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

项目总结 由于使用微型微型机的活体宽视野荧光成像的最新发展， 在显微镜下，现在可以在对环境输入的反应期间实时观察神经元网络，目标是- 行为定向，功能异常，具有前所未有的解剖学和表型特异性。巡回-- 下丘脑视交叉上核(SCN)中的CADIAN神经网络是一个理想的模型系统 来应用这些工具。SCN网络的独特之处在于它从 眼睛，将光的开始和日长编码到其网络功能中，并提供神经输出来调节 大多数生理功能和行为的时间。然而，对SCN的调查的一个主要限制 到目前为止，生理学一直需要分割大脑，摧毁大部分网络，并使其分离 从它的感觉输入或行为输出。用功能视觉研究完整的SCN网络的能力 传入和行为传出，将代表着该领域前所未有的飞跃。我们建议 细胞特异性光遗传调控SCN神经生理学实时成像工具包的研制 活动。这需要通过对基因构建体、病毒载体、成像设备、 小鼠模型和外科手术程序。一旦优化，我们将使用这些强大的方法来研究 昼夜节律生物学以前无法寻址的关键重要特征：网络处理 光输入导致昼夜节律变化的光输入。

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.