Circadian Photoentrainment in Mammals

 描述(申请人提供)：黑素蛋白表达和固有的光敏视网膜神经节细胞(mRGC或ipRGC)对于非成像(NIF)视觉反应是必不可少的，包括瞳孔光反射、生物钟的光携带、成像视觉中的亮度感知、活动的光调制、睡眠和松果体褪黑素合成。尽管mRGC具有这些多效性作用，但它们的中央投射、接受mRGC输入的遗传定义的细胞类型以及突触超微结构的全面图谱是不完整的。这一应用将创新性地利用小鼠遗传学和病毒载体，在小鼠视网膜的mRGC中特异性地表达荧光和电子显微镜报告程序mini SOG。微型SOG的固有荧光将指导mRGC亚型的选择，以用于后续的连续块面电子显微镜(SBEM)分析。对mini SOG标记的神经突起和胞体的自动分割将加速不同亚型mRGC的快速3D重建，这些MRGC将被用作支架来重建mRGC的全面视网膜内连接图。利用工程狂犬病病毒进行单突触逆行传播，mRGC细胞类型及其构成光输入电路的突触前视网膜神经元将被映射到不同的目标大脑区域。中的mRGC连接 通过结合电子显微镜、微型SOG和SBEM，将在超微结构水平上进一步绘制目标脑区的图谱。总而言之，该应用程序将生成mRGC亚型的细胞网络，识别在大脑中接受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.