Intrinsic Anatomy of the Circadian Rhythm System

昼夜节律系统的内在解剖

• 批准号: 6615128
• 负责人: LAWRENCE P MORIN
• 金额: $ 15.05万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615128
• 关键词: biological clocks; cell type; circadian rhythms; colchicine; gamma aminobutyrate; gene expression; hamsters; histology; immunofluorescence technique; laboratory mouse; light microscopy; neuroanatomy; neurotransmitters; photobiology; suprachiasmatic nucleus

DESCRIPTION (provided by applicant): The clock governing such mammalian circadian rhythms as the sleep-wake or activity-rest cycles is located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, several studies have demonstrated that areas within a single SCN oscillate out of phase with respect to one another. In order to properly understand the spatial issues relating to phasic activity at the cellular level in the SCN, it is necessary to have a careful description of the SCN itself. Despite the very large volume of literature on the topic, there has been no complete, three dimensional evaluation of the SCN intrinsic anatomy in any species. The vast majority of studies have used adjacent tissue sections to compare relative spatial locations of cell types within the nucleus; many fewer experiments have employed double label methods to determine whether individual cells contain two distinct neuromodulators. The proposed work will use immunohistofluorescence techniques to study combinations of three labels in order to determine their specific locations in the SCN. Importantly, all the work will be performed with animals pretreated with colchicine.Preliminary data show that all SCN neurons are darkly immunoreactive for GABA. This neuromodulator will serve to define the nucleus and anchor the locations of cells immunoreactive (IR) to other neuromodulators. There are neuron groups in the hamster SCN immunoreactive to GAGA, substance P, calbindin, calretinin, vasoactive intestinal polypeptide, vasopressin, cholecystokinin, and gastrin releasing peptide. The distribution of GABA cells encompasses the entire nucleus, vasopressin and cholecystokinin neurons occupy a dorsomedial location; substance P, gastrin releasing peptide, calbindin and calretinin cells are found in a central subnucleus; and vasoactive intestinal polypeptide neurons are found in a ventrally located area. There appears to be fairly extensive overlap among certain of the cell distributions. The exact locations of the cell types and their locations of overlap and co-localization of neuromodulator will be determined, and function will be assessed by evaluating co-localization of light-induced protein markers. In addition, the locations of the various cell types will be determined with respect to the terminal fields of the three main SCN-afferent projections, the NPY-IR geniculohypothalamic tract from the intergeniculate leaflet, the serotonin-lR projection from the median raphe nucleus and the retinohypothalamic projection identified by cholera toxin B fragment-IR following an eye injection. Presynaptic terminal identity will be confirmed by the presence of synaptophysin-IR. Work on the hamster will be extended to the mouse and we will determine which cell types exhibit light-induced gene expression in response to nocturnal light exposure. The results will provide the anatomical framework necessary for interpretation of future physiological and molecular experiments.

描述（由申请人提供）：控制哺乳动物昼夜节律（如睡眠-觉醒或活动-休息周期）的时钟位于下丘脑视交叉上核（SCN）。最近，一些研究表明，在一个单一的SCN内的区域振荡的相位相对于彼此。为了正确理解与SCN中细胞水平的阶段性活动相关的空间问题，有必要仔细描述SCN本身。尽管关于该主题的文献非常多，但在任何物种中都没有对SCN内在解剖结构进行完整的三维评价。绝大多数研究使用相邻的组织切片来比较细胞核内细胞类型的相对空间位置;很少有实验采用双标记方法来确定单个细胞是否含有两种不同的神经调质。拟议的工作将使用荧光技术来研究三种标记的组合，以确定它们在SCN中的具体位置。重要的是，所有的工作都将在用秋水仙碱预处理的动物身上进行，初步的数据显示所有的SCN神经元都对GABA呈暗免疫反应。这种神经调质将用于限定细胞核并将免疫反应性（IR）细胞的位置锚于其他神经调质。在仓鼠SCN内存在对GAGA、P物质、钙结合蛋白、钙视网膜蛋白、血管活性肠肽、加压素、胆囊收缩素和胃泌素释放肽免疫反应的神经元群。GABA细胞的分布包括整个核，加压素和胆囊收缩素神经元占据背内侧位置; P物质，胃泌素释放肽，钙结合素和钙视网膜蛋白细胞被发现在中央亚核;和血管活性肠多肽神经元被发现在腹侧位于区域。在某些细胞分布中似乎有相当广泛的重叠。将确定细胞类型的确切位置及其重叠位置和神经调质的共定位，并通过评价光诱导蛋白标志物的共定位来评估功能。此外，各种细胞类型的位置将相对于三个主要SCN-传入投射的终末区域来确定，所述三个主要SCN-传入投射是来自膝状体间小叶的NPY-1 R膝状体下丘脑束、来自中缝核的NPY-IR投射和通过眼睛注射后的霍乱毒素B片段-IR鉴定的视网膜下丘脑投射。突触前末梢的身份将确认突触素-IR的存在。仓鼠的工作将扩展到小鼠，我们将确定哪些细胞类型表现出光诱导的基因表达在夜间光照。这些结果将为未来的生理和分子实验提供必要的解剖学框架。