BioLuminescent OptoGenetics (BL-OG): A Novel and Versatile Strategy for Neuromodulation

生物发光光遗传学 (BL-OG)：一种新颖且多功能的神经调节策略

基本信息

批准号：
9356587
负责人：
UTE H HOCHGESCHWENDER
金额：
$ 72.87万
依托单位：
CENTRAL MICHIGAN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-30 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9356587
关键词：
Acoustics Action Potentials Address Animals Anions Area Autistic Disorder Automobile Driving BRAIN initiative Basic Science Behavior Behavioral Benchmarking Biochemical Biological Bioluminescence Biophysics Blood - brain barrier anatomy Brain Cells Chemicals Chimeric Proteins Clinical Communities Complement Complex Data Development Dimensions Electromagnetics Elements Epilepsy Fiber Optics Forms Controls Goals Human In Vitro Injection of therapeutic agent Light Link Luciferases Memory Loss Mental Depression Mental disorders Methods Mission Mutagenesis Neocortex Neurons Neurosciences Opsin Outcome Output Peripheral Population Production Proteins Protocols documentation Proton Pump Public Health Reagent Regulation Research Research Personnel Route Schizophrenia Site Technology Testing Thalamic structure Therapeutic Time United States National Institutes of Health Validation Variant Viral Vector Work addiction brain cell comparative design designer receptors exclusively activated by designer drugs experimental study flexibility genetic approach in vivo light emission luciferin minimally invasive multi-electrode arrays mutant nervous system disorder neural circuit neuronal circuitry neuroregulation new technology novel optogenetics receptor relating to nervous system response sensor small molecule tool

项目摘要

New tools to selectively regulate neurons have revolutionized causal experimentation. Optogenetics provides an array of elements for specific biophysical control, while designer chemogenetic receptors provide a minimally invasive method to control circuits in vivo by peripheral injection. We have developed a strategy for selective regulation of activity in specific cells that integrates opto- and chemo-genetic approaches, and thus allows manipulation of neuronal activity over a range of spatial and temporal scales in the same experimental animal. Light-sensing molecules (opsins) are activated by biologically produced light through luciferases upon peripheral injection of a small molecule, which crosses the blood-brain barrier. Such BioLuminescence-driven OptoGenetics (‘BL-OG’) is a minimally invasive method like chemogenetics, but one that leverages the full array of bioluminescent and optogenetic options. Importantly, BL-OG allows conventional fiber optic activation while at the same time providing chemogenetic access to the same sensors. This opens, in principle, the entire optogenetic toolbox for complementation by a chemogenetic dimension. Further, because different forms of luciferases use non-cross reactive luciferins, multiple distinct effects can be independently and conjointly controlled in the same animal. We demonstrated proof of concept for this technology by using fusion proteins that directly link Gaussia luciferase (GLuc) to opsins, creating luminescent opsins (luminopsin, LMO). Here, we describe our next steps to increase the benefit of this technology for the field. We will expand the range of BL-OG options, increase their potency, and systematically quantify BL-OG impact in vitro and in vivo. In Aim I, we will generate new luciferases with increased light emission and luciferase/luciferin pairs with non- overlapping substrates to allow multiplexing. In Aim II, we will develop an extended toolkit of luciferase-opsin combinations and test their efficacy in vitro. In Aim III, we will validate and quantify the efficacy of bioluminescence activation of neural circuits in vivo by and directly compare stimulation of LMOs versus fiber optics versus DREADDs. Reflecting the basic science and clinical importance of BL-OG and the expertise of the investigators, we will use defined networks in neocortex and thalamus targeted with viral vectors expressing activating and silencing LMOs and DREADDs. The overall outcome of our work will be the optimization and validation of a novel, highly flexible tool set for bimodal optogenetic and chemogenetic interrogation of neuronal circuits in living animals. The proposed work will give the neuroscience community new molecules and comparative data to aid in making an informed decision when choosing among the various tools that may meet their specific experimental needs.

选择性调节神经元的新工具彻底改变了光遗传学的因果实验。一系列用于特定生物物理控制的元素，而设计的化学遗传受体则提供了我们开发了一种通过外周注射控制体内回路的微创方法。整合光遗传学和化学遗传学方法的特定细胞活性的选择性调节，从而允许在同一实验中在一定范围的空间和时间尺度上操纵神经活动动物的光敏分子（视蛋白）被生物产生的光通过荧光素酶激活。外周注射一种小分子，这种小分子可以穿过血脑屏障。 OptoGenetics（“BL-OG”）是一种类似于化学遗传学的微创方法，但它充分利用了重要的是，BL-OG 允许传统的光纤激活。同时提供对相同传感器的化学遗传学访问，原则上，这打开了整个系统。此外，由于不同形式的化学遗传学维度的补充。荧光素酶使用非交叉反应性荧光素，可以独立或联合产生多种不同的效果我们通过使用融合蛋白证明了这项技术的概念。直接将高斯荧光素酶 (GLuc) 与视蛋白连接，产生发光视蛋白（luminopsin，LMO）。在这里，我们描述了我们将扩大该技术在该领域的优势的下一步措施。 BL-OG 选项的范围，提高其效力，并最终量化 BL-OG 在体外和体内的影响。在目标 I 中，我们将产生具有增加光发射的新荧光素酶以及具有非荧光素酶/荧光素对。在目标 II 中，我们将开发荧光素酶-视蛋白的扩展工具包。在 Aim III 中，我们将验证和量化其功效。通过直接比较 LMO 与纤维的刺激来激活体内神经回路光学与 DREADD 的比较反映了 BL-OG 的基础科学和临床重要性以及专业知识。研究人员，我们将在新皮质和丘脑中使用以病毒载体为目标的特定网络表达激活和沉默 LMO 和 DREADD 我们工作的总体成果将是用于双模光遗传学和化学遗传学的新型、高度灵活的工具集的优化和验证拟议的工作将为神经科学界提供对活体动物神经回路的询问。新分子和比较数据有助于在各种选择时做出明智的决定可以满足他们特定实验需求的工具。