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

一种强大的离子型激活剂，用于全脑操纵神经元功能

基本信息

批准号：
9145668
负责人：
Andrew D Ellington
金额：
$ 22.54万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9145668
关键词：
Adverse effects Animals Attention Behavior Behavioral Binding Blood - brain barrier anatomy Brain Brain Mapping Cations Cells Clinical Cognition Cognitive Development Directed Molecular Evolution Disabled Persons Dose Eating Disorders Endogenous Factors Engineering Epilepsy Evaluation Family Fiber Fibroblasts Fluorescence G-Protein-Coupled Receptors Gated Ion Channel Health Hour Human In Vitro Investigation Kinetics Laboratories Libraries Ligands Light Lighting Location Mechanics Mental Depression Methodology Methods Modeling Modification Molecular Genetics Mutagenesis Mutation Nature Neuraxis Neurons Nucleotides Optical Methods P2X-receptor Pharmaceutical Preparations Pharmacogenetics Physiologic pulse Population Positioning Attribute Property Protein Engineering Proteins Psyche structure Publishing Purinoceptor Regulation Scheme Signal Pathway Signal Transduction Site Specificity Stimulus Structure System Techniques Technology Testing Time Variant Visible Radiation Work Yeasts analog base brain circuitry calcium indicator cell type cellular engineering chronic pain clinical application desensitization design flexibility high throughput screening human disease implantation in vivo interest light gated member neural circuit neurochemistry neurotransmission novel nucleotide analog nucleotide receptor optical fiber optogenetics patch clamp photoactivation receptor response scaffold small molecule tool

项目摘要

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.

描述（由应用程序提供）：该建议体现了理性的设计，高吞吐量筛选以及新型神经元执行器的体外表征。我们努力的起点是一个离子型通道，发起了光遗传革命。我们有信心，我们概述的高度原始和全面的开发计划将产生一套新的变革性工具，以进行功能性大脑分析。近十年来，脑回路的功能分析一直依赖于允许神经元活性在具有细胞类型特异性的完整大脑中扰动的方法。遗传编码的神经元致动物的范围从嵌合G蛋白偶联受体（GPCR）和正交配体到光门控的离子辅导通道。尽管这些工具有助于发现认知和行为状态的细胞底物，但仍然存在重大局限性。光纤植入具有破坏性，并且照明受到机械约束的限制，并且要求提前确定目标位点。 GPCR通常是无效的，表现出较差的临时控制，并且可以在神经元中产生长期功能变化。我们建议开发和测试一种神经元激活剂，该神经元激活剂体现了现有方法的强大特征。基于嘌呤能P2X受体，该离子型通道将显示高单位电导，可以忽略不计的脱敏和可调门控。它的小分子配体很容易越过血脑屏障。通道和配体结构中的互补修饰将有助于产生一个正交受体配体对家族，以独立控制大脑中多个细胞种群，同时消除与内源性因素的串扰。这种和其他药物遗传学方法的优势在于，靶神经元的位置不必先验。但是，应需要精确的临时调节，配体可以是化学障碍（笼子），从而实现短暂的局部光激活。我们有信心我们的新型合成嘌呤能激活剂（SPARK）将推进功能性脑映射，从而对代表已知神经化学类别的离散神经元种群进行强有力的控制，或使用基于开拓性活动的分子基因方法选择。 Spark是现有方法的及时，高效且灵活的替代品。在神经元信号通路的体内机理询问中持续进展至关重要。我们设想了一系列的工具，这些工具将在整个物种中进行大脑部署，以控制神经元的不同集合，揭示电路连接性和信号层次结构。但是，与现有技术一样，许多工作仍有许多工作要做，不仅要设计合成受体，而且还为合成和筛选正交配体的良好耐受性，易于管理，并且很容易地到达大脑中的目标位点。我们将在酵母和成纤维细胞中跨实验系统进行工作，以确定最有前途的执行候选者，以在动物部署之前在体外进行广泛的测试和优化。