Electrophysiological detection of acetylcholinesterase activity using concentration clamp on physically isolated Aplysia neurons.

In this study, we have used electrophysiological techniques to evaluate the acetylcholinesterase (AChE) activity on single neurons physically isolated from the pedal ganglia of Aplysia californica and kurodai. Acetylcholine (ACh) was applied to cells having Na-dependent response using a concentration clamp technique which allows a step-like complete change of external medium around the cell (diameter more than 120 micron) within 30 msec. When the neuron was exposed to ACh (50 microM) initially by rapid and brief (400-800 msec) flow and continuously after stopping the perfusion, the induced current rose to a peak and then decayed in the presence of ACh. However, the current increased again when the rapid flow of the same ACh solution around the cell was resumed. The increase in the current by the resumption of the perfusion was not seen when carbachol was substituted for ACh or when extraordinarily high concentration (10 mM) of ACh was used. Furthermore, this increase in the current was blocked by the antiAChE agent, edrophonium, in a dose-dependent manner. These results suggest that acetylcholinesterase (AChE) causes a local depletion of ACh at the membrane surface where the rate of hydrolysis of ACh exceeds the rate of ACh diffusion from bulk solution and the increase in the current by the resumption of the perfusion resulted from the restoration of ACh concentration at the membrane surface. This electrophysiological indication of AChE activity may be a useful tool for the study of AChE by other than biochemical means.