Unstable whole cell patch clamp

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azylbetal
azylbetal's picture
Unstable whole cell patch clamp

Hi all,

I'm conducting whole cell patch clamp experiments in acute slices of accessory olfactory bulb (mitral cells)  in mice.

I'm achieving a G-ohm seal and going to whole cell configuration without problems, but in almost every cell I get an abrupt drop in resistance (3-8 fold), 3-20 minutes after breaching the membrane. After this happens the recording gets very noisy, and usually I have to drop the cell. I attached an example recording (in VC) during which it happened. It contains a pulse train, but the phenomenon isn't related to stimuli (it also happens "spontaneously").

In some occasions the resistance goes back to it's initial value, just as abruptly as it dropped. This leads me to the conclusion that nothing is actually happening to the cell, but rather to the pipette connection with the membrane.
Other researchers in the lab are using the same setup for WC patch of (much larger) cerebellar Purkinje cells, and they don't experience the full extent of the problem as I do.

Needless to say, this presents a big obstacle for my research, and I already tried a lot of proposed solutions -
* Changing pipette width (usually I use ~8MOhm, tried higher and lower).
* Changing pipette approach angle (a higher angle seemed to make the situation a bit better, but not enough)
* Changing intracellular and extracellular solutions

Any suggestions would be highly appreciated.

hypa_dude
hypa_dude's picture
I've had a problem very

I've had a problem very similar to this early on. A few things make a difference:

1. Using thick-walled glass (O.D. 1.5 I.D. 0.86) instead of thin walled glass (1.5 O.D. 1.17 I.D.)
2. Fire polishing and slygarding electrode tips. there is a good jove article on this.
3. Eliminating vibrations on the manipulators.  Even if I can't see vibrations, tightening screws with threadlock, making sure the manipulators don't have anything leaning or pulling on them (pressure tubes, cables, etc) and putting on a little vibration dampening (like dynamat) helps.
4. intracellular solution pH and osmolarity.  I know this is a fundamental but one thing I noticed is that the pH of the intracellular solution takes at least a few hours of mixing to stablize after adding all the indgredients and before freezing.  This is incredibly annoying but I've found this is absolutely critical and will make a bigger difference in small cells.
5. After the gigohm seal has formed, pulling back a micron before breaking in.
6.  When I approach, I try to go straight in, I've found that going  down can contort the slice and put elastic pressure on it.  Backing the electode in and out  a few times about 50 microns or so can relieve this pressure.

I hope this helps.

azylbetal
azylbetal's picture
Thanks a lot for the detailed

Thanks a lot for the detailed response!

So far, it seems that fire-polishing did the most substantial improvement.

Sergiy
Sergiy's picture
Hi.

Hi.
Recently I started to work with hippocampal slices. I use Axopath 200B, Clampex 9.2. After I reached gigaseal it becomes extremly unstable, baseline begins to shake finely (it's not a 50 Hz, it's a tiny rundom, unsystematic shaking with almost indistinguishable amplitude), and after a few seconds baseline drops down, I'm loosing the seal at all. It happens every time I've got a seal. Could please somebody explain why is seal unstable and what to do to get stable seal and whole-cell?

Another problem is that I cannot define the real resistance of my pipettes. If to take two pipettes made from one capillary, on my setup one of them has 11 MOhm but on the another rig (also with Axopath 200B and Clampex 9.2) the second pipette from this pair shows only 6-7 MOhm. I used the same model of the cell on these both setups and it showed equal parameters on both. Where could be the problem?

I would be greatly appreciated for any suggestions.