IMPROVED MICROPERFUSION CHAMBER FOR MULTIPLE AND RAPID SOLUTION EXCHANGE IN ADHERENT SINGLE CELLS

A new method for fast perfusion of single adherent cells during whole-cell and single-channel patch-clamp measurements is described. The main advantages over previous methods are: (1) the solution surrounding a single cell in a Petri dish (or cover slip) can be exchanged without contamination of the entire cell population, and (2) only small quantities of test solution (50 mu l) are required. The method consists of inserting a patch pipette into a micro-perfusion chamber (MPC) where whole-cell and single-channel currents can be studied. A small volume (2-3 mu l) surrounding adherent single cells in a Petri dish is separated by enclosing it in a ring of silicone rubber which is then pressed to the bottom of the dish. A drug-containing test solution (50 mu l) is supplied to a funnel at the chamber inflow. The MPC volume isolated from the main solution in the Petri dish is rapidly changed when suction is applied to the camber outflow. The speed of solution exchange in the MPC (16 +/- 5 ms, n = 12) was estimated by observing changes in the tip potential of 1-M Omega patch pipettes during rapid chamber perfusion with solutions of different ionic composition. The seal of the MPC with the surface of a Petri dish was approximated by measuring the electrical resistance between the MPC interior and a reference electrode placed in the Petri dish outside the MPC (range 300-500 M Omega). Additionally, a radioactive calcium channel ligand [H-3]isradipine was added to the MPC and the apperance of radioactivity in the Petri dish (outside the MPC) subsequently measured. We could not observe a significant leak of radioactivity via the seal between the MPC and the Petri dish. Because no significant contamination of the bath outside the chamber occurs, repeated drug applications in the same Petri dish (with different locations of the MPC) are justified for many applications.