Stable whole-cell on hippocampal slice

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Sergiy
Sergiy's picture
Stable whole-cell on hippocampal slice

Hi all,

Recently I started to work with hippocampal slices. I use Axopatch 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 random, 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 on hippocampal slices?

Another problem is that I cannot define the REAL resistance of my glass 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 Axopatch 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?

Any suggestions concerning either of problems will be greatly appreciated.

Simon8
Simon8's picture
Are you sure that the

Are you sure that the solution sare the same? Because pipette resistance also depends on the solutions you use.

As for the Drop of baseline you might want to have a look here:
http://www.scientistsolutions.com/t19490-unstable+whole+cell+patch+clamp.html

P.S.: I see you already posted there. Did you do all the things hypa_dudesuggested?

Sergiy
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Thank you for your reply.

Thank you for your reply.

I tried almost all suggestions posted by hypa_dude. I'll try tip polishing else.

Jack Galvan
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 Hi dude,

 Hi dude,
I would suggest you to extra check out your grounding inside the slice chamber... maybe you need to rechloride the pellet and the wire of your headstage.

Sergiy
Sergiy's picture
Thank you all for your

Thank you all for your suggestions. Now it's working.

Simon8
Simon8's picture
Do you have any hint for us,

Do you have any hint for us, what caused the problem?

Sergiy
Sergiy's picture
Concerning unstable whole

Concerning unstable whole-cell: I switched from glass microelectrodes of 7 - 8 to 3 - 4 MOhms and sometimes use polishing.
Also it is useful (as Jack Galvan told) to chloride ground and recording electrodes quite often (at least once per week).

About discrepancies in glass microelectrode resistances: Actually, it seems that there was no this problem. It disappered and now both setups shows similar values for similar pipettes. Or, maybe, it resolved after chloriding of setup electrodes.

Thank you for your participation in resolving of my problems.

Sergiy
Sergiy's picture
Hi guys,

Hi guys,

After preparation brain slices I try to patch their cells but it seems that membranes are very delicate and sensitive to the negative pressure: to establish a gigaseal is not a problem but to make a whole-cell - that is a big deal! When I try to rupture membrane I lose my seal immediately.

I tried unpolished and fire polished pipettes, pipettes with different resistances (from 3.5 up to 7 MOhm) but nothing had effect. Solutions are fine.

I suspect that something wrong in slice preparation procedure.

Can anybody suggest what to do to get the preparation with membranes good enough to be stable for patching them?

Thank you.

Simon8
Simon8's picture
The Prepartion should be fast

The Prepartion should be fast and the tissue should be kept near 0°C at least during cutting.
If you cut your slices on a vibratome, make sure to cut slow and with high amplitude to ensure smooth cutting.

What do your cells look like (how do you look at them? Normal Phase Contrast oder Infrared Camera?)? InInfrared they should look solid and not glassy and transparent.

It would help alot, if you post your detailed prcedure during preparation to see, whats wrong maybe.

Sergiy
Sergiy's picture
Hi Simon8,

Hi Simon8,

Thank you for your reply.

Yes, I use the ice cold solution for preparation the brain slices as emphasized in all the literature. The brain isolation I do as fast as possible.

I use the speed 0.12 mm/s on my vibratome (Leica). I don't regulate the amplitude (it is all the time the same).

I can not tell about the view of the cells because I see them only like uniform mass with different brain slice regions of transparency (like blur spots).

I would be grateful for your suggestions.

JunEphys
JunEphys's picture
 Visualization is also

 Visualization is also critical according to my experience. what about your perfusion speed? Are you sure your cells are healthy?

Sergiy
Sergiy's picture
Hi JunEphys,

Hi JunEphys,

Thank you for your suggestion. I think perfusion speed and cells are OK. Successful rate increased before summer. Hopefully, this trend will keep after it. :-)