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

项目概要/摘要 引入光遗传学方法来控制限定的神经元子集中的活性，使得新的神经元能够被激活。 深入了解与药物成瘾有关的解剖学上不同的大脑区域的功能作用。然而，在这方面， 现有的光遗传学工具的局限性使得难以解决给定细胞内的局部连接是如何发生的。 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 一种基本上独特类型的光遗传学工具，用于沉默局部微电路内的限定突触组， 最终了解它们是如何控制成瘾相关行为的。我们的方法利用了 突触前G蛋白偶联受体（GPCR）抑制突触传递。相比于现有 虽然我们的方法使用光传感GPCR的直接光学激活，但我们的方法涉及光传感GPCR的光学激活。 在突触间隙的突触后侧上的单独构建体，其反过来激活突触前GPCR。 在我们的两个目标中，我们将开发能够使用高表达的跨细胞GPCR激活的光遗传学工具。 在细胞系中进行通量测定，然后使用神经元实验来完善这些工具， 控制GPCR激活。我们将联合收割机结合光遗传学和电生理学读数， 培养物和脑切片以验证它们可逆地抑制突触传递的能力。成功完成 该提案的一部分将建立新的工具，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与成瘾有关的关键大脑区域，如 腹侧被盖区和前额叶皮层，局部整合从 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.