Holding current is creeping up (becoming more negative)

10 posts / 0 new
Last post
PaulFarrow
PaulFarrow's picture
Holding current is creeping up (becoming more negative)

Hi guys,

I am having real trouble patching hippocampal CA1 neurones. I am blind patching and every time I patch onto a cell the holding current rises to about -300 pA. It's driving me insane. The input resistance also decreases simultaneously. It seems to happen directly after my cell 'opens up' (ie. my series resistance becomes ridiculously low; 5 MOhms). Has anyone experienced anything similar. Is it osmolarity, temperature, movement of pippette... I am losing my mind. Please contact me.

Fraser Moss
Fraser Moss's picture
 First things first.

 First things first.

Are you ever actually achieving a Giga seal or is the thing blowing out while you patch onto the cell?

Have you actually used an osmometer to check the balance of the osmolarity of your pipette and extracellular solutions?

Have you measured the diameter of your pipette?  the resistance might look ok in solution but it still could be just too big.  Alternatively it could be too small and the suction you are applying to the cells to go whole cell is just sucking the hell out of the membrane and the cell blows.

If you are worried about pipette movement just put a graticle in the eyepiece and measure the drift of your pipette over time, or before and after you apply the suction to go whole cell.

Are you just being too rough with the suction when you go whole cell?  On the axon instruments amplifiers there is a "zap" function that you can use alone or in combination with suction to try and break into the cells more gently. 

PaulFarrow
PaulFarrow's picture
in responce to each question:

in responce to each question:

Always get a very good gigaseal

yes, I have used an osmometer to check both solutions. They are around 275 and 300 respectively I think

I haven't measured the diameter of my pippette, but other people here are using the pippettes and don't have the same problem

I am blind patching so I won't be able to use a graticle at the resolution I'm scoping at

Not being too rough, I am fairly experienced at going whole-cell and the decline in input resistance is too slow to be anything like that. It usually declines over an hour period. I think it might be the cell getting 'leaky' but I am using a fairly standard intracellular solution,

Any more thoughts?

Fraser Moss
Fraser Moss's picture
 do you have ATP in your

 do you have ATP in your pipette?

PaulFarrow
PaulFarrow's picture
yeah, 4 mM MgATP

yeah, 4 mM MgATP

Fraser Moss
Fraser Moss's picture
What is the major counter ion

What is the major counter ion in your pipette solution?
Cl-, Gluconate or MeSO4?
Apparently KMeSO4 in the pipette preserves neuronal excitability more effectively than K-gluconate.
 
Velumian, A. A., Zhang, L., Pennefather, P., and Carlen, P. L. Reversible inhibition of IK, IAHP, Ih and ICa by internally applied gluconate in rat hippocampal pyramidal neurones. Pflügers Arch. 433: 343-350, 1997

Zhang, L., Weiner, J. L., Valiante, T. A., Velumian, A. A., Watson, P. L., Jahromi, S. S., Schertzer, S., Pennefather, P., and Carlen, P. L. Whole-cell recording of the Ca2+-dependent slow afterhyperpolarization in hippocampal neurones: effects of internally applied anions. Pflügers Arch. 426: 247-253, 1994

PaulFarrow
PaulFarrow's picture
I'm using CsMeSO3 to obtain

I'm using CsMeSO3 to obtain better voltage clamp. Do you think I should switch to CsMeSO4?

Fraser Moss
Fraser Moss's picture
 No i don't think that will

 No i don't think that will make a difference.

you say that it is taking an hour for the holding current to creep up.  For how long is the patch actually useful?

An hour patch is pretty good for most people.  Do you experiments require super long durations of patching 1 cell to get the data you need?

PaulFarrow
PaulFarrow's picture
My patch experiments are two

My patch experiments are two hours long (and I have to wait for a baseline). Besides, even if they were one hour, my holding current is going up (inout resistance, down) continuously for that hour. So it makes me wonder what is happening to the cell and whether what I am recording is physiologically relevant.

Fraser Moss
Fraser Moss's picture
Clearly there is an effect of

Clearly there is an effect of the dialysis of the cell contents by the contents of the patch pipette over 2 hours.

If you think this is the real problem, them some controlled experiments similar to this done in the papers I cited above will be necessary to determine what component of your pipette solution it is that is responsible for the decrease in the input resistance.

The other factor could just be the health of your slice.  The older the animals are the harder it is to prepare good slices that last a long time.  There is a wealth of papers out there describing the preparation of slices fore patch clamp recording and I expect that you will just have to try a "suck it and see" approach for each protocol to determine which parameters prolong the life of the slice long enough for you to perform the experiments.  

Are other members of your lab able to hold a patch from a slice prepared the same way for the 2hrs required for your experiments?
 
If so are they able to do so using your set-up?

Another idea I had last night was to check all your suction lines and the state of repair of your pipette holder.  If there is a leak/crack anywhere this may well effect the stability of your patch.  Try changing all the O-rings in the pipette holder and try borrowing a pipette holder from someone else's set up who can maintain good patch to see if that helps.