A robust ionotropic activator for brain-wide manipulation of neuronal function

 描述(由申请人提供)：这项建议体现了新型神经元致动器的合理设计、高通量筛选和体外表征。我们努力的起点是一个启动了光遗传革命的电离通道。我们相信，我们概述的高度原创和全面的开发计划将产生一套新的变革性工具，用于脑功能分析。近十年来，对大脑回路的功能分析一直依赖于一些方法，这些方法允许在具有细胞类型特异性的完整大脑中干扰神经元活动。基因编码的神经元致动器的范围从具有正交配体的嵌合G蛋白偶联受体(GPCRs)到光门控离子通道。虽然这些工具帮助揭示了认知和行为状态的细胞底物，但仍然存在重大限制。光纤植入是破坏性的，而照明受到机械约束和提前识别目标位置的要求的限制。GPCR往往效率低下，表现出较差的时间控制，并能在神经元中产生长期的功能变化。我们建议开发和测试一种神经激活剂，它体现了现有方法中最强大的特征。基于嘌呤能的P2X受体，这种离子亲和性通道将表现出高的单位电导，可忽略的脱敏和可调的门控。它的小分子配体很容易通过血脑屏障。通道和配体结构的互补修饰将有助于产生一系列正交的受体-配体对，以独立控制大脑内的多个细胞群体，同时消除与内源性因素的串扰。这种方法和其他药物遗传学方法的优点是，靶神经元的位置不需要事先知道；然而，如果需要精确的时间调节，可以化学禁用(笼子)配体，使短暂的局部光激活成为可能。我们相信，我们的新型合成嘌呤能激活剂(SPARK)将促进大脑功能图谱的绘制，为代表已知神经化学类别或使用开创性的基于活动的分子遗传学方法选择的离散神经元群体提供强大的控制。Spark是现有方法的一种及时、高效和灵活的替代方法；它对于在活体神经信号通路的机械询问方面继续取得进展至关重要。我们设想了一整套工具，这些工具将跨物种在大脑范围内部署，以控制不同的神经元集合，揭示电路连接和信号层次结构。然而，就像现有的技术一样，不仅在设计合成受体方面，而且在合成和筛选耐受性好、易于管理、容易到达大脑靶点的正交配体方面，仍有很多工作要做。我们将在酵母和成纤维细胞的实验系统中开展工作，以确定最有希望的致动器候选方案，在应用于动物之前，将在体外进行广泛的测试和优化。

 DESCRIPTION (provided by applicant): This proposal embodies the rational design, high throughput screening, and in vitro characterization of novel neuronal actuators. The starting point for our endeavor is an ionotropic channel that launched the optogenetic revolution. We are confident that the highly original and comprehensive development scheme we have outlined will yield a new set of transformative tools for functional brain analysis. For nearly a decade, functional analysis of brain circuitry has relied on methods that allow neuronal activity to be perturbed in an intact brain with cell type-specificity. Genetically-encoded neuron actuators have ranged from chimeric G-protein coupled receptors (GPCRs) with orthogonal ligands to light-gated ionotropic channels. While these tools have helped uncover cellular substrates of cognitive and behavioral states, significant limitations remain. Optical fiber implantation is destructive, and illumination is limited by mechanical constraints and the requirement that the target site be identified in advance. GPCRs are often inefficient, display poor temporal control, and can produce long-term functional changes in neurons. We propose to develop and test a neuronal activator that embodies the strongest features of existing approaches. Based on the purinergic P2X receptor, this ionotropic channel will display high unitary conductance, negligible desensitization, and tunable gating. Its small molecule ligand will readily cross the blood-brain barrier. Complementary modifications in channel and ligand structure will help generate a family of orthogonal receptor-ligand pairs for independent control over multiple cell populations within the brain while eliminating crosstalk with endogenous factors. The strength of this and other pharmacogenetic approaches is that the locations of target neurons need not be known a priori; however, should precise temporal regulation be needed, the ligands can be chemically disabled (caged), enabling brief localized photoactivation. We are confident that our novel synthetic purinergic activator (SPArk) will advance functional brain mapping, providing robust control over discrete neuronal populations that represent known neurochemical classes or are selected using pioneering activity-based molecular-genetic methods. SPArk is a timely, highly efficient and flexible alternative to existing approaches; it is essential for continued progress in in vivo mechanistic interrogation of neuronal signaling pathways. We envision a panoply of tools that will be deployed brain-wide across species to control distinct ensembles of neurons, uncovering circuit connectivity and signaling hierarchies. However, just as with existing technologies, much work remains to be done not only to engineer the synthetic receptors, but also to synthesize and screen orthogonal ligands that are well-tolerated, easy to administer, and that readily reach target sites in the brain. We will work across experimental systems, in yeast and fibroblasts, to identify the most promising actuator candidates, to be subjected to extensive testing and optimization in vitro prior to deployment in animals.