Large-Scale Molecular Interrogation of Synaptic Transmission

描述（由申请人提供）：本申请提出了一种综合检测阵列的开发，用于定量测量培养神经元和急性脑切片中的突触功能，具有将这些检测扩大到高通量筛选的潜力。正如RFA-MH-11-40“神经生物学功能无偏分析的可扩展分析”所建议的那样，该资助不解决特定的生物学问题，但描述了大规模分析神经元功能的新工具。具体来说，我们的应用在八个特定目标中提出了一系列相关但独立的新分析系统，包括在小鼠中实现神经元基因控制表达的新方法，以及测量神经元突触功能特性的新技术，范围从谷氨酸受体运输分析的突触前和突触后钙信号传导到神经元兴奋或沉默的成像以及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，以满足对研究神经精神疾病（如自闭症和精神分裂症）的更好方法日益明显的需求。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（如神经毒素）可能具有突触功能，这表明突触可能代表了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，需要在不投入大量时间和精力的情况下扩大和量化新的方法。我们在这里描述的工具旨在满足这一需求，至少在一定程度上是这样，它们基于一系列技术创新。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出，以允许可扩展性。在此应用程序的支持下开发的所有工具都将免费并立即分发给社区，希望它们将成为在全国范围内执行的项目中大规模询问突触功能的标准化方法。这个应用程序试图解决一个迫切需要可扩展的分析系统的神经元功能，正如RFA-MH-11-40所阐明的。由于神经药理学和人类遗传学认为突触传递可能是许多重要脑部疾病（包括自闭症和精神分裂症）损伤的一个部位，因此提出的新检测系统关注突触传递。

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.