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

 描述（由申请人提供）：该提案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。我们奋进的起点是启动光遗传学革命的离子通道。我们相信，我们所概述的高度原创和全面的开发计划将为功能性大脑分析提供一套新的变革性工具。近十年来，大脑回路的功能分析一直依赖于允许神经元活动在具有细胞类型特异性的完整大脑中受到干扰的方法。遗传编码的神经元致动器已经从具有正交配体的嵌合G蛋白偶联受体（GPCR）到光门控离子通道。虽然这些工具有助于揭示认知和行为状态的细胞基质，但仍然存在重大局限性。光纤植入是破坏性的，并且照明受到机械约束和预先识别目标部位的要求的限制。GPCR通常效率低下，显示出较差的时间控制，并且可以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体现现有方法最强功能的神经元激活剂。基于嘌呤能P2 X受体，该离子通道将显示高单位电导、可忽略的脱敏和可调门控。其小分子配体将容易地穿过血脑屏障。通道和配体结构的互补修饰将有助于产生正交受体-配体对家族，用于独立控制脑内的多个细胞群体，同时消除与内源性因子的串扰。这种和其他药物遗传学方法的优势在于靶神经元的位置不需要先验已知;然而，如果需要精确的时间调节，则配体可以被化学禁用（笼），从而实现短暂的局部光活化。我们相信，我们的新型合成嘌呤能激活剂（SPArk）将推进功能性脑映射，对代表已知神经化学类别或使用开创性的基于活性的分子遗传方法选择的离散神经元群体提供强大的控制。SPArk是一种及时、高效和灵活的替代现有方法的方法;它对于神经元信号通路的体内机制询问的持续进展至关重要。我们设想了一整套工具，这些工具将在整个物种的大脑中部署，以控制不同的神经元集合，揭示电路连接和信号层次。然而，正如现有技术一样，仍有许多工作要做，不仅要设计合成受体，而且要合成和筛选耐受性良好、易于给药且易于到达大脑靶点的正交配体。我们将在酵母和成纤维细胞的实验系统中工作，以确定最有前途的致动器候选者，在动物中部署之前进行广泛的体外测试和优化。

 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.