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

大脑功能是由复杂协调的神经元网络执行的，其操作模式是 对一系列神经调节剂高度敏感。更具体地说，神经调节剂，如多巴胺， 去甲肾上腺素、5-羟色胺和乙酰胆碱对全局大脑过程有戏剧性的控制，如 觉醒、注意力、情感或认知知觉。神经调节器信号的改变与 神经和精神障碍，如帕金森氏症、精神分裂症、抑郁症和成瘾。 同样，阿片神经肽在认知和行为的调节中起着重要的作用。而当 产生神经调节信号的解剖结构是众所周知的，但对空间上的 以及这些信号在被神经支配的大脑区域中的时间演变。这是因为电流测量 微透析法或循环伏安法等技术缺乏空间或时间分辨率(通常 分子特异性)来解析各自的信号。这一技术挑战一直是一个长期存在的障碍 对于神经调节如何改变神经回路功能从而影响行为的理解。至 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 荧光蛋白用于单胺类神经调节剂和阿片类药物的大规模光学测量 行为动物中的神经肽信号，通过将一个多学科的研究小组与 独特和互补的专业知识。这些感受器蛋白有可能给神经科学带来革命性的变化。 以类似于钙、谷氨酸和电压的遗传编码指示器的方式，这些指示器现在在 广泛使用。结合钙离子和电压成像，神经调节剂传感器将揭示这些 系统会影响细胞和电路功能。特别是，所提出的传感器将使微创、 高分辨率，长期，直接测量神经调节剂和神经肽信号在突触， 蜂窝层和系统层。长期以来，神经调节信号的传感器一直难以捉摸。我们的 该团队最近开发了第一代5-羟色胺(5-羟色胺)的遗传编码指标， 去甲肾上腺素(NE)和多巴胺(DA)可以报告纳摩尔到微摩尔的浓度变化 空间和时间分辨率。在此基础上，提出了以下具体目标：1)优化 并使用计算模型使单胺的遗传编码传感器多样化， 进化和高通量筛选；2)开发和优化阿片类药物的遗传编码传感器 使用新型蛋白质支架的神经肽；3)系统地验证新型神经调节剂和 急性脑片和行为动物的神经肽感受器。总而言之，这项工作将提供 神经科学社区，具有广泛的特性良好的多颜色指示器，用于探测 神经调节剂和神经肽在调节神经回路功能和行为中的作用 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.