That is probably due to diffusion potential of the pipette tip where Cs and K have different diffusion rate relatively to external (NaCl-based) solution (ACSF or Ringer or others). It should be equilibrating by time, otherwise there is other source of the drift.
For example, as other source of a drif it may be Ag-AgCl-wire: I observed dramatic jump of the offset potential when changing K-gluconate to K-gluconate with addition of 1 mM polyamines (putrescine, spermidine and others)-I refer it to silver wire sensitivity to polyamines in pipette holder. The drift was slowed down after few minutes.
Thanks, Dr. Statchkov. Does that mean less diffusion potential needs less time to be equilibrated? Since I found that Cs internal with 20 mM Cl- was much faster to be stabalized than with 8 mM Cl-. And would you mind to tell me why you prefer to use polyamines in the internal? I noticed some researcher are using it, but I don't know the theory of this.
Dear Jianli,
1. You will certainly better solve your drift problems if you will put more chloride in the ICS: you observed that 20 mM is already better, try 40 mM (if it is possible for your natural chloride level because some cells have low endogenous chloride such as adult neurons (~7-10 mM), but some high such as juvenal (up to 20-30 mM). Check, please the literature for details on chloride content.
2. You will also get less problems if you will fill the pipettes with your original Cs-Gluconate only the tip of the pipette (about 3-8 milometers), but the rest of the glass pipette fill with high chloride ICS, so that Ag-AgCl wire (which is inserted only into the back end of the glass tubing-pipette will be in high chloride level and will equilibrating potential faster.
3. Polyamines that we use are necessary to work with glial cells (astrocytes, Mueller cells, etc.) because the cells contain naturally endogenous polyamines, spermine, spermidine for example (Skatchkov et al., 2000; Biedermann et al., 1998; Laube and Veh, 1997) and we want to keep intracellular conditions close to natural. The other reason was: doing so, we discovered that the properties of astrocytic communication, cell-to-cell signaling, was so much increased and improved by polyamines (Benedikt et al., 2012).
Did I answer you all?
Cordially, Dr. Skatchkov
Dear Jianli,
can you answer briefly, if any of my suggestions helped?
Cordially, Serguei.
Dear Dr. Skatchkov,
I will not have experiment for the next three weeks. I will try it after SfN meeting. And will let you know what I get. Thanks a lot for your concern. And I'm very sorry for spelling your last name wrong twice.
With Best!
THANKS! I wait
DO not worry, you are correct, my first name is Serguei and last is Skatchkov
Cordially, Serguei.
Dear Dr. Skatchkov,
Sorry for the late response. I figured out that the major cause for the drifting in my recording is the 10 mM QX314 in the internal. I have no idea why QX314 has this effect. I decrease its concentration to 1 mM, which is enough to block action potentials, it's much better. Even though increase concentration of Cl- does help.
Happy holidays!
Jianli
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