A novel holder allowing internal perfusion of patch-clamp pipettes

We describe a simple pipette holder which allows, within a single experiment, multiple exchanges of the solution inside "gigaseal" glass pipettes commonly used for electrical studies of single cells or isolated membrane patches. The design minimizes electrostatic and mechanical perturbations associated with perfusion by integrating into the holder a reservoir which is connected to a perfusion pipette fabricated from flexible, resilient quartz tubing. The tip of the perfusion pipette can be pulled to any diameter and positioned precisely within the main patch-pipette by sliding the reservoir along a guide in the holder. An open reservoir for suction driven solution exchange, and a closed reservoir for pressure driven solution exchange were developed. For the open system, the speed of solution exchange was studied as a function of the tip diameter of the perfusion pipette (approximately 22 s for a 40 micron tip diameter). Both systems were characterized using atrial myocytes (a) by examining the effects of intracellular applications of cAMP or of the catalytic subunit of protein kinase A on calcium currents in the whole cell recording mode and (b) by studying the effects of local applications of acetylcholine (ACh) on single channel currents in the isolated membrane patch mode.