Trans-synaptic optogenetics: reversible temporal control of activity at defined synaptic connections

项目摘要/摘要 用于控制特定神经元亚群活动的光遗传方法的引入使新的 对与药物成瘾有关的解剖上不同的大脑区域的功能作用的洞察。然而， 现有光遗传工具的局限性使得解决给定区域内的局部连接变得困难 大脑区域使其能够整合和处理来自其他区域的多个输入。在这里，我们建议生成 一种基本独特类型的光遗传工具，用于使局部微电路中定义的突触组沉默， 最终了解他们是如何控制与成瘾相关的行为的。我们的方法利用了 突触前G蛋白偶联受体(GPCRs)抑制突触传递。与现有的 使用光敏GPCR的直接光学激活的方法，我们的方法涉及光学激活 在突触裂隙的突触后侧有一个单独的结构，这反过来又激活了突触前GPCR。 在我们的两个目标中，我们将开发能够使用高密度脂蛋白激活跨细胞gpr的光遗传工具。 在细胞系中进行吞噬能力分析，然后利用神经元实验来改进这些工具，以实现跨突触 控制gpr的激活。我们将在初级神经元中结合光遗传学和电生理读数 培养和脑切片来验证它们可逆地抑制突触传递的能力。成功完成 该提案将建立新的工具，对神经传递进行前所未有的突触分辨率控制。 这项技术将使新的实验能够剖析大脑关键区域是如何参与成瘾的，比如 伏隔核、腹侧被盖区和前额叶皮质，局部整合来自 不同的大脑区域。

Project Summary/Abstract The introduction of optogenetic methods for controlling activity in defined subsets of neurons has enabled new insights into the functional roles of anatomically distinct brain regions implicated in drug addiction. However, limitations with existing optogenetic tools have made it difficult to address how local connections within a given brain region enable it to integrate and process multiple inputs from other regions. Here we propose to generate a fundamentally unique type of optogenetic tool for silencing defined sets of synapses within local microcircuits, to ultimately understand how they control addiction-related behaviors. Our approach leverages the ability of presynaptic G protein coupled receptors (GPCRs) to inhibit synaptic transmission. In contrast to existing approaches that use direct optical activation of light-sensing GPCRs, our approach involves optical activation of a separate construct on the postsynaptic side of the synaptic cleft, which in turn activates the presynaptic GPCR. In our two aims, we will develop optogenetic tools capable of trans-cellular GPCR activation using high throughput assays in cell lines, and then use experiments in neurons to refine these tools to enable trans-synaptic control of GPCR activation. We will combine optogenetics with electrophysiological readouts in primary neuronal cultures and brain slices to validate their ability to reversibly inhibit synaptic transmission. Successful completion of the proposal will establish new tools with unprecedented synaptic-resolution control over neurotransmission. This technology will enable new experiments to dissect how key brain regions involved in addiction, like the nucleus accumbens, ventral tegmenetal area, and prefrontal cortex, locally integrate information received from diverse brain regions.