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Parallel on-chip analysis of single vesicle neurotransmitter release

Real-time investigations of neurotransmitter release provide a direct insight into synaptic communication. In this work we present parallel on-chip in vitro measurements of modulation in individual neurotransmitter release events from cells, treated with a pharmacological reagent.

By Alexey Yakushenko, Enno Kätelhön and Bernhard Wolfrum

Published: Anal. Chem., 2013, 85, pp 5483-5490, May 6, 2013

Parallel recordings of neurotransmitter release Parallel recordings of neurotransmitter release from PC12 cells on a MEA. (a) 64-Channel MEA. Insets: (i) Live-dead staining of PC12 cells on MEA 2 h after plating. Calcein (green) indicates live and ethidium homodimer (red) indicates dead cells (ii) zoom on a single electrode, scale bars 200, 200 and 50 µm. (b) Simultaneous amperometric recordings of spontaneous exocytotic events from 10 electrodes. Each trace represents one electrode, scale bar 50 pA.

Neurotransmitters are the chemical messenger molecules in synaptic communication that transduce information between cells. Malfunctioning of this transduction mechanism is related to mental disorders such as depression or schizophrenia. Several drugs, including the norepinephrine-dopamine reuptake blocker nomifensine, aim at relieving associated symptoms by affecting local neurotransmitter concentrations in the brain. Investigation of these processes at a level of a chemical transduction may shed light on the underlying nature of these diseases. To this end direct electrochemical detection of redox-active neurotransmitters is typically performed with carbon-fiber microelectrodes (CFMs) in combination with a patch-clamp amplifier. The successful implementation of CFMs to measure neurotransmitter release from large dense core vesicles of different cell types, as well as from small synaptic vesicles (SSV) of midbrain neurons, established the method as a standard for the detection of single vesicle release. However, despite being the state-of-the-art tool for studies of secretory events, CFMs are limited to investigations of only a few cells due to labor-intensive manual handling of each individual probe.

Effect of dopamine transporter blocker nomifensine Effect of dopamine transporter blocker nomifensine on the reuptake of released dopamine. Histogram of peak half-times for control (blue, mean = 1.40 ± 0.07 ms, Nelectrodes = 42) and 10 µM nomifensine (red, mean = 2.12 ± 0.26 ms, Nelectrodes = 37), errors are SEM.

A logical solution to address this challenge is the introduction of a functional substrate that can record from many cells in parallel without the need of manual electrode positioning. In this work, we report on the development and implementation of a platform for parallel recordings of vesicular neurotransmitter release in real time. The system operates with MEA chips comprising 64 simultaneously addressable microelectrodes with a minimum electrode diameter of 3 µm. It allows electrochemical measurements in the sub-picoAmpere regime with automated analysis of single vesicle release characteristics. We further demonstrate the potential of the system by distinguishing concentration-dependent effects of the dopamine reuptake blocker nomifensine on the vesicular neurotransmitter release in vitro. We observe minute but statistically significant drug-induced changes by exploiting the acquired large datasets. The results are interpreted by an analytical model that takes into account geometric aspects of the cell-substrate interface. Overall, due to the high number of recording sites, large bandwidth, and the ability to record single-vesicle signals, our system provides all the toolkits needed for electrochemical measurements of neurotransmitter release in drug-screening applications.