Patch amperometry: high-resolution measurements of single-vesicle fusion and release

Patch amperometry is a new technique for the observation of single-vesicle exocytosis. Exocytosis of single vesicles as small as 50 nm in diameter can be detected by cell-attached patch-clamp admittance measurements(1-4) indicating fusion of vesicles with the plasma membrane or by amperometry with a carbon fiber electrode (CFE)(5-9) indicating release of oxidizable molecules such as catecholamines. The admittance measurement provides the membrane capacitance that increases in proportion to the membrane area because of the incorporation of the vesicle into the patch membrane. It also reveals the fusion pore conductance during an exocytotic event, giving an estimate of fusion pore dimensions. Amperometry provides the amount and time course of release of molecules that are readily oxidizable such as dopamine, norepinephrine or serotonin. This technique is capable of detecting as little as a few thousand motecules(8,9). it also resolves the flux of catecholamines through a narrow fusion pore in a so-called foot signal that precedes rapid release indicated by an amperometric spike(6). Patch amperometry combines high-resolution patch capacitance measurements with amperometry by placing the amperometric detector inside the patch pipet(10). The method provides precise information on single-vesicle size and quantal content, fusion pore conductance and permeability of the pore for catecholamines(10-14). Thus, it is a unique tool to investigate the mechanisms that modulate quantal size and the effect of molecular manipulations affecting the properties of the fusion pore. Here we provide step-by-step instructions for the application of this method.