Cell-type-specific analysis of the suprachiasmatic nucleus

在哺乳动物中，行为和生理过程表现出24小时的节律，这些节律是由 位于下丘脑视交叉上核(SCN)的昼夜节律振荡器。南华早报 在分布的昼夜节律振荡器层次结构的顶端充当主起搏器 遍及全身。尽管SCN是大脑中一个相对较小的核，包含 每侧约有10,000个神经元，由多种细胞类型组成。两大类 神经肽能神经元、VIP(血管活性肠肽)和AVP(精氨酸 加压素)，分别富含在SCN的“核心”和“外壳”区域。它是 已知血管活性肠肽和血管紧张素转换酶神经元可以起不同的作用，然而，它还没有 有可能在细胞水平上实时研究SCN中经遗传鉴定的细胞类型。 我们已经开发出新一代生物发光昼夜节律报告小鼠 依赖于Cre-lox重组。使用特定于细胞类型的Cre驱动程序，这些Cre-lox依赖于 记者可以在受限的和遗传定义的细胞群体中被激活，从而使昼夜节律 这些细胞的特性可以与其他类型的细胞分开研究。在这项提案中，我们 将分析SCN内VIP和AVP神经元的细胞类型特定的昼夜节律特性 使用具有点击甲虫的ColorSwitch PER2：：荧光素酶报告的神经元网络 红色(CBR)荧光素酶与PER2融合，在PER2上切换为点击甲虫绿(CBG)荧光素酶 Cre-lox重组。有了细胞型受限记者，我们可以第一次研究 SCN内每种神经肽类神经元的昼夜节律特性。 在这里，我们将研究VIP和AVP神经元在控制昼夜行为中的作用 在这些细胞中同时使用功能丧失和功能获得的昼夜节律突变的表型 上课。我们还将使用VIP和AVP神经元的光遗传控制来分析动力学 在SCN神经网络内重置。最后，我们将使用SCN的单细胞rna-seq 对SCN细胞类型进行分类，并在标记的VIP和AVP神经元中确定 这两种细胞所特有的分子特征和途径。加在一起，这些 实验将提供关于昼夜节律特性和动力学的关键新信息 SCN为了促进我们对哺乳动物昼夜节律系统的了解，这是至关重要的 为了了解人类的昼夜节律紊乱如何导致与 神经疾病、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.