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

对内部和外部暗示做出反应的动作调节代表了人类和动物最复杂的行为特征之一。电生理学已经明确指出，前额叶皮质(PFC)在控制这种行为反应中起着关键作用。尽管光遗传技术帮助破译了PFC中潜在的复杂神经回路的许多方面，但与此同时，这种方法也受到了空间重叠细胞和来自邻近脑区细胞体的传入轴突的无意共同刺激的阻碍。为了克服这一重大瓶颈，我们提出了基于植物光敏色素光受体的光遗传工具的开发和实现，用于红光调控的基因表达和监测前额投影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以比常规可能的更高的空间和时间精度被限制在感兴趣的靶组织中。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部署，我们将使用蛋白质工程来获得变异体，这些变异体使用广泛存在的胆绿素作为发色团，而不是植物特有的发色团，并对可见光谱中的不同颜色做出反应。这些新工具将被嵌入细胞培养的基因调控电路中，并在脉冲控制实验期间嵌入大鼠PFC的神经元中。一个光基因控制的顺行追踪系统将允许对PFC内和外的投影模式进行精确的功能标测。为了能够同时提供来自多个地点的几种浅色和电子记录，将开发定制的神经探头，作为这一综合合作项目的一部分。因此，作为一个团队，我们将提供和优化基因编码的光调节执行器和神经探测器，它们结合起来将有助于阐明支持由内部和外部线索触发的运动控制的神经电路。具体地说，我们将集中在前缘和下缘皮质之间的相互作用以及它们向皮质下区域的投射的影响。因此，我们希望解开前额叶对皮质-皮质下直接和间接运动通路的影响。

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.