Cell-type-specific analysis of the suprachiasmatic nucleus

视交叉上核的细胞类型特异性分析

• 批准号: 9750837
• 负责人: JOSEPH S TAKAHASHI
• 金额: $ 35.44万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-25 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9750837
• 关键词: ARNTL gene; Argipressin; Automobile Driving; Behavior; Behavioral; Body Temperature; Brain; Cell Nucleus; Cells; Characteristics; Circadian Dysregulation; Circadian Rhythms; Cognition; Cognition Disorders; Coupled; Endocrine Physiology; Expression Profiling; Feeding behaviors; Gene Expression; Gene Expression Profile; Generations; Genetic Recombination; Health; Human; Hypothalamic structure; Label; Lead; Light; Luciferases; Malignant Neoplasms; Mammals; Measures; Mental Health; Mental disorders; Metabolism; Methods; Molecular; Molecular Profiling; Morbidity - disease rate; Mus; Mutation; Neurons; Neuropeptides; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Order Coleoptera; Pacemakers; Pathway interactions; Pattern; Phase; Phenotype; Photoperiod; Physiologic pulse; Physiological Processes; Population; Predisposition; Property; Reporter; Rest; Role; Side; Signal Transduction; Sleep; System; Testing; Time; Transcript; Vasoactive Intestinal Peptide; Work; cell type; circadian; circadian pacemaker; experimental study; flexibility; gain of function; gene discovery; light effects; loss of function; molecular phenotype; nervous system disorder; neuronal excitability; optogenetics; single-cell RNA sequencing; suprachiasmatic nucleus; transcriptome sequencing

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.

在哺乳动物中，行为和物理过程显示受控制的24小时节奏 由位于下丘脑上的核（SCN）中的昼夜节律振荡器。 SCN 在分发的圆形振荡器层次结构的顶部充当大师起搏器 整个身体。尽管SCN是大脑中相对较小的核，其中包含 每侧约有10,000个神经元，由许多细胞类型组成。两个主要类别 含神经肽的神经元，VIP（血管活性肠多肽）和AVP（精氨酸） 加压素）分别富含SCN的“核心”和“壳”区域。这是 知道VIP和AVP神经元可以得到不同的功能，但是，它并非 可以在细胞水平实时研究SCN中的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记者小鼠 Cre-lox推荐依赖性。使用细胞型特异性CRE驱动器，这些Cre-Lox依赖 记者可以在受限和普遍定义的细胞群体中激活，以便昼夜节律 这些细胞的性质可以与其他细胞类型分开研究。在这个建议中，我们 将分析SCN中VIP和AVP神经元的细胞类型特异性昼夜节律特异性 神经元网络使用ColorsWitch Per2 :: Luciferase Reporter，它具有单击甲虫 红色（CBR）荧光素酶融合到PER2上，切换到单击甲虫绿（CBG）荧光素酶 Cre-lox重组。使用细胞类型受限的记者，我们可以首次研究 SCN中每种神经肽类别的昼夜节律特性。 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 在这些细胞中使用功能丧失和功能获得的昼夜节律突变的表型 课程。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神经元网络中重置。最后，我们将使用SCN的单细胞RNA-seq 细胞对SCN细胞类型进行分类以及标记为VIP和AVP神经元的细胞，以确定 这两种细胞类型的分子特征和途径。在一起，这些 实验将提供有关昼夜节目和动态的关键新信息 SCN为了促进我们的理解哺乳动物的昼夜节律系统，这是至关重要的 了解人类中的昼夜节律干扰如何有助于与之相关的发病率 神经系统疾病，认知，心理健康，肥胖，2型糖尿病和癌症。