Large-Scale Molecular Interrogation of Synaptic Transmission

描述（由申请人提供）：本申请提出开发一系列集成的测定以定量测量培养的神经元和急性脑切片中的突触功能，并具有扩大这些测定用于高通量筛选的潜力.正如RFA-MH-11-40“用于神经生物学功能无偏分析的可扩展测定”所建议的那样，该资助并没有解决特定的生物学问题，而是描述了用于神经元功能大规模分析的新工具。具体而言，我们的申请提出了八个具体目标的一系列相关的，但独立的新的检测系统，包括新的方法，实现控制表达的神经元基因在小鼠中，和新的技术，用于测量神经元突触功能的特性，从突触前和突触后的钙信号的分析谷氨酸受体贩运成像的神经元兴奋或沉默和各种形式的神经元应激。通过这些检测，我们的总体目标是开发工具，以满足日益明显的对更好方法的需求，以研究神经精神疾病，如自闭症和精神分裂症。越来越多的人类遗传学文献描述了这些疾病的许多候选致病基因，其中一些相关基因（如neurexins）可能具有突触功能，这表明突触可能代表这些疾病中至少一部分病例的发病热点。然而，分析候选疾病基因已经被证明是困难的，目前的方法需要在耗时和昂贵的实验中对单个基因进行长期研究。因此，需要新的方法，可以扩大规模和量化，而不需要大量的时间和精力的投资。我们在这里描述的工具是为了解决这一需求，至少在一定程度上，是基于一系列的技术创新。这些工具可以应用于培养的神经元、急性切片或小鼠体内实验，并且主要使用光学检测方法作为读数以允许可扩展性。在该应用程序的主持下开发的所有工具将免费并立即分发给社区，希望它们将成为在全国范围内进行的项目中大规模询问突触功能的标准化方法。本申请试图解决对用于分析神经元功能的可扩展测定系统的迫切需求，如RFA-MH-11-40所阐述的。拟议的新检测系统专注于突触传递，因为神经药理学和人类遗传学将突触传递确定为许多重要脑部疾病（包括自闭症和精神分裂症）的可能损伤部位。 公共卫生相关性：本申请试图解决对用于分析神经元功能的可扩展测定系统的迫切需求，如RFA-MH-11-40所阐述的。拟议的新检测系统专注于突触传递，因为神经药理学和人类遗传学将突触传递确定为许多重要脑部疾病（包括自闭症和精神分裂症）的可能损伤部位。

DESCRIPTION (provided by applicant): This application proposes development of an integrated array of assays to quantitatively measure synaptic function in cultured neurons and acute brain slices, with the potential for scaling up these assays for high- throughput screens. As suggested by RFA-MH-11-40 "Scalable Assays for Unbiased Analysis of Neurobiological Function", this grant does not address a specific biological question, but describes new tools for large-scale analysis of neuronal function. Specifically, our applications proposes in eight specific aims a series of related but independent new assay systems, including new methods of achieving controlled expression of neuronal genes in mice, and new techniques for measuring properties of synaptic function in neurons, ranging from pre- and postsynaptic calcium-signaling over analysis of glutamate receptor trafficking to imaging of neuronal excitation or silencing and various forms of neuronal stress. With these assays, our overall goal is to develop tools to meet the increasingly obvious need for better approaches to study neuro-psychiatric disorders such as autism and schizophrenia. A growing human genetics literature describes many candidate pathogenic genes for these disorders, with a synaptic function likely for some of the implicated genes such as neurexins, suggesting that synapses could represent a pathogenetic hotspot for at least a subset of cases in these diseases. Analyzing candidate disease genes, however, has proven difficult with current approaches that require long-term studies of single genes in time-consuming and expensive experiments. Thus, new approaches that can be scaled up and quantitated without enormous investments in time and effort are needed. The tools we describe here are meant to address this need, at least in part, and are based on a series of technical innovations. The tools can be applied to cultured neurons, acute slices, or in vivo experiments in mice, and primarily use optical detection methods as readout to allow scalability. All of the tools developed under the auspices of this application will be freely and immediately distributed to the community, with the hope that they will become standardized approaches for large-scale interrogation of synaptic function in projects performed throughout the country. This application attempts to address an urgent need for scalable assay systems for analysis of neuronal function, as enunciated by the RFA-MH-11-40. The proposed new assay systems focus on synaptic transmission because neuropharmacology and human genetics identified synaptic transmission as a possible site of impairment in many important brain diseases, including autism and schizophrenia. PUBLIC HEALTH RELEVANCE: This application attempts to address an urgent need for scalable assay systems for analysis of neuronal function, as enunciated by the RFA-MH-11-40. The proposed new assay systems focus on synaptic transmission because neuropharmacology and human genetics identified synaptic transmission as a possible site of impairment in many important brain diseases, including autism and schizophrenia.