Circadian Photoentrainment in Mammals

 描述（由申请人提供）：表达黑视素和固有光敏视网膜神经节细胞（mRGC或ipRGC）对于非成像（NIF）视觉反应是不可或缺的，包括瞳孔光反射、昼夜节律钟的光诱导、成像视觉中的亮度感知、活动、睡眠和松果体褪黑激素合成的光调制。尽管mRGC具有这些多效性作用，但其中央投射、接受mRGC输入的遗传定义的细胞类型和突触超微结构的全面地图是不完整的。本申请将创新性地利用小鼠遗传学和病毒载体在小鼠视网膜mRGC中特异性表达荧光和电子显微镜报告基因miniSOG。miniSOG的固有荧光将指导mRGC亚型的选择，用于通过连续块面电子显微镜（SBEM）进行后续分析。miniSOG标记的神经突和索马体的自动分割将加速不同亚型mRGC的快速3D重建，其将用作支架来重建mRGC的全面视网膜内连接图。使用工程狂犬病病毒进行单突触逆行传递，将映射mRGC细胞类型及其突触前视网膜神经元，其构成不同目标脑区域的光输入回路。研究资助拨款委员会在 结合电子显微镜报告基因miniSOG和SBEM，将进一步在超微结构水平上定位靶脑区。该应用程序将生成mRGC亚型的细胞网络，识别大脑中接收mRGC输入的细胞类型，并建立可用于解开任何遗传定义的神经元连接的工具和技术。

 DESCRIPTION (provided by applicant): Melanopsin-expressing and intrinsically photosensitive retinal ganglion cells (mRGC or ipRGC) are indispensable for non-image forming (NIF) visual responses including pupillary light reflex, photoentrainment of the circadian clock, brightness perception in image-forming vision, light modulation of -activity, -sleep, and - pineal melatonin synthesis. Despite these pleiotropic roles of mRGCs, a comprehensive map of their central projections, genetically defined cell types that receive mRGC input, and synaptic ultra-structure are incomplete. This application will make innovative use of mouse genetics and viral vectors to specifically express fluorescent and electron microscopy reporter miniSOG in the mRGCs of mouse retina. Inherent fluorescence of miniSOG will guide selection of mRGC subtypes for subsequent analyses by serial blockface electron microscopy (SBEM). Automatic segmentation of miniSOG labeled neurites and soma will accelerate rapid 3D reconstruction of different subtypes of mRGCs, which will be used as scaffolds to reconstruct a comprehensive intra-retinal connectivity map of mRGCs. Using engineered rabies virus for monosynaptic retrograde transmission, mRGC cell types and their pre-synaptic retinal neurons constituting light input circuits to different target brain regions will be mapped. The mRGC connectivity in the target brain regions will be further mapped at ultrastructure level by combining electron microscopy reporter miniSOG and SBEM. All together the application will generate cellular networks of mRGC subtypes, identify cell types that receive mRGC inputs in the brain, and establish tools and techniques that can be used to unravel the connectivity of any genetically defined neuron.