Na+ current recordings

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Bubli
Bubli's picture
Na+ current recordings

Hi all,

I am currently trying to record sodium currents from acutely dissociated neurons (dissociate them, let them settle for an hour and then record withing 4 hours). 

I can seal my cells but when I go wholecell  I lose the seal across my cell within 2 mins.  I know my recording setup is stable b/c I am able to record from these cells using regular solutions for at least 30 mins.

Does anyone have any suggestions?

The FFM
The FFM's picture
There is a contradiction in

There is a contradiction in your question as it is presently written.

You say that "these cells" are stable for more than 30 minutes in "regular" solutions, but this is after you state you cannot go whole cell for more than two minutes.  Are you in fact unable to mainatin your seal longer than 2 minutes in a solution different from your regular solutions?

Can you repost your question again. I think there is some important information missing or you need to rephrase somewhere.

Please include the following information in your next post:

the recipies for your extracellular solutions and pipette solutions (including osmolarities - measured, not calulated)

typical resistance of your patch pipette in solution before you make your seal
The tip diameter of the patch pipette - measure it on a microforge if you have not done so already
Do you fire polish?
Do you balance the osmolarities of all your solutions?

what are the conditions under which you can maintain a 30 minute whole cell patch vs a the condition that you cannot hold it for less than 2 minutes?

thanks

Bubli
Bubli's picture
Hi there, sorry about the

Hi there, sorry about the confusion!

The current experiment is to record VG-Na+ currents.  The solutions that I'm currently using are as follows, external recording solution (in mM):  50 NaCl, 100 TEA-Cl, 1 MgCl2, 2 CaCl2, 1 CsCl, 1 BaCl2, 0.3 CdCl2, 10 HEPES and 10 glucose (pH 7.3 with NaOH) and internal recording solution (in mM): 10 NaCl, 120 CsMeS04, 1 MgCl2, 10 HEPES, 10 EGTA  and 2 Na2ATP  (pH 7.3 with CsOH).

The measured osmolarity for the external recording solution is 300 mmol/kg and 291 mmol/kg for the internal recording solution.

The typical resistance of my patch pipette range from 6-7 MOhms.  I do firepolish my tips, however, I have not measured the tip diameter.

I don't understand the question about balancing the osmolarity of my solutions.  So, I probably don't  do that.

Using the above conditions I cannot maintain the cells for more than two minutes.  When I look at my cells the look like they have swelled and burst.  In short they look dead and on the oscilloscope the seal drops from GOhms to 10-20 MOhms.

For the conditions where I can maintain these cells  for 30 minutes (even after going whole cell and maintaing a GOhm seal) I use the following solutions:
External recording solution (in mM):  140 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 HEPEs and 10 glucose
Internal recording solution (in mM):  130 K-gluconate, 10 KCl, 10 HEPES, 5 EGTA, 1 MgCl2, 0.1 CaCl2, 4 Na2ATP
Using these sets of solutions I am able to obtain both voltage clamp and current clamp data.