Amperometric Detection of Neuropeptide Release

描述（由申请人提供）：本研究的长期目标是开发一种可以监测已识别神经肽释放的新技术。这些递质分子遍布整个神经系统，调节着各种各样的生理过程。其释放模式的变化与包括癫痫和高血压在内的一系列疾病有关。然而，要充分认识这些递质在神经系统的生理和病理功能中所起的作用，需要确定控制它们释放的因素。由于大量不同的神经肽，很难在生理时间尺度上监测特定肽的释放。安培法是一种满足灵敏度和时间分辨率要求的技术，但由于许多多肽不具有氧化性，因此不能广泛应用。因此，我们建议开发短的可氧化肽标签，可以插入到任何神经肽原激素。在“标记”激素原的翻译之后，这些可氧化肽将作为致密核心颗粒特定群体的独特分子标记。标签的分泌（以及在同一激素原上的其他肽）可以用安培法测量。这种方法的一个优点是它可以潜在地应用于任何已测序的神经肽。为了检验该技术的实用性，我们建议在多种细胞类型中进行测试，并最终开发一种转基因小鼠，该小鼠在含有神经肽的致密核心颗粒的有限种群中携带可氧化标签。我们建议开发的试剂将允许神经科学家使用该技术研究广泛的临床相关问题。

DESCRIPTION (provided by applicant): The long-term goal of this research is to develop a new technique that can monitor the release of identified neuropeptides. These transmitter molecules are found throughout the nervous system and regulate a wide variety of physiological processes. Changes in their patterns of release have been implicated in a spectrum of diseases including epilepsy and hypertension. However to fully appreciate the roles that these transmitters play in the physiological and pathological functioning of the nervous system requires that the factors controlling their release be identified. Due partially to the large number of different neuropeptides it is difficult to monitor the release of specific peptides on a physiological time scale. Amperometry is a technique that fulfills the important requirements of sensitivity and temporal resolution but can't be widely applied since many peptides are not oxidizable. We therefore propose to develop short oxidizable peptide tags that can be inserted into any neuropeptide prohormone. Following translation of the "tagged" prohormone these oxidizable peptides will act as unique molecular markers for a specific population of dense core granules. Secretion of the tag (and thus the other peptides on the same prohormone) can then be measured using amperometry. One advantage of this approach is that potentially it can be applied to any sequenced neuropeptide. To examine the utility of the technique we propose to test it in a variety of cell types and ultimately to develop a transgenic mouse that harbors the oxidizable tag in a restricted population of neuropeptide-containing dense core granules. The reagents that we propose to develop would allow the technique to be used by neuroscientists investigating a wide range of clinically relevant questions.