Neuronal Imaging using Fluorescent Sensors for Neurotransmitters

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 DESCRIPTION (provided by applicant): Neurotransmitters are critical to the regulation of the central and peripheral nervous systems and command a number of functions such as learning, memory, sleep, and movement. Discerning the machinery involved in vesicular fusion, the spatiotemporal mechanisms of synaptic release, and the chemical activity of neurotransmitters is vital to understanding both normal and atypical neuronal processes. The ability to effectively measure neurotransmitters in neurons is an essential tool in the study of neurophysiology and neuropsychiatric disorders. Furthermore, understanding the mechanisms of vesicle fusion and transmitter release via exocytosis is of broad medical significance because it will not only aid in the development of therapies for diseases where release of neurotransmitters is compromised, but it will also advance our understanding of FDA-approved treatments that modulate transmitter release, such as Botulinum Toxin A and B. This project involves the preparation and evaluation of fluorescent chemical sensors for catecholamines with a view toward the fluorescent detection of neurotransmitters. These sensors are related to the NeuroSensor class of probes, which have already been used to detect norepinephrine in isolated chromaffin cells. The major advantage of the proposed sensors is that they will be selective for just one type of neurotransmitter. In addition to these selective sensors, pH dependent variants will be produced and tested for analyte binding as well as pH sensitivity. Here, the sensors will bind the neurotransmitter, but only fluoresce upon undergoing the pH jump typically associated with exocytosis. The sensors will be used to study mechanisms of exocytosis via fluorescence imaging in combination with amperometric measurements.