A transgenic platform for highly specific and remote control of neuronal activity

描述（由申请人提供）：神经生物学的一个核心挑战是在活体动物中控制特定神经回路的激活或沉默，以研究这些回路的功能。为此，已经设计了许多技术，包括光遗传学和化学遗传学方法。在后者中，我们开发了一种由惰性药物激活的合成信号系统，我们将其命名为DREADD（由设计药物独家激活的设计受体）。DREADD是突变的G蛋白偶联受体，其失去了对其天然配体的响应能力，并且其响应于具有纳摩尔效力的药物惰性的药物样化合物（CNO）。目前，现有的DREADD涵盖Gi、Gq和Gs信号通路。这些修饰的GPCR可用作远程控制体内离散神经元群体的活性的理想工具。我们已经在几项概念验证研究中证明了这种方法的强大功能。为了充分探索DREADD技术的体内应用，我们在此提出通过创建hM 3Dq、rM 3Ds以及G偶联的hM 4Di小鼠品系来产生广泛适用的转基因平台，所述小鼠品系允许Cre重组酶介导的这些途径选择性DREADD的限制性表达。具体地，我们将产生C57 BL/6小鼠系，其在ROSA 26基因座内携带泛在表达的启动子，该启动子允许在去除floxed STOP（Aim 1）后Cre介导的Dq和Ds以及Di的表达。然后，我们将在Cre介导的切除floxed STOP（Aim 2）后，在与诱导Dq和Ds以及Di的神经元特异性表达的Cre驱动程序杂交后表征这些细胞系。我们的分析将确保每个系在存在Cre的情况下具有稳健的表达，并且在不存在Cre的情况下没有相应DREADD的背景表达，并且每个DREADD的Cre诱导的表达与Cre驱动因子的表达模式一致。我们将在体外表征DREADD系的功能，以确保Cre介导的每个DREADD的表达在CNO应用时导致神经元激活或沉默;激活或沉默仅在CNO存在时发生，而不是在其不存在时;并且仅在表达Cre的细胞中而不是在对照细胞中。最后，我们将通过评估增加剂量的外周施用CNO对DREADD：Cre小鼠的行为影响来测试体内功能。在我们的项目结束时交付的将是三个完全特征化的DREADD鼠标线准备与任何Cre驱动器线交配。随着越来越多的Cre驱动线成为可用这些DREADD线将适用于研究广泛的研究问题。因此，我们的项目将通过建立一个通用的平台，在转基因小鼠的神经元活动和信号的高度特异性和远程控制，推进神经元电路分析的努力。 公共卫生相关性：异常神经回路可能是许多神经精神疾病的核心。该提案的目标是推进分析方法，并由此增加对哺乳动物大脑中神经回路如何工作的理解。因此，这一建议，开发一个广泛适用的转基因平台，分析神经回路的目标领域的根本重要性，以公共卫生。

DESCRIPTION (provided by applicant): A central challenge in neurobiology is the controlled activation or silencing of defined neural circuits in the living animal to investigate the functions of these circuits. A number of technologies have been devised to this end, including optogenetic and chemical genetic approaches. Among the latter we have developed a synthetic signaling system activated by an inert drug, which we named DREADD (Designer Receptors Exclusively Activated by a Designer Drug). DREADDs are mutant G protein coupled receptors which lost the ability to respond to their natural ligand and which respond to a pharmacologically inert, drug-like compound (CNO) with nanomolar potency. Currently, the existing DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools for remotely controlling the activity of discrete populations of neurons in vivo. We have demonstrated the power of this approach in several proof-of-concept studies. In order to fully explore the in vivo applications of the DREADD technology we propose here to generate a widely applicable transgenic platform by creating hM3Dq, rM3Ds, as well as the G-coupled hM4Di strains of mice which allow for the Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. Specifically, we will generate C57BL/6 mouse lines carrying within the ROSA26 locus a ubiquitously expressed promoter allowing Cre-mediated expression of Dq and Ds, as well as Di, upon removal of a floxed STOP (Aim 1). We will then characterize these lines after crossing to a Cre driver inducing neuron-specific expression of Dq and Ds, as well as Di, after Cre-mediated excision of the floxed STOP (Aim 2). Our analyses will insure that each line has robust expression in the presence and no background expression of the respective DREADD in the absence of Cre, and that the Cre-induced expression of each DREADD is consistent with the pattern of expression of the Cre driver. We will characterize the functionality of the DREADD lines in vitro to insure that Cre-mediated expression of each DREADD leads to neuronal activation or silencing upon CNO application; that activation or silencing occurs only in the presence of CNO, but not in its absence; and only in cells expressing Cre and not in control cells. Finally, we will test functionality in vivo by assessing behavioral effects of DREADD:Cre mice with increasing doses of peripherally administered CNO. The deliverables at the end of our project will be three fully characterized DREADD mouse lines ready for mating with any Cre driver line. With the ever growing number of Cre driver lines becoming available these DREADD lines will be applicable to studying a wide array of research questions. Our project will thus advance neuronal circuit analysis efforts by establishing a generic platform for the highly specific and remote control of neuronal activity and signaling in transgenic mice. PUBLIC HEALTH RELEVANCE: Abnormal neural circuitry is likely at the core of many neuropsychiatric diseases. The goal of this proposal is to advance the methods of analysis, and the resulting increased understanding, of how neural circuits in the mammalian brain work. Thus this proposal for developing a widely applicable transgenic platform for the analysis of neural circuitry targets areas of fundamental importance to public health.