Real-time amperometric measurements of zeptomole quantities of dopamine released from neurons

Amperometry with carbon-fiber microelectrodes provides a unique way to measure very small chemical concentration changes at the surface of biological cells. In this work, an investigation of dopamine release from individual neurons isolated from the mouse retina is described. The mice were genetically modified so that, in cells that expressed the protein responsible for catecholamine synthesis, tyrosine hydroxylase, the marker protein, placental alkaline phosphatase, was also expressed. This modification allowed for identification of the dopamine-containing cells among the many present in the freshly dissociated retina. Release of dopamine was evoked by chemical secretagogues delivered from micropipets that were calibrated with respect to response time and concentration delivered. Amperometric measurements were recorded with low-noise patch clamp amplifiers, and the primary noise source was found to be the electrode capacitance. Dopamine release occurred in the form of transient concentration spikes, consistent with release from small intracellular vesicles. With optimized filtering of the data, the quantity secreted during each release event could be determined. The average quantity determined at one cell was 52 zmol. However, the spikes were quite variable in size and the amount released per event ranged from 8 to 170 zmol. These measurements allow an estimation of the concentration of released transmitter in a synapse.