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。为此目的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，它们失去了对天然配体的反应能力，而对具有纳摩尔效力的药理学惰性药物样化合物（CNO）产生反应。目前，已有的dreadd涵盖了Gi、Gq和Gs信号通路。这些修饰的gpcr可以作为远程控制体内离散神经元群体活动的理想工具。我们已经在几个概念验证研究中展示了这种方法的强大功能。为了充分探索DREADD技术在体内的应用，我们提出通过构建小鼠hM3Dq、rM3Ds以及g偶联hM4Di品系来建立一个广泛适用的转基因平台，这些品系允许Cre重组酶介导的这些通路选择性DREADD的限制性表达。具体来说，我们将生成在ROSA26位点内携带一个无处不在表达的启动子的C57BL/6小鼠系，在去除一个固定的STOP后，允许cre介导的Dq和Ds以及Di的表达（目的1）。然后，我们将在Cre介导的封闭STOP切除后，通过Cre驱动诱导Dq和Ds以及Di的神经元特异性表达后对这些系进行表征（目的2）。我们的分析将确保在存在Cre的情况下，每个菌株都有稳健的表达，而在不存在Cre的情况下，各自的DREADD没有背景表达，并且Cre诱导的每个DREADD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细胞系的功能，以确保cre介导的每种DREADD表达在CNO应用后导致神经元激活或沉默；激活或沉默只发生在CNO存在时，而不发生在CNO不存在时；并且只在表达Cre的细胞中存在，而在对照细胞中没有。最后，我们将通过增加外周给药CNO剂量来评估DREADD:Cre小鼠的行为影响，从而在体内测试功能。项目结束时的可交付成果将是三个完全具有特征的DREADD小鼠系，可以与任何Cre驱动系配对。随着越来越多的Cre驱动线可用，这些DREADD线将适用于研究广泛的研究问题。因此，我们的项目将通过在转基因小鼠中建立一个高度特异性和远程控制神经元活动和信号的通用平台来推进神经元电路分析工作。

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.