Real-Time Amperometric Measurements of Zeptomole Quantities of Dopamine Released from Neurons

Abstract

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.

Synaptic Vesicle Transporter Expression Regulates Vesicle Phenotype and Quantal Size

“…Before analysis, the current wave was digitally refiltered using a smoothing f unction at 8 kHz. Whereas increased filtering uncovered significantly more events in recordings from dispersed acutely dissociated retinal amacrine cells (Hochstetler et al, 2000), in our preparation we observed few such extra events of sufficient amplitude to reach the 5.0ϫ rms noise threshold with filtering as low as 1 kHz (data not shown).…”

Contribution of Kv1.2 Voltage-gated Potassium Channel to D2 Autoreceptor Regulation of Axonal Dopamine Overflow

“…One recording and stimulation site per slice was tested and each slice served as its own precondition control. Fast scan cyclic voltammetry was used to measure electrically evoked DA overflow (34,35). The potential of the carbon fiber electrode was held at Ϫ0.4 V versus Ag/AgCl between scans and was linearly ramped from Ϫ0.4 to ϩ1.0 V and then back to Ϫ0.4 V at a rate of 300 V/s.…”

The Effects of Vesicular Volume on Secretion through the Fusion Pore in Exocytotic Release from PC12 Cells

“…Indeed, amperometric measurements of current produced during oxidation of released secretory products from adrenal chromaffin cells (Wightman et al, 1991), mast cells (Alvarez de Toledo et al, 1993), PC12 cells (Chen et al, 1994), and mammalian neurons (Jaffe et al, 1998;Hochstetler et al, 2000) have shown that a single fusion event results in a single spike-like current. In contrast, early patch-clamp investigations of exocytosis on mast cells have suggested that neurotransmitter release proceeds in at least two main stages (Breckenridge and Almers, 1987;Zimmerberg et al, 1987).…”