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，而设计的化学发生受体提供了 通过外周注射控制体内电路的微创方法。我们已经制定了一项战略， 对特定细胞活动的选择性调节，结合了光和化学遗传方法，因此 允许在同一实验中在一系列空间和时间尺度上操纵神经元活动 动物。感光分子(视蛋白)是由生物产生的光通过荧光素酶激活的。 外周注射一种小分子，它能穿过血脑屏障。这样的生物发光驱动 光遗传学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选项。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生成途径。原则上，这打开了整个 光遗传工具箱，用于通过化学发生维度进行互补。此外，因为不同形式的 荧光素酶使用非交叉反应的荧光素，多个不同的作用可以独立和联合 在同一只动物体内控制。我们通过使用融合蛋白证明了这项技术的概念。 它直接将赤子荧光素酶(GLuc)与视黄素联系起来，产生发光视蛋白(LMO)。 在这里，我们描述下一步增加这项技术在该领域的好处。我们将扩大 BLOG的选择范围，增加它们的效力，并系统地量化BLOG在体外和体内的影响。 在目标I中，我们将产生新的发光增加的荧光素酶和荧光素酶/荧光素对与非 重叠衬底以实现多路复用。在AIM II中，我们将开发一套扩展的荧光素酶视蛋白工具包 并在体外测试它们的疗效。在目标III中，我们将验证和量化 刺激LMOS和纤维对体内神经回路的生物发光激活及其直接比较 光学与DREADS。反映了BL-OG的基础科学和临床重要性，以及 研究人员，我们将使用病毒载体靶向的新皮质和丘脑中的特定网络 表达、激活和沉默LMO和DREADD。我们工作的总体结果将是 一种新的、高度灵活的双模式光遗传和化学遗传工具集的优化和验证 对活体动物的神经回路的询问。这项拟议的工作将给神经科学界 新的分子和比较数据，帮助在各种选择中做出明智的决定 可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.