Selective Control of Synaptically-Connected Circuit Elements by Interluminescence

通过间发光选择性控制突触连接的电路元件

• 批准号: 10165226
• 负责人: UTE H HOCHGESCHWENDER
• 金额: $ 320.85万
• 依托单位: CENTRAL MICHIGAN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10165226
• 关键词: Address; Adopted; Animals; BRAIN initiative; Behavior; Brain; Cell Culture Techniques; Cells; Communication; Communities; Complex; Data; Dependence; Development; Electrophysiology (science); Elements; Engineering; Enzymes; Epilepsy; Generations; Goals; Hippocampus (Brain); In Vitro; Individual; Kinetics; Light; Luciferases; Memory Loss; Mental Depression; Mental disorders; Methods; Mission; Neurons; Neurosciences; Opsin; Optics; Output; Pathway interactions; Performance; Photons; Physiological; Postsynaptic Membrane; Presynaptic Terminals; Production; Property; Proteins; Public Health; Regulation; Research; Resolution; Schizophrenia; Sensitivity and Specificity; Sensory; Slice; Specificity; Synapses; Synaptic Cleft; Synaptic Transmission; Technology; Testing; Thalamic structure; Therapeutic; Time; United States National Institutes of Health; Validation; Variant; Work; addiction; autism spectrum disorder; awake; barrel cortex; cell type; density; experimental study; imaging modality; in vivo; in vivo evaluation; innovation; luciferin; nervous system disorder; neural network; new technology; novel; novel strategies; optogenetics; postsynaptic; postsynaptic neurons; presynaptic; response; small molecule; tool; vesicle-associated membrane protein

PROJECT SUMMARY/ABSTRACT A wealth of new tools can directly control output of specific neurons on fast (e.g., optogenetic) or sustained (e.g., chemogenetic) time scales. In contrast, almost no methods exist for selectively modulating communication between defined cells at the synaptic level, which is key to understanding how functional connectivity creates percepts, engrams and actions. Here, we advance a novel strategy for selectively modulating synaptic transmission, Interluminescence. This approach uses bioluminescent light from a presynaptic axon terminal, generated by a luciferase, to modulate an opsin in its postsynaptic target under experimenter-controlled introduction of a small molecule (luciferin). A challenge in developing Interluminescence is generating sufficient photon density across the synapse. To address this challenge, we developed two separate methods that target the luciferase to the synaptic cleft. These two Interluminescent methods offer distinct features for experimenter needs. To provide sustained and synapse-specific regulation, the ‘Persist-Int’ strategy places a luciferase in the synaptic cleft tethered to the presynaptic terminal, and an opsin in the opposing postsynaptic membrane. In this configuration, light generation creates sustained and activity-independent modulation. In the complementary ‘Act-Int’ strategy, luciferase is released into the synaptic cleft in response to presynaptic activity, a synapse-specific form of activity-dependent modulation. In addition to their distinctive features, these Interluminescence methods are unique in providing synapse-specific pre- to post-synaptic regulation under experimenter control. Two further steps are crucial to deliver a reliable toolset that can be readily adopted, making Interluminescence useful to a broad community. First, we need to characterize in detail the impact of Interluminescence in individual neurons. To this end we will conduct experiments in cell culture and brain slices, recording from individual postsynaptic neurons and endogenous brain circuits in vitro (Aim IA-B). Second, we need to examine the impact of Interluminescence in vivo. Here we will test Interluminescence in anesthetized and awake animals, in the well-characterized barrel cortex (Aim IC). In two additional Aims, we will begin to elaborate this platform technology by testing a novel activity-dependent luciferase regulation mechanism (Aim II), and by engineering light emitting components to further increase the temporal and spatial resolution of Interluminescence (Aim III). The validated technology will enable pursuing a new class of research questions, both basic and translational, that currently cannot be addressed with available technology.

项目总结/摘要 大量的新工具可以快速直接控制特定神经元的输出（例如，光遗传学）或持续（例如， 化学发生）时间尺度。相反，几乎不存在用于选择性地调制通信的方法 在突触水平上定义的细胞之间，这是理解功能连接如何创建的关键 感知、记忆和行动。在这里，我们提出了一种选择性调节突触的新策略， 透射，内发光。这种方法使用来自突触前轴突末端的生物发光光， 由荧光素酶产生，在实验者控制下调节其突触后靶点中的视蛋白 引入小分子（Escherichin）。开发Interluminescence的一个挑战是产生足够的 突触上的光子密度为了应对这一挑战，我们开发了两种不同的方法， 荧光素酶到突触间隙这两种间发光方法提供了不同的特征， 实验者需要为了提供持续的和突触特异性的调节，“持久-内在”策略将 一个荧光素酶在突触间隙拴到突触前末端，和一个视蛋白在相反的 突触后膜在这种配置中，光的产生创造了持续的和独立于活动的 调变在互补的“Act-Int”策略中，荧光素酶被释放到突触间隙中， 这是对突触前活动的反应，是一种突触特异性的活动依赖性调制形式。除了它们 这些Interluminescence方法具有独特的功能，在提供突触特异性的预 突触后调节实验者控制。另外两个步骤对于交付可靠的工具集至关重要 它可以很容易地被采用，使Interluminescence对广泛的社区有用。首先我们要 详细描述了个体神经元中内发光的影响。为此，我们将 细胞培养和脑切片实验，记录单个突触后神经元和内源性 体外脑回路（Aim IA-B）。第二，我们需要检查内发光在体内的影响。这里我们 将在充分表征的桶状皮质（Aim IC）中测试麻醉和清醒动物的内发光。 在另外两个目标中，我们将开始通过测试一种新的活动依赖的 荧光素酶调节机制（Aim II），并通过工程化发光组件，以进一步增加 间发光时间和空间分辨率（Aim III）。经过验证的技术将使追求 一类新的研究问题，包括基本的和翻译的，目前还不能解决现有的 技术.