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

选择性调节神经元的新工具彻底改变了因果实验。光遗传学提供了 一系列用于特定生物物理控制的元件，而设计者化学遗传受体提供了一种 通过外周注射控制体内回路的微创方法。我们制定了一项战略， 选择性调节特定细胞的活性，整合光遗传和化学遗传方法， 允许在相同的实验中在一定范围的空间和时间尺度上操纵神经元活动 动物光敏感分子（视蛋白）被生物产生的光激活， 外周注射小分子，其穿过血脑屏障。这种生物发光驱动的 OptoGenetics（'BL-OG'）是一种像化学遗传学一样的微创方法，但它充分利用了 一系列生物发光和光遗传学的选择。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发生通路。这在原则上打开了整个 用于通过化学遗传学维度互补的光遗传学工具箱。此外，由于不同形式的 酶使用非交叉反应性的酶，多种不同的作用可以独立地或联合地进行。 控制在同一个动物身上。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）连接到视蛋白，产生发光视蛋白（发光蛋白，LMO）。 在这里，我们描述了我们的下一步，以增加该领域的这项技术的好处。我们将扩大 一系列BL-OG选项，增加其效力，并系统地量化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的光发射和与非荧光素酶成对的荧光素酶/荧光素酶的新的荧光素酶。 重叠衬底以允许多路复用。在目标II中，我们将开发一个扩展的工具包， 组合并在体外测试其功效。在目标III中，我们将验证和量化 通过直接比较LMO与纤维的刺激来生物发光激活体内神经回路 光学与DREADDs的对比反映了BL-OG的基础科学和临床重要性以及 研究人员说，我们将使用病毒载体靶向的新皮层和丘脑中的定义网络， 表达激活和沉默LMO和DREADD。我们工作的总体成果将是： 用于双峰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 活体动物神经回路的研究。这项工作将给神经科学界 新的分子和比较数据，以帮助在各种选择中做出明智的决定 可以满足其特定实验需求的工具。

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.