Explore fundamental aspects of neurotransmission with multifunctional nanosensor

描述(由申请人提供) 摘要：大脑是一个由数十亿个神经元组成的极其复杂的网络。突触是神经元之间的接触点，几十年来一直是人们感兴趣的中心。从拉姆·卡哈尔开始，科学家们一直在借用其他学科的工具来研究这种微小但至关重要的结构。真正的突破是在电生理学的引入之后取得的，电生理学允许在极高的信噪比下测量神经元的活动。突触传递是通过突触前神经递质从突触小泡释放并随后激活突触后受体来进行的。到目前为止，突触传递的改变通常被归因于突触后受体的改变。然而，新出现的证据表明，受体并不是自主作用的，突触小泡确实对突触的可塑性做出了重大贡献。尽管有很大的兴趣和努力，但缺乏直接和精确的测量阻碍了对突触小泡的进一步研究，从而阻碍了对这种相互调制的完整理解。借鉴纳米技术，我设计了一种使用量子点的单囊泡成像方法，首次提供了与膜片钳记录相当甚至超过的卓越时空分辨率。利用这个原始工具，我揭示了哺乳动物中央突触中囊泡重复使用的非传统模式的存在、动力学和调控机制。在这里，我认为突触调制是由突触前和突触后的一系列协调的、同样深刻的变化组成的。为了测试它，我将开发由量子点和适配子(一种新型合成寡核苷酸)制成的“智能”纳米传感器。这些纳米传感器可以精确地定位指定的突触结构或蛋白质，并同时报告突触前和突触后的原位协调变化。为了展示这一革命性工具的力量，我将利用它来解决突触生理学中一些基本和长期存在的问题，例如提取后小泡再酸化和再灌流的耦合，突触裂隙周围神经递质浓度的波动，以及突触前囊泡和突触后受体的适应。因为Qdots是活体成像的理想选择，我将筛选能够将这些纳米传感器传递到完整大脑中指定突触的适配子，这样就可以在定义的神经元电路中研究突触传递。这个项目的成功保证了我们对神经传递的理解有一个巨大的飞跃。这项工作所取得的技术和科学进步可以很容易地移植到其他生物医学领域，因为囊泡周转和表面受体运输广泛涉及几乎所有的生物过程。此外，Qdots和适配子都是模块单位，可以与各种诊断或治疗分子相连。因此，这个项目的影响超出了神经科学的范畴。。 公共卫生相关性：突触是脑神经网络中传递信息的最基本的元素，它负责我们的认知和日常行为。通过发明和部署多功能纳米棒，我们将研究哺乳动物中枢神经系统中突触传递的最基本方面，包括突触小泡、神经递质和受体。由于这些突触成分在大多数神经疾病中明显异常，我们的发现将弥合基础研究和临床研究之间的差距，以更好地了解、检测、表征和治疗神经疾病。

DESCRIPTION (Provided by the applicant) Abstract: The brain is an extraordinarily complex network made of billions of neurons. The synapse, the contact point between neurons, has been the centre of interest for decades. Starting with Ram¿n Cajal, scientists have been borrowing tools from other disciplines to study this tiny, but crucial structure. The breakthrough truly came after the introduction of electrophysiology, which allows the measurement of neuronal activity at an extremely high signal-to-noise ratio. It became clear that synaptic transmission is conducted via the presynaptic release of neurotransmitters from synaptic vesicles and the subsequent activation of postsynaptic receptors. So far, changes in synaptic transmission have generally been attributed to the modification of postsynaptic receptors. Nevertheless, newly emerged evidence has demonstrated that the receptors do not act autonomously and synaptic vesicles indeed make significant contribution to synaptic plasticity. Despite great interest and effort, the lack of a direct and precise measurement obstructs further investigation of synaptic vesicles and thus impedes a complete comprehension of such mutual modulation. Borrowing from nanotechnology, I devised a single-vesicle imaging approach using quantum dot (Qdot), which for the first time provides superior spatiotemporal resolution matching or even exceeding that of patch-clamp recording. With this original tool, I unveiled the existence, kinetics and regulatory mechanisms of an unconventional mode of vesicle reuse in mammalian central synapses. Here, I propose that synaptic modulation is composed of a sequence of coordinated and equally profound changes at both pre- and postsynaptic terminals. To test it, I will develop ""smart"" nanosensors made of Qdots and aptamers, a new class of synthetic oligonucleic acids. These nanosensors can precisely target designated synaptic structures or proteins and simultaneously report orchestrated pre- and postsynaptic changes in situ. To demonstrate the power of this revolutionary tool, I will deploy it to address some fundamental and long-standing questions in synaptic physiology, such as the coupling of vesicle reacidification and refilling after retrieval, the fluctuation of neurotransmitter concentration around the synaptic cleft, and the acculturation of presynaptic vesicles and postsynaptic receptors. Because Qdots are ideal for in vivo imaging, I will screen for aptamers that can deliver these nanosensors to designated synapses in the intact brain such that synaptic transmission can be studied in defined neuronal circuits. The success of this project promises a quantum leap in our understanding of neurotransmission. The technical and scientific advances made from this work can be readily transplanted to other biomedical fields because vesicles turnover and surface receptor trafficking are widely involved in almost all biological processes. Moreover, both Qdots and aptamers are modular units that can be linked with all kinds of diagnostic or therapeutic molecules. Therefore, this project has a broad impact beyond neuroscience. . Public Health Relevance: Synapse is the most essential element for transmitting information in brain neuronal network which is responsible for our cognition and daily behavior. By inventing and deploying multifunctional nanoporbes, we will investigate the most fundamental aspects of synaptic transmission including synaptic vesicles, neurotransmitters and receptors in mammalian central nervous system. As these components of synapses evidently became abnormal in most neurological disorders, our finding will bridge the gap between basic and clinic research for better understanding, detection, characterization, and treatment of neurological disorders.