophysiological problem before the physiology...

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Bruder Jakob
Bruder Jakob's picture
Technical electrophysiological problem before the physiology...

Hello,

I have just finished mounting a set-up and configuring the amplifier (Heka, EPC7) with the model cell. However, upon entering the patch pipette (1mm, borosilicate, filled with 2M NaCl) into the bath (filled with NaCl 0.9%) despite the voltage being at 0mV, I obtain a big current offset where the current signal is out of range and saturates (flatline), and cannot be brought back to 0 with the Vhold knob or the Vp offset knob.
Furthermore, looking at the test pulse of the amplifier window (of the Patchmaster software) in the set-up and on-cell configurations, the current is now at 0mV but the voltage is this time out of range and cannot be zeroed.
Therefore, the 2 signals cannot be zeroed at once, as should be the case when starting an experiment.

Usually, when offset problems occur, I know that it can be due to the microelectrode, or the intra-cellular solution, or the bath ground. However, this time these do not seem to be implicated since they have been changed many times.

Here is a list of all parameters we have changed and findings:

-Amplifier: changed with another EPC7 amplifier - signals still out of range
-Headstage: changed with another EPC7 headstage - signals still out of range
-AgCl microelectrode: tried to chloride it with Chlorox, pure sodium hypochlorit, HCl and current - signals still out of range
-Pipettes: changed the resistance high , low - signals still out of range
-Intracellular solution: started with NaCl 2M, then saline... signals still out of range
-Bath ground: platinum multi wire cable touching the bath solution and connected to the headstage by a gold pin connector, and copper single wire cable touching the bath solution and connected to the headstage by a silver pin connector. - signals still out of range
-Bath solution: started with saline, NaCl 2M, PBS. Deionized water was the only solution to bring signals approximately to 0mV and pA, which is normal as it does not conduct. This suggests to me that the system must be ok and that the problem lies in the bath solution.
-We also tried directly connecting a 5 Ohm resistance onto the BNC connector of the Heka headstage touching the bath solution instead of the holder and pipette. This permit us to zeroe both signals suggesting that the problem lies within the holder/pipette/intracellular solution/microelectrode system...contrary to the previous point.
-The set-up has been grounded and signals are clear with the model cell. There does not seem to be any excessive noise.

So what can be the problem???

Thank you for help

Fraser Moss
Fraser Moss's picture
Is your recording chamber

Is your recording chamber completely clean?

We recently had a problem that we could not maintain a steady holding voltage and determined that it was because the chamber had not been cleaned properly and there were some crystals of one ionic salt or another that was redissolving in the bath and messing with the solution compositions.