What is the theory behind perforated patch recording?

Answer: Perforated patch is a technique used by electrophysiologists to record neuronal activity with minimal disruption of the intracellular components.


In standard whole cell patch clamp electrophysiology, a tiny glass pipette is used to record neuronal activity. The glass pipette is about 1 micron in diameter. This glass pipette is physically pushed up against the body of a cell, and a small suction is applied. This negative pressure allows a small patch of the cell membrane to adhere to the opening of the pipette. The resistance between the cell surface and the electrode at this point is a gigaohm. With a sharp pull of suction, the cell membrane is ruptured, thus allowing the solution inside the pipette to dialyze and mix with the intracellular solution, and vice versa.

This mixing of solutions can be seen as a disadvantage. The volume of the solution inside the pipette is several orders of magnitude greater than the cell volume. Therefore, as dialysis rapidly proceeds over the next few minutes, the intracellular solution will be completely replaced by the solution inside the pipette. Although this allows for total control over the intracellular solution, it no longer means that the experimenter is observing the cell. Rather, instead of observing how the cell would normally behave, the scientist is changing the conditions.

One way to minimize this artifact is to record neuron activity in a perforated patch configuration. In perforated patch, the solution inside the pipette contains a compound such as amphotericin B or gramicidin. These compounds have antifungal or antibiotic properties. They are able to destroy other cells because they puncture (perforate) holes in the cell membrane, making the cell lose its ability to regulate water and the balance of ions such as sodium and potassium.

In a perforated patch recording, the pipette is moved directly in contact with the cell surface, just as with traditional whole cell patch clamp. When the cell is tightly up against the pipette (gigaohm seal), the gramicidin diffuses through the pipette, and reaches the cell soma. Once there, it begins to perforate the cell membrane only at the site of the patch. Eventually, after a sufficient number of pores have been formed, the pipette is able to read the membrane potential of the inside of the cell.

Perforated patch is useful for researchers who are worried that the whole cell patch clamp approach will change the properties of the cell. For example, there are several intracellular signaling molecules that regulate some function of cellular activity. These intracellular molecules, such as cyclic AMP or guanylyl cyclase, are dialyzed into the tremendous volume of the pipette during a whole cell patch clamp recording. However, these intracellular components remain within the cell membrane during perforated patch recording.

Additionally, perforated patch is stable over long periods of time. In whole cell recordings, the scientist must concern themselves with changes in access resistance, the measure of how accurately the patch is able to influence and read the changes that occur to the cell. Because it is so stable, the perforated patch configuration does not have this worry.