A platform for high throughput, cell type-restricted in vivo knockdown of pre- or postsynaptic gene expression

科学中最具挑战性的问题之一是了解大脑中神经细胞表达的分子如何使思想和行动发生。解决这一挑战对于理解大脑是如何工作的至关重要。它还将为神经和精神疾病的治疗方法以及生物启发的计算技术的未来发展奠定基础。然而，尽管大脑中的细胞与其他器官中的细胞非常相似，但由于大脑的巨大复杂性，弄清楚细胞中的分子如何对大脑功能做出贡献是非常具有挑战性的。传统上，人们可以通过寻找与分子结合的药物来研究分子的功能，或者通过对动物进行基因工程来删除分子。然而，许多大脑功能涉及到在彼此邻近的不同类型的细胞之间传递信号。用传统的方法，通常很难并且非常耗时地计算出哪种细胞类型介导了分子效应。此外，使用这些方法，单个模型动物通常只能用于研究一个分子靶点。为了解决这些问题，我们建议开发一种系统，用于相对低成本和有效地研究任何给定分子在特定神经元群体和与其相互作用的已识别神经元之间的信号传递中所起的作用。该系统将使用病毒将两种类型的基因序列引入神经元。第一个编码短干扰RNA。这些短的基因序列包含识别位点，使它们能够“击倒”目标分子。它们可以被设计成击倒几乎任何感兴趣的分子的表达。第二种类型的基因序列编码的蛋白质既可以作为荧光标记，也可以作为光敏神经元激活剂。这些蛋白质可以用来识别和控制受感染的神经元。我们系统的特殊能力来自于一种新的方法，我们将使用这种方法来控制表达这两种类型基因序列的细胞。这一系统使得这两种类型的序列的表达都需要标记分子Cre和FLP的存在。通过将病毒引入Cre和FLP标记感兴趣的细胞群体的动物中，我们可以靶向病毒传递的蛋白质的表达并干扰这些群体的RNA。我们建议通过开发旨在研究大脑内嗅觉皮质区域神经回路的新工具来验证这一新方法。这一区域对空间认知很重要。专注于这一领域使我们能够利用成熟的实验分析，同时生成应该使我们能够解决以前非常具有挑战性的问题的工具。我们的第一个目标将是生成和表征工具，这些工具仅在单一类型的细胞中击倒特定离子通道的表达。我们的第二个目标将是制造工具，在两个不同的神经元群体之间的连接的上游或下游抑制受体的表达。这些工具还将允许上游神经元专门用光激活，下游神经元通过它们的荧光来识别，以便指导后续的电子记录。我们的第三个目标是开展初步工作，将我们的方法扩展到在同一动物中并行研究多个分子靶点。在完成该项目后，我们的目标是引入并验证一个新的工具集，用于高通量和低成本地研究信号分子在定义的神经元群体之间的连接中的作用。我们旨在建立的平台将对细胞群体之间信号传递的分子机制的基础和应用研究具有普遍用途。应用包括研究年轻和衰老大脑的认知功能机制，以及开发药物开发的新模式。

One of the most challenging problems in science is to understand how the molecules expressed by nerve cells in the brain enable thoughts and actions to take place. Addressing this challenge is of fundamental importance for understanding how brains work. It will also underpin future development of therapies for neurological and psychiatric disorders, and of biologically inspired computing technologies. However, while cells in the brain are very much like those in other organs, figuring out how molecules in cells contribute to brain functions is exceptionally challenging because of the brain's great complexity. Conventionally one could study the function of a molecule by finding drugs that bind to it, or by engineering animals from which the molecule is deleted. However, many brain functions involve signals being passed between different types of cell that are found nearby one another. With conventional approaches it is usually difficult and very time consuming to figure out which cell type mediates a molecules effects. Moreover, with these approaches a single model animal can usually only be used to study one molecular target.To address these issues we propose to develop a system for relatively low cost and efficient investigation of the role of any given molecule in signalling between a particular population of neurons and identified neurons with which they interact. This system will use viruses to introduce two types of genetic sequence into neurons. The first encodes short interfering RNAs. These are short genetic sequences that contain recognition sites that enable them to "knockdown" targeted molecules. They can be designed to knockdown expression of almost any molecule of interest. The second type of genetic sequence encodes proteins that act either as fluorescent labels or as light-sensitive neuronal activators. These proteins can be used to identify and control infected neurons. The particular power of our system comes from a novel approach we will use to control the cells in which these two types of genetic sequence are expressed. This system makes expression of both types of sequence require the presence of marker molecules called Cre and Flp. By introducing viruses into animals in which Cre and Flp label cell populations of interest, we can target expression of the virally delivered proteins and interfering RNAs to these populations.We propose to validate this new approach by developing new tools aimed at studying neural circuits in a brain area called the entorhinal cortex. This region is important for spatial cognition. Focussing on this region allows us to take advantage of well established experimental assays, while generating tools that should enable us to address previously very challenging questions. Our first aim will be to generate and characterise tools that knockdown expression of particular ion channels in only a single type of cell. Our second aim will be to make tools that knockdown expression of receptors at either the up- or downstream side of a connection between two distinct populations of neurons. The tools will also allow the upstream neurons to be activated specifically with light and the downstream neurons to be identified by their fluorescence in order to guide subsequent electrical recordings. Our third aim will be to carry out preliminary work to extend our approach to investigation of multiple molecular targets in parallel in the same animal.On completion of the project we aim to have introduced and validated a new toolset for high throughput and low cost investigation of the roles of signalling molecules at connections between defined neuronal populations. The platforms that we aim to establish will be of general utility for fundamental and applied research into molecular mechanisms of signalling between cell populations. Applications include investigation of mechanisms of cognitive function in the young and ageing brain, and development of novel models for drug development.