Red-light-regulated actuators for spatiotemporal control of opsin expression and modulation of cell-cell interactions within the prefrontal circuit during impulse control

红光调节执行器，用于在脉冲控制过程中时空控制视蛋白表达和调节前额叶电路内的细胞间相互作用

• 批准号: 315304909
• 负责人: Professorin Dr. Ilka Diester
• 金额: --
• 依托单位: Institut für Mikrosystemtechnik (IMTEK)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/315304909?language=en
• 关键词: Red; light; regulated; actuators; spatiotemporal

The regulation of movements in response to internal and external cues represents one of the most sophisticated behavorial hallmarks of humans and animals. Electrophysiology has pinpointed the prefrontal cortex (PFC) as crucial in governing this behavorial response. Although optogenetic technology has helped in deciphering many aspects of the underlying intricate neural circuits in the PFC, at the same time this approach has been hampered by inadvertent co-stimulation of spatially overlapping cells and incoming axons emanating from cell bodies in neighboring brain areas. To overcome this significant bottleneck, we propose the development and implementation of optogenetic tools based on plant phytochrome photoreceptors for red-light-regulated gene expression and monitoring of anterograde communication between prefrontal projection and target cells. In this way, expression of rhodopsin-based optogenetic actuators can be restricted to target tissues of interest with much higher spatial and temporal accuracy than conventionally possible. To facilitate and diversify the optogenetic deployment of plant phytochrome-derived systems in vertebrates, we will use protein engineering to obtain variants that use as chromophore the widely occurring biliverdin as opposed to a plant-specific chromophore, and that respond to different colors across the visible spectrum. These novel tools will be embedded in gene-regulatory circuits in cell culture and in neurons of the rat PFC during impulse-control experiments. An optogenetically controlled anterograde tracing system will allow the precise functional mapping of projection patterns within and outgoing from the PFC. To afford simultaneous delivery of several light colors and electrical recordings from multiple sites, customized neural probes will be developed as part of this integrative collaborative project. As a team, we will thus provide and optimize genetically encoded light-regulated actuators and neural probes that in combination will serve to elucidate the neural circuitry underpinning movement control triggered by internal and external cues. Specifically, we will focus on the impact of the interaction between the prelimbic and infralimbic cortex as well as their projections to subcortical areas. Thereby, we hope to disentangle the prefrontal influence on the direct and indirect cortico-subcortical motor pathway.

根据内部和外部线索对运动进行调节是人类和动物最复杂的行为特征之一。电生理学指出前额皮质（PFC）在控制这种行为反应中起着至关重要的作用。尽管光遗传学技术有助于解读PFC中潜在的复杂神经回路的许多方面，但与此同时，这种方法也受到空间重叠细胞和邻近脑区细胞体发出的传入轴突的无意共刺激的阻碍。为了克服这一重大瓶颈，我们提出了基于植物光敏色素光感受器的光遗传学工具的开发和实施，用于红光调节基因表达和监测前额叶投射与靶细胞之间的顺行通讯。通过这种方式，基于视紫红质的光遗传致动器的表达可以被限制在目标组织中，具有比传统方法更高的空间和时间精度。为了促进和多样化植物光敏色素衍生系统在脊椎动物中的光遗传学部署，我们将使用蛋白质工程来获得使用广泛存在的胆绿素作为发色团的变体，而不是植物特异性发色团，并且在可见光谱中响应不同的颜色。这些新工具将在脉冲控制实验中嵌入细胞培养和大鼠PFC神经元的基因调控回路中。光遗传学控制的顺行追踪系统将允许pfc内外投射模式的精确功能映射。为了同时提供来自多个地点的几种光色和电记录，定制的神经探针将被开发作为该综合合作项目的一部分。因此，作为一个团队，我们将提供并优化基因编码的光调节致动器和神经探针，它们的结合将有助于阐明由内部和外部线索触发的运动控制的神经回路。具体来说，我们将关注边缘前皮层和边缘下皮层之间的相互作用以及它们对皮层下区域的投影的影响。因此，我们希望解开前额叶对直接和间接皮层-皮层下运动通路的影响。