SUPRACHIASMATIC NUCLEI/NEUROBIOLOGY OF A CIRCADIAN CLOCK

昼夜节律是一种与生俱来的计时能力的明显表现 系统，即，“生物钟”，其被同步（夹带）到 24小时昼夜循环的环境光。 临床和 人类生物钟的实际意义现在已经得到了充分的认识。 的 这项研究计划的总体目标是了解 昼夜节律起搏的内源性振荡机制 哺乳动物的视交叉上核（SCN）。 在目前的赠款期间， 在此期间，我们开始研究基因表达的改变是否可能 与光的夹带效应有关。 我们发现那道光 调控c-Fos和Fos相关转录因子的表达 腹外侧SCN和膝状体间的调节蛋白 小叶（IGL）。 在这个竞争性的延续申请中， 实验结果导致的假设， 转录水平上的事件是光致发光机制的一部分， 将昼夜节律起搏器夹带到明暗周期，以及更多 具体地，SCN中Fos表达可以是 夹带机理 提出了研究四个问题的方案 针对这个假设。 (1)光线和昼夜节律对表达的影响是什么 的Fos，其相互作用与六月，并对他们的结合DNA？ Fos/Jun表达模式将通过免疫组织化学进行评价 原位杂交、免疫反应性和mRNA鉴定 通过Western和北方印迹分析， 通过迁移率变动（凝胶阻滞）测定的序列特异性DNA结合。 (2)Fos的本地化对 视网膜受体腹外侧SCN和IGL？可能的共定位 Fos对合成已知神经肽的神经元的依赖性 来自IGL的视觉输入时SCN中光诱导的Fos激活 将被评估。 (3)影响起搏器牵拉的刺激是否改变了Fos的表达？ SCN？注射后观察Fos表达的变化 N-甲基-D-天冬氨酸拮抗剂MK-801，每日注射 褪黑激素和胎儿SCN中。 (4)实验性降低SCN中细胞内Fos水平是否 抑制光的夹带作用？反义寡核苷酸 与大鼠c-fos mRNA的5'末端互补的蛋白质将通过以下方式引入： 立体定向微渗透泵输注SCN及其作用 光诱导的明显行为节律性的相移将是 测定了

Circadian rhythms are the overt manifestation of an innate timekeeping system, i.e., a "circadian clock," that is synchronized (entrained) to the 24-hr day-night cycle by environmental light. The clinical and practical significance of the human clock is now well recognized. The overall objective of this research program is to understand the endogenous oscillatory mechanism of the circadian pacemaker in the suprachiasmatic nuclei (SCN) of mammals. During the current grant period, we began to study whether an alteration of gene expression might be involved in the entraining effect of light. We found that light regulates the expression of c-Fos and Fos-related transcriptional regulatory proteins in the ventrolateral SCN and in the intergeniculate leaflet (IGL) of rats. In this competing continuation application, experimental results are presented that lead to the hypothesis that events at the transcriptional level are part of the mechanism for photic entrainment of the circadian pacemaker to the light-dark cycle, and, more specifically, that Fos expression in the SCN may be a link in the entrainment mechanism. Protocols are proposed to study four questions directed to this hypothesis. (1) What are the effects of light and circadian phase on the expression of Fos, on its interaction with Jun, and on their binding to DNA? Patterns of Fos/Jun expression will be evaluated by immunohistochemistry and in situ hybridization, identification of the immunoreactive and mRNA species by Western and Northern blot analysis, and detection of sequence-specific DNA binding by mobility shift (gel retardation) assay. (2) What are the implications of the localization of Fos to the retino-recipient ventrolateral SCN and IGL? The possible colocalization of Fos to neurons that synthesize known neuropeptides and the dependence of light-induced Fos activation in the SCN on visual input from the IGL will be assessed. (3) Do stimuli that affect pacemaker entrainment modify Fos expression in the SCN? Changes of Fos expression will be investigated after injection of the N-methyl-D-aspartate antagonist MK-801, after daily injections of melatonin, and in the fetal SCN. (4) Will experimental reduction of intracellular Fos levels in the SCN inhibit the entraining action of light? An antisense oligonucleotide complementary to the 5' end of rat c-fos mRNA will be introduced by stereotaxic infusion in to the SCN via mini-osmotic pump, and its effects on light-induced phase shifts of overt behavioral rhythmicity will be measured.