Intrinsic Anatomy of the Circadian Rhythm System

描述(申请人提供)：控制哺乳动物睡眠-觉醒或活动-休息周期等昼夜节律的时钟位于下丘脑视交叉上核(SCN)。最近，几项研究表明，单个SCN内的区域彼此之间存在异相振荡。为了正确理解SCN中与细胞水平的相性活动有关的空间问题，有必要对SCN本身进行仔细的描述。尽管关于这一主题的文献数量非常多，但还没有对任何物种的SCN内在解剖学进行完整的三维评估。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；使用双重标记方法来确定单个细胞是否包含两种不同的神经调节剂的实验要少得多。这项拟议的工作将使用免疫组织化学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，所有的工作都将在秋水仙碱预处理的动物身上进行。初步数据显示，所有的SCN神经元都是GABA的黑色免疫反应阳性。这种神经调节剂将用于确定细胞核，并将细胞免疫反应(IR)的位置固定在其他神经调节剂上。仓鼠SCN内有GagA、P物质、Calbindin、Calretinin、血管活性肠肽、加压素、缩胆囊素和胃泌素释放肽免疫反应的神经元群。GABA细胞分布于整个核团，加压素和缩胆囊素神经元位于背内侧，P物质、胃泌素释放肽、钙结合蛋白和钙视黄素细胞位于中央亚核，血管活性肠多肽神经元位于腹侧。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与神经调节剂重叠和共定位的位置，并将通过评估光诱导蛋白标记物的共定位来评估功能。此外，各种细胞类型的位置将根据三个主要的SCN传入投射的终场来确定，即来自膝间小叶的NPY-IR膝状体-下丘脑束，来自中缝正中核的5-羟色胺-LR投射，以及由霍乱毒素B片段-IR识别的视网状下丘脑投射。突触前终末的身份将通过突触素-IR的存在来确认。对仓鼠的研究将扩展到小鼠，我们将确定哪些细胞类型表现出光诱导的基因表达，以响应夜间的光暴露。这些结果将为解释未来的生理和分子实验提供必要的解剖学框架。

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.