Hello everyone
I recently started to do a perforated patch-clamp in VC
my objective is to record for a long time electrically evoked EPSC in the CA1
to do that I'm patching in the CA1 pyramidal layer (mouse hippo acute slice) while electrically stim in the Schaffer collater near the CA2 CA3 region
my problem is the access to the cell is still low I think around 170-150 although I'm using 100µg/ml with a 5MOhm pipette. Also, the holding current is varying sometimes a lot from zero to -0.03
The EPSC is good and I can record around 50-100pA but the signal is not steady over time which is the main reason I switched from whole-cell to PP
Does anyone have an idea how to make it more stable especially the access and holding current?
With the gramicidin, are you making up a fresh stock solution everyday? Also, are you vortexing and sonicating really well? If you aren't sufficiently sonicating, it may act like a more diluted gramicidin solution-- it seems like 100 ug/ml would be sufficient.
Dear Nour,
I would recommend lower resistance (larger tip diameter) of around 3 megaOhms. The smaller the electrode tip the more likely it will be blocked over a short period of time. Especially with perforated patch you can use larger tips, since you are not sucking the neuronal membrane open.
I would also recommend to use voltage pulses, instead of suction, to perforate the pores further. This way it works like electroporation and you have better and longer recordings without blockage of the tip. I could do reliable recordings for hours using such techniques in the whole-mount retina or brain slices.
For details, you can have a look at my 2008 retina paper in Pflugers Archive and 2013 methods paper on brain slices, both available here at RG.
best wishes,
Refik
Thank you for your reply
How much Vortexing and sonication is required? I usually obtain a residual free solution in methanol and do a brief sonification before diluting it in the IZ
Best
Nour
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