Large-Scale Molecular Interrogation of Synaptic Transmission

描述(由申请人提供)：本申请建议开发一套综合的分析方法，以定量测量培养神经元和急性脑片的突触功能，并有可能扩大这些分析方法的规模，用于高通量筛选。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，这项拨款并没有解决特定的生物学问题，而是描述了用于大规模神经功能分析的新工具。具体地说，我们的申请针对八个具体目标提出了一系列相关但独立的新测试系统，包括实现小鼠神经基因可控表达的新方法，以及测量神经元突触功能特性的新技术，从突触前和突触后的钙信号到谷氨酸受体转运的分析，到神经元兴奋或沉默的成像和各种形式的神经元应激。通过这些分析，我们的总体目标是开发工具，以满足越来越明显的需求，即更好的方法来研究神经精神障碍，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，其中一些相关基因可能具有突触功能，如Neurexins，这表明突触可能代表这些疾病中至少一部分病例的致病热点。然而，用目前的方法分析候选疾病基因已经被证明是困难的，这些方法需要在耗时和昂贵的实验中对单基因进行长期研究。因此，需要能够扩大和量化的新方法，而不需要在时间和精力上投入大量资金。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、急性切片或小鼠的活体实验，并主要使用光学检测方法作为读数，以实现可扩展性。在该应用程序的支持下开发的所有工具将立即免费分发给社区，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。如RFA-MH-11-40所述，该应用程序试图解决对用于分析神经元功能的可扩展分析系统的迫切需求。拟议的新测试系统专注于突触传递，因为神经药理学和人类遗传学发现突触传递可能是许多重要大脑疾病的损害部位，包括自闭症和精神分裂症。

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.