Hi all,
I am trying to implement the perforated patch-clamp method to assess intrinsic excitability in mPFC slices from adult rats (PND 70-90).
Maintaining enough positive pressure in my electrode holder has always been a limiting factor in my rig, while working with the regular whole-cell patch clamp. My first attempts to work with perforated patch clamp using the regular Amphotericin B solubilization in DMSO (followed by tip filling the electrode with antibiotic free internal solution and back-filling with the internal containing Amphotericin B), are being especially frustrating given the requirement to keep positive air pressure to a minimum, due to the limitation I always have in my rig.
In the references below, I saw this alternative method to solubilize the antibiotics.
"An alternative method used the bipolar molecule N-methyl-d-glucamine (NMDG) to help disperse and solubilize nystatin or amphotericin B. A stock solution is made from a 0.1 M solution of NMDG dissolved in methanol (pH adjusted to about 7 with methanesulfonic acid in the presence of 0.01 M phenol red) to which nystatin (5 mg/ml) was added. Immediately before use, 50 ml of the stock solution is placed in a polyethylene test tube and dried completely with a stream of N2 gas. Pipette solution is added to the tube and briefly vortexed. The pipette solution can be filtered through a 0.22-um syringe filter; tip filling is not required (hence perforation is achieved more rapidly). Positive pressure can be applied during the approach to the cell, which makes this particularly useful for blind patch-clamping in tissues slices."
Endo K and Yawo H. J Physiol. 200 Article μ-Opioid receptor inhibits N-type Ca2+ channels in the calyx...
Ishibashi, Moorhouse and Nabekura. Patch Clamp Techniques: From Beginning to Advanced Protocols, 2012
Since it is compatible with applying positive pressure while approaching the cell, I am really tempted to give it a try. However, I haven't found any other citation of the method in the bibliography and I wonder if there is any reason for that.
Does anyone have experience with this alternative method? Any feedback and advice would be greatly appreciated!
Please, let me know if there is any further information that you need
Thank you so much in advance.
Best,
Ana
Never heard of dissolving amphotericin in NMDG methanesulfonate. The problem I see with that approach is that neither of the ions permeates amphotericin-formed channels. (You can read about the biophysical characteristics of antibiotic channels in papers from the 1970s.)
Using traditional perforated-patch recording with amphotericin you can reduce its concentration which will make it less of a problem with gigaohm seal formation. This is also good for prevention of spontaneous break-in (i.e. whole cell ).
Thank you so much for your answer, J. Ashot Kozak !
If I properly understand that method, I think the purpose of using NMDG methanesulfonate is to help solubilize the antibiotic, not to use them as active components of the internal solution in terms of supporting the physiology of the neuron. So I wasn't anticipitating they would have to permeate the amhotericin-formed channels. If that was the case, I totally agree with you.
Regarding the biophysical characteristics of this antibiotic channels, I was familiar with this sequence of permeability: Rb+ > K+ > Na+> Li+ > Cs+ and >> Cl- and with the facts that the pores are slighlty permeable to urea, glycerol and erythritol and impermeable to multivalent ions, GAPDH, NADP+, Glucose 6-P, sucrose. Are these the type of characteristics you were referring to?
As for the concentration of the antibiotic, I wasn't intending to use such a high concentration as the stated in this method. I am aiming to work with ≈ 200 ug/ml Amphotericin B, although I have tested a concentration range of [175 - 400ug/ml]. However, when I dissolve it in DMSO, it forms some precipitates when added to the internal solution -even after sonicating and vortexing for several minutes- that often block the patch electrode. The other major issue I am dealing with is very frequent transition to spontaneous break-in during the perforation.
Thank you very much again.
Ana, nevertheless having ions around that cannot pass through the antibiotic channels may result in pore block.
One possibility for precipitate formation: your DMSO is oxidized. Try with fresh. Another thing to consider is switching to nystatin which works more slowly and might be better for preventing spontaneous break-in.
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