Selective Control of Synaptically-Connected Circuit Elements by Interluminescence

项目摘要/摘要 大量的新工具可以直接控制快速(例如，光遗传)或持续(例如， 化学发生学)时间尺度。相反，几乎不存在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 感官、铭文和行动。在这里，我们提出了一种选择性调节突触的新策略 透射性、互发性。该方法使用来自突触前轴突终末的生物发光， 由荧光素酶产生，在实验者控制下调节突触后靶点的视蛋白 引入一种小分子(荧光素)。发展相互发光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，分别针对 到突触裂隙的荧光素酶。这两种发光方法提供了截然不同的特征 实验者需要。为了提供持续的和突触特有的调控，“持续-整合”策略将 连接到突触前终末的突触裂隙中的荧光素酶，以及相对的突触裂隙中的视黄素 突触后膜。在这种配置中，光产生创造了持续的和独立于活动的 调制。在互补的‘Act-Int’策略中，荧光素酶被释放到突触间隙中 对突触前活动的反应，这是一种突触特有的依赖活动的调制形式。除了他们的 独特的特点，这些发光方法在提供突触特异性的预 在实验者控制下的突触后调节。另外两个步骤对于交付可靠的工具集至关重要 这可以很容易地被采用，使发光对广泛的社区有用。首先，我们需要 详细描述单个神经元中相互发光的影响。为此，我们将进行 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)。其次，我们需要检查体内相互发光的影响。在这里我们 将在麻醉和清醒动物的桶状皮质(Aim IC)中测试相互发光。 在另外两个目标中，我们将开始通过测试一种新的依赖于活动的平台技术来详细说明该平台技术 荧光素酶调节机制(AIM II)，并通过设计发光元件来进一步提高 相互发光的时间和空间分辨率(目标III)。经过验证的技术将使您能够追求 一类新的研究问题，既有基本的，也有翻译的，目前无法通过可用的 技术

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.