Genetically encoded sensors for the biogenic amines: watching neuromodulation in action

 描述(由申请人提供)：这项提案的目标是开发生物胺的遗传编码指标工具箱，生物胺是最重要的神经调节剂家族。所有神经系统都受到神经调制的影响，神经调制通过改变靶神经元的固有放电特性和调节它们的突触可塑性来重新配置神经回路的动力学。神经调节剂的动态变化与许多人类神经和精神疾病有关，包括帕金森氏症、精神分裂症和成瘾。生物胺是一组神经调节剂，所有动物的大脑都使用它来调节神经回路的发育、结构和功能。虽然人们对生物胺投射的解剖学特征和功能意义有了一定程度的了解，但这些分子控制行为的确切机制还不完全清楚。为了破译这些分子对大脑和行为施加影响的机制，我们必须以必要的空间和时间分辨率对神经调节器的瞬变进行敏感和具体的测量，包括广泛的(音量调制)和局部的(靶向调制)，最好是在INTAC电路中。现有的方法，包括微渗析和循环伏安法，是有用的，但不足以完成手头的这项任务。一种可能的解决方案是开发基于荧光蛋白的遗传编码指示器，并结合现代显微镜，允许以增强的空间和时间分辨率直接和特定地测量不同类型的神经调节剂。最近，我们成功地建立了开发神经活动的遗传编码指示器的技术平台，这导致了几个高质量的光学探针，用于同时成像活动物的大规模神经元种群。基于高度优化的传感器平台和在神经科学中传感器表征和应用的丰富经验，我们建议为生物胺类神经调节剂，特别是行为最普遍的神经调节剂多巴胺开发高质量的光学传感器工具包。我们的具体目标将从设计和筛选每个生物胺的传感器开始，使用计算重新设计和直接旋转相结合的方法。然后，我们将开发突触靶向策略，以显示树突和轴突中的传感器，以提高它们对突触成像的效用。我们最终将描述 这些传感器在活神经元和大鼠脑片中的性能，并展示了它们探测活动物中多巴胺瞬变的能力。用于这些分子的最先进的传感器将有助于在活的模式生物中无创、准确、直接和连续地在突触和回路水平上测量释放的神经调节剂。光学记录的这种技术进步将促进神经回路映射，并描绘出神经调节系统在调节神经回路和行为方面的动态图景。鉴于生物胺与神经系统疾病的明确相关性，这些传感器对人类干细胞和动物疾病模型(特别是帕金森氏病)的长期研究和评估候选疗法的效果特别有用。

 DESCRIPTION (provided by applicant): The goal of this proposal is to develop a toolbox of genetically encoded indicators for biogenic amines, the most important family of neuromodulators. All nervous systems are subject to neuromodulation, which reconfigure the dynamics of neural circuitry by transforming the intrinsic firing properties of targeted neurons and regulating their synaptic plasticity. The altered dynamics of the neuromodulators have been implicated in a number of human neurological and psychiatric diseases, including Parkinson's, schizophrenia and addiction. Biogenic amines are a group of neuromodulators used by all animal brains to regulate the development, structure and function of neural circuits. Although the anatomical characterization and functional significance of biogenic amine projections are understood to a moderate degree, the precise mechanisms by which these molecules exert control over behavior are not fully understood. To decipher the mechanisms by which these molecules exert their influence on the brain and behavior, we must perform sensitive and specific measurements of neuromodulator transients, both broadly (volume modulation) and locally (targeted modulation), with the requisite spatial and temporal resolution, ideally in intac circuits. Existing methods, encompassing microdialysis and cyclic voltammetry, are useful, but not adequate for this task at hand. One potential solution would be to develop genetically encoded indicators based on fluorescent proteins combined with modern microscopy allowing direct and specific measurement of diverse types of neuromodulators with enhanced spatial and temporal resolutions. Recently we have successfully established technology platform for the development of genetically encoded indicators of neural activity, which have led to several high-quality optical probes for simultaneous imaging of large-scale neuronal populations in living animals. Building upon highly optimized platform for sensor sensors and extensive experience in sensor characterization and application in neuroscience, we propose to develop a high-quality toolkit of optical sensors for the biogenic amine neuromodulators, especially for dopamine, the most behavioral pervasive neuromodulator. Our specific aims will start by designing and screening sensors for each of the biogenic amines using combined computational redesign and direct revolution. We will then develop synaptic targeting strategies to display the sensors in dendrites and axons to improve their utility for synaptic imaging. We will finally characterize the performance of these sensors in living neurons and in rat brain slices and demonstrate their capabilities of probing dynamics of dopamine transients in living animals. State-of-the-art sensors for these molecules will facilitate the non-invasive, precise, direct and continual measurement of released neuromodulators at both the synaptic and circuit levels in live model organisms. Such technology advance in optical recordings will facilitate neural circuitry mapping and paint a dynamic picture of neuromodulation systems in regulating neural circuitry and behavior. Given the clear relevance of the biogenic amines to the neurological diseases, these sensors are especially beneficial for long-term studies of human stem cell and animal disease models (specially the Parkinson's disease) and evaluating the effects of candidate therapeutics.