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

对内部和外部线索做出反应的运动调节代表了人类和动物最复杂的动物特征之一。电生理学已经确定了前额叶皮层（PFC）在控制这种捕食反应中的关键作用。虽然光遗传学技术有助于破译PFC中潜在的复杂神经回路的许多方面，但与此同时，这种方法受到无意中对空间重叠细胞和来自相邻脑区细胞体的传入轴突的共同刺激的阻碍。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信。以这种方式，基于视紫红质的光遗传学致动器的表达可以以比常规可能的高得多的空间和时间准确度限制于感兴趣的靶组织。为了促进脊椎动物中植物光敏色素衍生系统的光遗传学部署并使其多样化，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而不是植物特异性发色团的变体，并且这些变体响应于可见光谱中的不同颜色。这些新的工具将嵌入在细胞培养的基因调控电路和大鼠PFC的神经元在冲动控制实验。一个光遗传学控制的顺行追踪系统将允许精确的功能映射的投影模式内和从PFC传出。为了提供同时交付的几个光的颜色和电记录从多个网站，定制的神经探针将开发作为这个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致动器和神经探针，这些传感器将用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，我们将专注于前边缘和边缘下皮层之间的相互作用的影响，以及他们的预测皮质下区域。因此，我们希望解开前额叶对直接和间接的皮质-皮质下运动通路的影响。

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.