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质。它们被释放到突触外空间， 在那里它们长距离扩散并通过G蛋白偶联的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 以及神经系统中的血液流动。药理学和分子遗传学研究表明 神经肽信号传导的改变是脑功能障碍的原因，包括偏头痛，成瘾， 动机和压力。虽然在大脑中广泛表达，但对何时和何时表达知之甚少。 释放神经肽的地方实时监测清醒动物神经肽的释放 执行复杂的行为将是变革性的，使阐明的功能， 神经肽调节大脑神经回路。 为了响应RFA-MH-16-775，我们建议开发和验证一种创新的神经技术， 光学测量脑中细胞特异性和回路特异性过程中神经肽的释放。的 新技术是基于基于细胞的神经递质荧光工程报告，称为 CNiFERs，最初开发用于检测经典小分子的释放， 神经传递素CNiFER是克隆HEK 293细胞，其被工程化以表达特异性G蛋白。 偶联受体和遗传编码的基于荧光的细胞内钙传感器。CNiFERs是 植入大脑，在那里它们产生最小的炎症，并保持活力数天， 成功地用于测量体内多巴胺、去甲肾上腺素和乙酰胆碱的体积传输 在学习期间。 将开发三种神经肽CNiFER，并用于我们自己的实验台验证项目。 实验室：食欲素，这是重要的睡眠调节以及药物寻求和恢复， 生长抑素与抑郁、动机和学习有关，血管活性肠肽 肽，它被认为与神经可塑性和学习有关。对于合作项目，我们将进一步 构建四种额外的神经肽CNiFERs，用于检测强啡肽、促肾上腺皮质激素释放肽和促肾上腺皮质激素释放肽的释放。 因子、神经肽Y和P物质。每种神经肽CNiFER将在体外进行严格的 在它们用于研究体内神经肽释放的动力学和结果之前进行测试。

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.