Cell-type-specific analysis of the suprachiasmatic nucleus

在哺乳动物中，行为和生理过程显示24小时节律， 通过位于下丘脑视交叉上核（SCN）的昼夜节律振荡器。的SCN 作为一个主起搏器在一个昼夜节律振荡器分布的层次结构的顶部， 在整个身体。虽然SCN是大脑中相对较小的核团， 每侧约有一万个神经元，它由多种细胞类型组成。两大类 含神经肽神经元，VIP（血管活性肠肽）和AVP（精氨酸 加压素）分别富集在SCN的“核”和“壳”区域。是 已知VIP和AVP神经元可以维持不同的功能，然而，还没有被证实。 可以在细胞水平上真实的时间研究SCN中遗传鉴定的细胞类型。 我们已经开发了新一代的生物发光昼夜节律报告小鼠， 依赖于Cre-lox重组。使用细胞类型特异性Cre驱动程序，这些Cre-lox依赖性 报告基因可以在有限的和遗传上确定的细胞群中被激活， 这些细胞的性质可以与其他细胞类型分开研究。在本提案中，我们 将分析SCN内VIP和AVP神经元的细胞类型特异性昼夜节律特性 通过使用具有点击甲虫的ColorSwitch PER 2：：LUCIFERASE报告子， 与PER 2融合的红色（CBR）荧光素酶，当PER 2被激活时，该荧光素酶转换为点击甲虫绿色（CBG）荧光素酶。 Cre-lox重组有了细胞类型限制性报告基因，我们可以第一次研究 SCN中神经元的这些神经肽类中的每一种的昼夜节律特性。 在此，我们将研究VIP和AVP神经元在控制昼夜行为中的作用 在这些细胞中使用功能丧失和功能获得的昼夜节律突变的表型 班我们还将使用VIP和AVP神经元的光遗传学控制来分析动力学 在SCN神经元网络内重置。最后，我们将使用SCN的单细胞RNA-seq 细胞来对SCN细胞类型进行分类，并在标记的VIP和AVP神经元中进行分类，以确定 这两种细胞类型的分子特征和途径。所有这些 实验将提供重要的新信息的昼夜节律特性和动态的 SCN对于促进我们对哺乳动物昼夜节律系统的理解， 了解人类的昼夜节律紊乱如何导致与 神经系统疾病、认知、心理健康、肥胖、2型糖尿病和癌症。

In mammals, behavioral and physiological processes display 24-hr rhythms that are controlled by circadian oscillators located in the hypothalamic suprachiasmatic nucleus (SCN). The SCN acts as a master pacemaker at the top of a hierarchy of circadian oscillators distributed throughout the body. Although the SCN is a relatively small nucleus in the brain containing about 10,000 neurons on each side, it is composed of many cell types. Two major classes of neuropeptide-containing neurons, VIP (vasoactive intestinal polypeptide) and AVP (arginine vasopressin), are enriched in the “core” and the “shell” regions of the SCN, respectively. It is known that the VIP and AVP neurons can subserve different functions, however, it has not been possible to study genetically identified cell types in the SCN in real time at the cellular level. We have developed a new generation of bioluminescent circadian reporter mice that are Cre-lox recombination dependent. Using cell-type-specific Cre drivers, these Cre-lox dependent reporters can be activated in restricted and genetically defined cell populations so that circadian properties of these cells can be studied separately from other cell types. In this proposal, we will analyze the cell-type specific circadian properties of VIP and AVP neurons within the SCN neuronal network by using a ColorSwitch PER2::LUCIFERASE reporter that has a click beetle red (CBR) luciferase fused to PER2 that switches to a click beetle green (CBG) luciferase upon Cre-lox recombination. With the cell-type restricted reporter, we can study for the first time the circadian properties of each of these neuropeptide classes of neurons in the SCN. Here we will study the role of VIP and AVP neurons in controlling circadian behavioral phenotypes using both loss-of-function and gain-of-function circadian mutations in these cell classes. We will also use optogenetic control of VIP and AVP neurons to analyze the dynamics of resetting within the SCN neuronal network. Finally, we will use single-cell RNA-seq of SCN cells to classify SCN cell types and in labeled VIP and AVP neurons in order to determine the molecular signatures and pathways characteristic of these two cell types. Together, these experiments will provide critical new information on the circadian properties and dynamics of the SCN in order to promote our understanding the circadian system in mammals, which is critical for understanding how circadian disruption in humans contributes to morbidity associated with neurological disorders, cognition, mental health, obesity, type 2 diabetes and cancer.