We propose to develop and utilize new electroanalytical methods to study single exocytotic events. Exocytosis is of central importance in neuronal transmission, release of hormones and neuromodulators, and immune response and plays an essential role in mediating brain function, emotional and behavioral responses, and many other physiological processes. As such, a detailed understanding of single exocytotic events is needed for better treatments for central nervous system diseases. Electroanalytical methods have played vital roles in this bioanalytical task in the past three decades owning to their unique characteristics, including very high spatial, temporal resolutions, and excellent chemical resolution. However, current electroanalytical methods have significant challenges. For example, the quantal size and release kinetics of the exocytotic event is likely affected by it location relative to the electrode. Array-based electrochemical imaging on single-cells has strong crosstalk. In addition, current methods do not allow for analysis of intracellular vesicles. We propose to address these challenges by developing new electroanalytical techniques and microelectrodes. We emphasize the use of a nanoband electrode integrated with on-chip microelectrodes to increase accuracy and eliminate crosstalk. We also develop new cyclic voltammetric methods to increase temporal resolution and further eliminate crosstalk in voltammetric imaging. Additionally, we use a nanopore electrode to analyze single intracellular vesicles. Building on our strong expertise in single-cell exocytosis, microelectrodes, and nanopores, we propose to accomplish our goal by pursuing three specific aims:
Aim 1. To develop and utilize an on-chip microelectrode integrated with a nanoband electrode to analyze single exocytotic events. We will analyze single exocytotic events using an integrated on-chip microelectrode. We will use computer simulation to achieve a deeper understanding of dopamine transport at the electrode/cell interface. We will optimize electrode geometry to improve accuracy and reproducibility in single-cell amperometry. In addition, we will improve their stability and sensitivity through electrode design and chemical functionalization.
Aim 2. To eliminated crosstalk and increase temporal resolution in electrochemical imaging utilizing new microelectrode arrays and voltage waveforms. We will image single-cell exocytosis with eliminated crosstalk using amperometry and voltammetry. We will employ new microelectrode arrays and use integrated nanoband electrodes to eliminate crosstalk in amperometry. We will then apply new voltage waveforms in fast-scan CV to improve temporal resolution and eliminate crosstalk in voltammetric imaging.
Aim 3. To further develop and utilize a nanopore-based method to simultaneously analyze the sizes and dopamine contents of single intracellular vesicles. A new nanopore electrode has been developed using a quartz nanopore and a microelectrode placed in close proximity to the pore orifice. This microprobe is especially useful for simultaneously determining the sizes and dopamine contents of single intracellular vesicles. We will further develop this new technique and use it to analyze vesicles from model cells. We will use this technique to characterize vesicles from single cells treated by pharmacological reagents. This work will provide new analytical strategies for better understanding single exocytotic events. Our proposed methods have key advantages and can provide new information inaccessible with current techniques. The integration of a nanoband electrode increases the accuracy in determining quantal size and kinetics. New voltage waveforms and microarrays can improve single-cell imaging to better study exocytotic heterogeneity. A nanopore electrode can analyze single intracellular vesicles. We anticipate these new methods and probes will find extensive use in analyzing exocytosis and will be quickly adapted by other groups in the community and become their everyday tools.

Public Health Relevance

This work will address key challenges in current analytical methods and provides new strategies to investigate the release of dopamine and many other electroactive neurotransmitters from single cells with ultrahigh spatial, temporal, and chemical resolutions. Exocytosis is a fundamental cellular process in neuronal communication and plays central role in numerous brain diseases, such as Parkinson's disease and Huntington's disease. As such, a more fully understanding of exocytosis will likely improve our knowledge of neuronal communication and will likely lead to better treatments to many neurological diseases and disorders.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Edmonds, Charles G
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University of Washington
Schools of Arts and Sciences
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Oja, Stephen M; Guerrette, Joshua P; David, Michelle R et al. (2014) Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator. Anal Chem 86:6040-8