Fluorescent biosensors for imaging neurotransmitters: observing synapses in actio

描述（由申请人提供）：神经科学中最大的挑战之一是破译神经回路的逻辑，并将其与学习，记忆和行为联系起来。神经回路是一个动态的网络，它包含了各种空间和时间尺度上的神经元活动。因此，神经回路的分析需要跨时间和大脑结构的神经元活动的广泛和密集的采样。现代显微镜和基于蛋白质的荧光传感器的最新突破使这一目标触手可及。例如，应用遗传编码的钙指示剂，如GCaMP 3，结合双光子显微镜，已经促进了在清醒的行为动物中在多个时间尺度上大规模记录遗传鉴定的群体中的神经活动。这些应用极大地推进了我们对神经回路的动力学及其对行为的控制的理解，这是理解复杂大脑功能的关键的第一步。基于钙成像的势头，加速未来神经回路动力学分析的迫切需要是开发更广泛的光学传感器套件，以扩大可以测量的神经元活动的种类。一个特别感兴趣的领域是突触传递，这是大脑中信息处理的一个关键事件，难以用目前可用的光学工具访问。在我们能够发展出突触传递的动态图像之前，有两个关键问题需要解决。首先，我们必须了解突触连接是如何与其活动联系在一起的;其次，我们必须确定不同类型的神经递质如何在一个定义的回路中相互平衡。因此，我计划开发两类新的基于蛋白质的荧光传感器，使用最近才出现的方法，从这两个不同的角度监测突触传递。对于本提案中概述的第一个项目，我将开发专门设计用于同时 记录突触活动和连接。最近，我一直在参与开发一种遗传编码的神经递质传感器（iGluSnfr），以直接测量释放的谷氨酸。这种传感器，第一次，提供了潜在的监测兴奋性 突触活动的时间和空间。然而，它报告突触连接的能力，这是存储在神经回路中的一条重要信息，目前还缺乏。因此，我将开发将iGluSnfr分成突触前和突触后成分的策略。这种设计传感器将允许同时记录突触活动和连接，从而提供一种方法来找到在定义的电路中依赖于活动的突触。对于本提案中概述的第二个项目，我将开发一种新的传感器来直接监测突触处神经元之间的抑制性通信。众所周知，基于释放的神经递质的种类，神经元之间的通信可以是兴奋性的或抑制性的。在特定的神经回路中，兴奋性和抑制性突触的不平衡与一系列的 神经系统疾病，包括抑郁症、成瘾、自闭症、精神分裂症和癫痫。然而，用于直接监测具有所需时空分辨率的抑制信号的光学传感器仍然缺失。我将利用计算建模来重新设计iGluSnFr来感知抑制性神经递质，例如？氨基丁酸（GABA）。类似地，将在项目一中开发的分裂策略将进一步用于将GABA传感器分裂成突触前和突触后组件。总之，拟议研究的成功结果将提供急需的成像工具，使神经科学家能够在细胞，组织和整个动物水平上获得兴奋性和抑制性突触的全面视图。

DESCRIPTION (provided by applicant): One of the greatest challenges in neuroscience is to decipher the logic of the neural circuitry and link it to learning, memory, and behavior. Neural circuitry is a dynamic network that incorporates neuronal activity at a variety of spatial and temporal scales. Therefore, analysis of neural circuitry demands broad and dense sampling of neuronal activity across time and brain structures. Recent breakthroughs in modern microscope and protein based fluorescence sensors have brought this goal within reach. For example, application of genetically encoded calcium indicators, such as GCaMP3, combined with two-photon microscopy, has facilitated the large- scale recording of neural activity in a genetically-identified population at multiple time scales in awake, behaving animals. These applications have greatly advanced our understanding of the dynamics of neural circuitry and its control of behavior-a critical first step toward understanding complex brain function. Building upon the momentum of calcium imaging, the immediate need to accelerate future analyses of the dynamics of neural circuitry is to develop a broader suite of optical sensors to expand the kinds of neuronal activity that can be measured. One particular area of interest is synaptic transmission, a critical event of information processing in the brain that is difficult to access wth the optical tools currently available. There are two key questions that need to be addressed before we can develop a dynamic picture of synaptic transmission. First, we must understand how synaptic connectivity is linked to its activity; second, we must determine how different types of neurotransmitters balance with each other in a defined circuitry. Therefore, I plan to develop two classes of novel protein-based fluorescent sensors, using methods that have emerged only recently, to enable monitoring of synaptic transmission from these two different angles. For the first project outlined in this proposal, I will develop sensors specially designed for simultaneous recording of both synaptic activity and connectivity. Recently, I have been involved in developing a genetically-encoded neurotransmitter sensor (iGluSnfr) to directly measure released glutamate. This sensor, for the first time, offers the potential for monitoring excitatory synaptic activity in time and space. However, its ability to report synaptic connectivity, a piece f important information stored in the neural circuitry, is currently lacking. Therefore, I will develp strategies to split iGluSnfr into pre- and post-synaptic components. This designer sensor will permit simultaneous recording of both synaptic activity and connectivity, thus providing a way to find the synapses that are activity-dependent in a defined circuitry. For the second project outlined in this proposal, I will develop a new sensor to direct monitor inhibitory communication between neurons at synapses. It is known that based on the kind of neurotransmitters released, the communication between neurons can be either excitatory or inhibitory. Imbalanced excitatory and inhibitory synapses in specific neural circuitry have been implicated in an array of neurological disorders, including depression, addiction, autism, schizophrenia and epilepsy. Yet, optical sensors for directly monitoring inhibitory signals with needed spatiotemporal resolution are still missing. I will leverage computational modeling to redesign iGluSnFr to sense inhibitory neurotransmitters, such as ?-aminobutyric acid (GABA). Similarly, the splitting strategy to be developed in project one will be further utilized to split the GABA sensor into pre- and post-synaptic components. Taken together, a successful outcome of the proposed research would provide much needed imaging tools to enable neuroscientists to obtain a comprehensive view of both excitatory and inhibitory synapses in action at the cellular, tissue, and whole-animal level.