Robust, high-resolution, whole cell patch-clamp capacitance measurements using square wave stimulation

High-resolution, whole cell capacitance measurements are usually performed using sine wave stimulation using a single frequency or a sum of two freque ncies. We present here a high-resolution technique for whole-cell capacitan ce measurements based on square-wave stimulation. The square wave represent s a sum of sinusoidal frequencies at odd harmonics of the base frequency, t he amplitude of which is highest for the base frequency and decreases as th e frequency increases. The resulting currents can be analyzed by fitting th e current relaxations with exponentials, or by a phase-sensitive detector t echnique. This method provides a resolution undistinguishable from that of single-frequency sine wave stimulation, and allows for clear separation of changes in capacitance, membrane conductance, and access resistance. In add ition, it allows for the analysis of more complex equivalent circuits as as sociated with the presence of narrow fusion pores during degranulation, tra cking many equivalent circuit parameters simultaneously. The method is inse nsitive to changes in the reversal potential, pipette capacitance, or widel y varying cell circuit parameters. It thus provides important advantages in terms of robustness for measuring cell capacitances, and allows analysis o f complicated changes of the equivalent circuits.