Realization of Optical Cell-based Reporters for in vivo Detection of Neuropeptides

项目摘要 神经肽是大脑中必不可少的神经调节剂。它们被释放到突触外空间， 在那里它们会远距离扩散，并通过G蛋白偶联神经肽受体发出信号。 神经肽通过血管张力的变化控制认知、感觉运动处理和能量学 神经系统中的血液流动。药理学和分子遗传学研究表明 神经肽信号的改变是导致大脑功能障碍的一个因素，包括偏头痛、成瘾、 动力和压力。虽然在大脑中广泛表达，但令人惊讶的是，人们对其何时和何时表达知之甚少 神经肽在那里被释放。实时监测清醒动物体内神经肽的释放 执行复杂的行为将是变革性的，使得能够阐明 调节大脑神经回路的神经肽。 作为对RFA-MH-16-775的响应，我们建议开发和验证一种创新的神经技术 光学测量神经肽在大脑中细胞特定和电路特定的过程中的释放。这个 新技术是基于细胞神经递质的荧光工程记者，简称 CNiFERs，最初是为检测经典小分子的释放而开发的 神经递质。CNiFER是一种克隆的HEK293细胞，它被设计成表达特定的G蛋白 偶联受体和基因编码的基于荧光的细胞内钙传感器。CNiFER是 植入大脑，在那里它们产生最小的炎症并保持几天的存活，并且已经 用于测量多巴胺、去甲肾上腺素和乙酰胆碱在体内的体积传递 在学习过程中。 三个神经肽CNiFERs将被开发并用于我们自己的试验台验证项目 实验室：食欲素，它在睡眠调节以及药物寻找和恢复方面都很重要， 与抑郁、动机和学习有关的生长抑素，以及血管活性肠病 与神经可塑性和学习有关的多肽。对于合作项目，我们将进一步 构建四个额外的神经肽CNiFERs用于检测强啡肽、促肾上腺皮质激素释放 因子、神经肽Y和P物质。每个神经肽CNiFER将在体外受到严格的 在使用它们之前进行测试，以研究神经肽在体内释放的动力学和后果。

Project Summary Neuropeptides are essential neuromodulators in the brain. They are released into the extrasynaptic space, where they diffuse over long distances and signal through G protein coupled neuropeptide receptors. Neuropeptides control cognition, sensorimotor processing, and energetics through changes in vascular tone and blood flow in the nervous system. Pharmacological and molecular genetic studies have implicated alterations in neuropeptide signaling as a contributor to brain dysfunctions, including migraines, addiction, motivation and stress. Although widely expressed in the brain, remarkably little is known about when and where neuropeptides are released. Monitoring the release of neuropeptides in real-time in awake animals performing complex behaviors would be transformative, enabling the elucidation of the function of neuropeptides in regulating neural circuits in the brain. In response to RFA-MH-16-775, we propose to develop and validate an innovative neurotechnique for optically measuring release of neuropeptides in a cell-specific and circuit-specific processes in the brain. The new technology is based on cell-based neurotransmitter fluorescent engineered reporters, referred to as CNiFERs, which were original developed for detecting the release of classical, small molecule neurotransmitters. A CNiFER is a clonal HEK293 cell that is engineered to express a specific G-protein coupled receptor and a genetically encoded fluorescence-based intracellular calcium sensor. CNiFERs are implanted in the brain, where they produce minimal inflammation and remain viable for days, and have been used successfully to measure volume transmission of dopamine, norepinephrine and acetylcholine in vivo during learning. Three neuropeptide CNiFERs will be developed and used for test-bed validation projects within our own laboratories: Orexin, which is important in sleep regulation as well as drug seeking and reinstatement, Somatostatin which has been implicated in depression, motivation and learning, and Vasoactive Intestinal Peptide, which as been implicated in neuroplasticity and learning. For collaborative projects, we will further construct four additional neuropeptide CNiFERs for detecting release of Dynorphin, Corticotropin-Releasing Factor, Neuropeptide Y and Substance P. Each neuropeptide CNiFER will be subjected to rigorous in vitro testing prior to their use to study the dynamics and consequences of release of neuropeptides in vivo.