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.

项目摘要/摘要 大量新工具可以直接控制特定神经元在快速（例如，光遗传学）或持续的输出（例如， 化学发生时间尺度。相反，几乎没有选择性调节通信的方法 在突触层的定义单元格之间，这是了解功能连接如何创建的关键 感知，恩格拉姆和行动。在这里，我们提出了一种选择性调节突触的新型策略 传播，发光。这种方法使用来自突触前轴突末端的生物发光光， 由荧光素酶生成，以在专家控制下调节其后突触靶标的opsin 引入小分子（荧光素）。开发发光的挑战是产生足够的 突触的光子密度。为了应对这一挑战，我们开发了两种针对的方法 荧光素酶到突触裂缝。这两种发光方法为 实验者需要。为了提供持续和突触的特定法规，“持久的”策略场所 在突触前终端的突触裂缝中的荧光素酶，在对方中的opsin 突触后膜。在这种配置中，光生创造了持续且与活动无关的 调制。在完整的“行动”策略中，荧光素酶被释放到突触中 对突触前活性的反应，突触特异性的活性依赖性调制形式。除了他们 独特的特征，这些发光方法在提供突触特异性的pre- 实验者控制下的突触后调节。另外两个步骤对于提供可靠的工具集至关重要 可以很容易地采用这种融合，使其对广泛的社区有用。首先，我们需要 详细介绍了单个神经元中发光的影响。为此，我们将进行 细胞培养和脑切片的实验，记录来自单个突触后神经元和内源性的实验 体外脑电路（AIM IA-B）。其次，我们需要检查流动性在体内的影响。我们在这里 将测试特征良好的枪管皮层中的麻醉和清醒动物中的发光（AIM IC）。 在其他两个目标中，我们将通过测试一种新型活动来阐述这项平台技术 荧光素酶调节机制（AIM II），并通过工程发光发射组件来进一步增加 发光的暂时和空间分辨率（AIM III）。经过验证的技术将使 基本和翻译的新的研究问题，目前无法与可用 技术。