Visualizing the circadian neural network in vivo: A toolkit for real-time imaging and optogenetic manipulation of the suprachiasmatic nucleus

项目摘要 由于最近发展的体内宽场荧光成像使用小型化的mi， 在显微镜下，神经网络现在可以在对环境输入、目标输入和目标输入的反应过程中以真实的时间观察到。 定向行为，以及具有前所未有的解剖学和表型特异性的异常功能。那个... 下丘脑视交叉上核（SCN）的cadian神经网络是一个理想的模型系统， 应用这些工具。SCN网络是独特的，因为它从突触接受直接的单突触感觉输入。 眼睛，编码光开始和日长到其网络功能，并提供神经元输出，调节 大多数生理功能和行为的时间。然而，SCN研究的一个主要局限性是， 迄今为止，生理学一直需要将大脑切片，破坏大部分网络，并将其分离 从它的感官输入或行为输出。用功能性视觉研究完整SCN网络的能力 传入和行为传出，将代表该领域前所未有的飞跃。我们提出 开发一个工具包，用于SCN神经生理学的细胞特异性光遗传学控制的实时成像 活动这需要通过系统测试遗传构建体、病毒载体、成像设备， 小鼠模型和外科手术。一旦优化，我们将使用这些强大的方法来研究一个 昼夜节律生物学的一个至关重要的特征，这是以前无法解决的： 导致昼夜行为节律变化的光输入。

PROJECT SUMMARY Due to the very recent development of in vivo widefield fluorescence imaging using a miniaturized mi- croscope, neuronal networks can now be observed in real time during responses to environmental input, goal- directed behaviors, and abnormal function with unprecedented anatomical and phenotypic specificity. The cir- cadian neural network in the hypothalamic suprachiasmatic nucleus (SCN) is an ideal model system in which to apply these tools. The SCN network is unique in that it receives direct monosynaptic sensory input from the eyes, encodes light onset and daylength into its network function, and provides neuronal output that regulates the timing of most physiological functions and behavior. However, a major limitation in investigations of SCN physiology to-date has been the need to section the brain, destroying most of the network, and divorcing it from its sensory inputs or behavioral outputs. The ability to study the intact SCN network with functional visual afferents and behavioral efferents, would represent an unprecedented leap forward for the field. We propose to develop a toolkit for the real-time imaging with cell-specific optogenetic control of SCN neurophysiological activity. This requires optimization via systematic testing of genetic constructs, viral vectors, imaging devices, mouse models, and surgical procedures. Once optimized, we will use these powerful approaches to study a critically important feature of circadian biology which was previously unaddressable: the network processing of light input that leads to shifts of the circadian behavioral rhythm.