Genetically encoded indicators for large-scale sensing of neuromodulatory signaling in behaving animals

大脑功能是由错综复杂的神经元网络执行的，其运作模式是 对一系列神经调质高度敏感更具体地说，神经调节剂如多巴胺， 去甲肾上腺素、血清素和乙酰胆碱对整个大脑过程发挥着巨大的控制作用， 唤醒、注意力、情感或认知知觉。改变的神经调质信号与 神经和精神疾病，如帕金森氏病、精神分裂症、抑郁症和成瘾。 同样，阿片类神经肽在认知和行为的调节中发挥重要作用。而 产生神经调节信号的解剖结构是众所周知的，但关于空间结构知之甚少。 以及这些信号在受神经支配的大脑区域中的时间演变。这是因为电流测量 诸如微透析或循环伏安法的技术缺乏空间或时间分辨率（并且通常 分子特异性）以分辨各自的信号。这种技术挑战一直是一个长期存在的障碍 有助于我们理解神经调节如何改变神经回路功能以影响行为。到 为了应对这一挑战，该项目将开发、验证和传播新的基因编码的 用于单胺神经调节剂和阿片样物质的大规模光学测量的荧光蛋白 神经肽信号在行为动物，汇集了多学科的研究团队， 独特和互补的专业知识。这些传感器蛋白有可能彻底改变神经科学 类似于钙、谷氨酸和电压的基因编码指标， 广泛使用。结合钙和电压成像，神经调质传感器将揭示这些 系统影响蜂窝和电路功能。特别地，所提出的传感器将能够实现微创， 高分辨率、长期和直接测量突触神经调质和神经肽信号传导， 蜂窝和系统级。神经调节信号的传感器在很长一段时间内仍然难以捉摸。我们 研究小组最近开发了第一代5-羟色胺（5-HT）的遗传编码指标， 去甲肾上腺素（NE）和多巴胺（DA），可以报告纳至微摩尔浓度的变化， 空间和时间分辨率。在这项工作的基础上，提出了以下具体目标：1）优化 和多样化的基因编码传感器的单胺使用计算建模， 进化和高通量筛选; 2）开发和优化阿片类药物的基因编码传感器 3）系统地验证新的神经调节剂， 神经肽传感器在急性脑切片和行为动物。总之，这项工作将提供 神经科学界拥有广泛的特征鲜明的多色指标，用于探测 神经调质和神经肽在调节神经回路功能和行为中的功能作用 健康和疾病。

Brain functions are executed by intricately coordinated networks of neurons, whose modes of operation are highly sensitive to a constellation of neuromodulators. More specifically, neuromodulators such as dopamine, norepinephrine, serotonin, and acetylcholine exert dramatic control over global brain processes such as arousal, attention, emotion, or cognitive perception. Altered neuromodulator signaling has been linked to neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, depression and addiction. Similarly, opioid neuropeptides play important roles in the modulation of cognition and behavior. While the anatomical structures that produce neuromodulatory signals are well known, little is known about the spatial and temporal evolution of these signals in the innervated brain regions. This is because current measurement techniques, such as microdialysis or cyclic voltammetry, lack the spatial or temporal resolution (and often the molecular specificity) to resolve respective signals. This technical challenge has been a long-standing barrier to our understanding of how neuromodulation alters neural circuit function in order to influence behavior. To address this challenge, this project will develop, validate, and disseminate novel genetically encoded fluorescent proteins for large-scale optical measurement of monoamine neuromodulator and opioid neuropeptide signaling in behaving animals, by bringing together a multi-disciplinary team of investigators with unique and complementary expertise. These sensor proteins have the potential to revolutionize neuroscience in a way similar to genetically encoded indicators for calcium, glutamate, and voltage, which are now in widespread use. Combined with calcium and voltage imaging, neuromodulator sensors will reveal how these systems impinge on cellular and circuit function. In particular, proposed sensors will enable minimally invasive, high-resolution, long-term, and direct measurement of neuromodulator and neuropeptide signaling at synaptic, cellular, and system levels. Sensors for neuromodulatory signaling have remained elusive for a long time. Our team recently developed a first generation of genetically encoded indicators for serotonin (5-HT), norepinephrine (NE), and dopamine (DA) that can report nano- to micromolar concentration changes with high spatial and temporal resolution. Building on this work, the following specific aims are proposed: 1) Optimize and diversify genetically encoded sensors for the monoamines using computational modeling, directed evolution and high-throughput screening; 2) Develop and optimize genetically encoded sensors for opiate neuropeptides using novel protein scaffolds; and 3) Systematically validate the novel neuromodulator and neuropeptide sensors in acute brain slices and behaving animals. Together, this work will provide the neuroscience community with a wide range of well-characterized multi-color indicators for probing the functional role of neuromodulators and neuropeptides in regulating neural circuit function and behavior in both health and disease.