Perforated Patch

Note: If you find information on this web page useful for your experiments, and if these experiments eventually lead to a publication, please, consider citing my paper from 2012 (see the citation immediately below). All perforated patch recordings in this paper were performed in the way described here, and can serve as a confirmation that this protocol works. Alternatively, you may treat this page as a "personal communication". Citation: Khakhalin AS, Aizenman CD. (2012). GABAergic Transmission and Chloride Equilibrium Potential Are Not Modulated by Pyruvate in the Developing Optic Tectum of Xenopus laevis Tadpoles. PLoS ONE 7(4): e34446.

In short, perforated patch recordings resemble those performed in whole cell or tight cell-attached modes. The perforating peptide (gramicidin) is added to the solution inside the pipette; after the gigaseal is achieved, one needs to wait several minutes for the peptide to get into the membrane and allow voltage control over the cell. The protocol is somewhat tricky however, so it worth describing the details.

Take a tube of internal solution. Some people prefer using slightly weird internals (such as ones with extremely low or extremely high Cl⁻ concentration, to be able to notice the moment when the perforated patch is broken and the recordings goes whole-cell [Akerman, Cline 2006]. Filter the internal into another Eppendorf tube (you have to do it before the gramicidin is added, otherwise it will be filtered out). The tubes on the table are usually quite dusty, so it is worth taking one from a rarely disturbed bag somewhere deep in the shelves. Add required amount of DMSO with gramicidin in it; it may cause partial precipitation of the peptide. Close and vortex (shake) the tube; then attach a piece of plastic foam to it (put the Eppendorf tube through the hole in the foam), and let it float in the sonicator; sonicate for ~2 min. Suck the contents of the tube into a whisker syringe. During the recording, keep the syringe with the gramicidin internal on ice. Prepare new solution every 3-4 hours (as allegedly gramicidin gradually deactivates while in solution).

As the gramicidin solution is not filtered, and as at high concentrations the peptide tends to precipitate, you cannot blow the bubbles through the pipette tip, and also you can't apply strong positive pressure while approaching a cell, for the same reason. There are two ways to tackle that. One approach is to filter the internal with a filter that has relatively large pores, however in this case the concentration of gramicidin in the solution will get lower, and you'll have to wait longer before getting enough access to the cell (possibly about 20-30 min). Alternatively, you can use a front-filling technique: just keep a small tube of filtered gramicidin-free internal, and dip the pipette tip in it for several seconds (10-20 s). Due to the action of capillary forces the tip will get filled with the solution (it is almost impossible to see the liquid inside the tip, but it should be there). Then the remaining of the pipette is filled with gramicidin-containing solution as usual. There will be some bubbles in the tip, and remember: this time you cannot blow them through, so hold the pipette in your hand vertically, its tip down, and gently knock it with a pen or a marker for 1-5 min, until the bubbles leave the tip. 5 minutes of knocking may sound like a pretty long time, but the result of this time investment is that you'll get a pipette with a clean unclogged tip, and full of gramicidin, which means higher yield and better recordings

Another reason not to apply positive pressure while approaching cells is that you don't want to spill the stuff all over their outer membranes - they will get leaky, and die if you do that. It is not advisable however to completely get rid of positive pressure, as in this case the electrode keeps sucking stuff from the solution (due to the capillary forces action), and the tip quickly becomes dirty. The best idea is to close the valve on the pressure-control syringe before installing an electrode into the holder: it will create some positive pressure, just enough to prevent the external solution from entering the tip, making it dirty. It will also make the patching easer: you won't probably see the dent on the membrane as you go close to it, but theoretically it will still be there, exactly as for normal patch-clamp protocol. At the same time the tip won't get clogged, as the flow of stuff from the tip will be really low.

After you patched a cell, establishing a Gigaseal, just wait for 5-10 min and see the access resistance gradually decrease, with postsynaptic events becoming visible. Usually in the beginning you would see a normal gigaseal (1-4 GΩ), which would gradually evolve to a stable perforated patch case, which looks like a "very small and resistant cell" (capacitance of ~5 pF, membrane resistance of 1-2 GΩ, access resistance of ~100 MΩ). After doing a recording, apply brief suction, going whole-cell: the capacitance should increase 2-3 times in a jump (up to about 15-20 pF), the access resistance could decrease slightly (down to 20-30 MΩ), while the cell membrane resistance would usually decrease gradually with gramicidin perforating it, killing the cell.