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

• 批准号: 9231901
• 负责人: UTE H HOCHGESCHWENDER
• 金额: $ 76.18万
• 依托单位: CENTRAL MICHIGAN UNIVERSITY
• 依托单位国家: 美国
• 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/9231901
• 关键词: Acoustics; Action Potentials; Address; Animals; Anions; Area; Autistic Disorder; Automobile Driving; BRAIN initiative; Basic Science; Behavior; Behavioral; Benchmarking; Biochemical; Bioluminescence; Blood - brain barrier anatomy; Brain; Cells; Chemicals; Chimeric Proteins; Clinical; Communities; Complex; Data; Development; Dimensions; Electromagnetics; Elements; Epilepsy; Fiber Optics; Forms Controls; Goals; Human; In Vitro; Indium; Injection of therapeutic agent; Life; 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; flexibility; genetic approach; in vivo; light emission; luciferin; meetings; minimally invasive; multi-electrode arrays; mutant; nervous system disorder; neural circuit; neuronal circuitry; neuroregulation; new technology; novel; optogenetics; receptor; relating to nervous system; research study; response; sensor; small molecule; tool

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已彻底改变了因果实验。光遗传学提供 特定生物物理对照的一系列元素，而设计师的化学生成受体则提供了 通过周围注射控制体内电路的最小侵入性方法。我们已经制定了一种策略 选择性调节特定细胞的活性调节，该细胞整合了选择和化学遗传学方法，因此 允许在同一实验中的一系列空间和临时尺度上操纵神经元活动 动物。通过生物学产生的光线通过荧光素酶激活光感应分子（OPSINS） 小分子的外围注入，该分子穿过血脑屏障。这样的生物驱动 光遗传学（“ BL-OG”）是一种像化学遗传学这样的微创方法，但一种利用了完整的方法 生物发光和光遗传学的阵列。重要的是，BL-OG允许传统的光纤激活 同时，为相同传感器提供化学发生通道。原则上，这打开了整个 通过化学遗传维度完成的光遗传学工具箱。此外，因为不同形式的 荧光素酶使用非交叉反应性荧光素，多种不同的效果可以独立和共同 在同一动物中控制。我们通过使用融合蛋白来证明该技术的概念证明 直接将高斯荧光素酶（GLUC）与Opsins联系起来，创建发光的Opsins（Luminopsin，LMO）。 在这里，我们描述了我们的下一步，以增加该技术对该领域的好处。我们将扩展 BL-OG选项范围，增加其效力，并系统地量化BL-OG在体外和体内的影响。 在AIM I中，我们将产生新的荧光素酶，并具有增加的光发射和荧光素酶/荧光素蛋白对，而非 - 重叠的基板以允许多路复用。在AIM II中，我们将开发一个扩展的Luciferase-oppin的工具包 组合并在体外测试其效率。在AIM III中，我们将验证和量化 通过体内神经回路的生物发光激活，并直接比较LMO与纤维的刺激 光学与恐惧。反映BL-OG的基础科学和临床重要性以及 研究人员，我们将使用针对病毒载体的新皮层和丘脑中的定义网络 表达激活和沉默的LMO和Dreadds。我们工作的总体结果将是 新型，高度柔韧的工具的优化和验证，用于双峰光遗传学和化学遗传 对活动物中神经元电路的询问。拟议的工作将为神经科学社区提供 新分子和比较数据，有助于在各个地方选择时做出明智的决定 可能满足其特定实验需求的工具。