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

描述(申请人提供)：神经生物学中的一个中心挑战是在活体动物中控制激活或沉默已定义的神经回路，以研究这些回路的功能。为此设计了许多技术，包括光遗传和化学遗传方法。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(由设计者药物唯一激活的设计者受体)。DREADD是一种突变的G蛋白偶联受体，它失去了对自然配体的反应能力，并对具有纳摩尔效力的药理惰性类药物化合物(CNO)产生反应。目前，现有的DREADD包括GI、GQ和Gs信号通路。这些修饰的GPCRs可以作为理想的工具来远程控制体内离散神经元群体的活动。我们已经在几个概念验证研究中证明了这种方法的力量。为了充分探索DREADD技术在体内的应用，我们建议通过建立hM3Dq、rM3D以及G偶联的hM4Di小鼠品系来建立一个广泛适用的转基因平台，从而允许Cre重组酶介导的这些途径选择性DREADD的限制性表达。具体地说，我们将产生在rosa26基因座上携带普遍表达的启动子的C57BL/6小鼠系，该启动子允许Cre介导的Dq和Dd的表达，以及Di的表达，当去掉一个带花环的止点时(目标1)。然后，我们将在与Cre驱动程序交叉后，在Cre介导的小花停止切除后，诱导神经元特异性地表达Dq和Ds，以及Di，然后对这些品系进行表征(目标2)。我们的分析将确保每一条线在存在时都有强劲的表达，在没有Cre的情况下没有各自的DREADD的背景表达，并且Cre诱导的每个DREADD的表达与Cre驱动程序的表达模式是一致的。我们将在体外鉴定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；只有在CNO存在的情况下才发生激活或沉默，而不是在没有CNO的情况下；并且只在表达Cre的细胞中发生，而在对照细胞中不发生。最后，我们将通过评估外周给药剂量增加的DREADD：CRE小鼠的行为影响来测试体内的功能。在我们的项目结束时，可交付的产品将是三个完全特征化的DREADD鼠标系列，准备与任何CRE驱动系列交配。随着越来越多的CRE驱动器线可用，这些DREADD线将适用于研究广泛的研究问题。因此，我们的项目将通过建立一个高度特异性和远程控制转基因小鼠神经元活动和信号的通用平台来推动神经元电路分析工作。 与公共卫生相关：异常的神经回路可能是许多神经精神疾病的核心。这项提议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，这项关于开发一个广泛适用的转基因平台来分析神经电路的建议针对的是对公共健康至关重要的领域。

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.