Neuronal Imaging using Fluorescent Sensors for Neurotransmitters

 描述（由申请人提供）：神经递质对中枢和外周神经系统的调节至关重要，并控制许多功能，如学习、记忆、睡眠和运动。识别囊泡融合中涉及的机制、突触释放的时空机制以及神经递质的化学活性对于理解正常和非典型神经元过程至关重要。有效测量神经元中神经递质的能力是神经生理学和神经精神疾病研究中的重要工具。此外，了解囊泡融合和通过胞吐释放递质的机制具有广泛的医学意义，因为它不仅有助于 开发神经递质释放受损的疾病的治疗方法，但它也将促进我们对FDA批准的调节递质释放的治疗方法的理解，如肉毒杆菌毒素A和B。本计画系以神经传递素之萤光侦测为目标，制作及评估儿茶酚胺萤光化学感测器。这些传感器与NeuroSensor类探针相关，NeuroSensor类探针已经用于检测分离的嗜铬细胞中的去甲肾上腺素。所提出的传感器的主要优点是，它们将只选择一种类型的神经递质。除了这些选择性传感器之外，还将生产pH依赖性变体，并测试分析物结合以及pH敏感性。在这里，传感器将结合神经递质，但只有在经历通常与胞吐作用相关的pH值跳变时才会发出荧光。该传感器将被用来研究通过荧光成像结合电流测量的胞吐机制。

 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.