SUPRACHIASMATIC NUCLEI-NEUROBIOLOGY OF A CIRCADIAN CLOCK

昼夜节律是天生守时的明显表现。 系统，即“生物钟”，它同步(携带)到 环境光24小时昼夜循环。临床和临床 人类时钟的实际意义现在得到了很好的认识。这个 本研究计划的总体目标是了解 心脏昼夜节律起搏器的内源性振荡机制 哺乳动物的视交叉上核。在目前的拨款期间 期间，我们开始研究基因表达的改变是否可能 参与到光的夹带效应中。我们发现了那道光 调节c-Fos及其相关转录因子的表达 SCN腹外侧区和膝间核内的调节蛋白 大鼠小叶(IGL)。在这个竞争的继续申请中， 给出的实验结果导致了这样的假设 转录水平上的事件是光的机制的一部分 昼夜节律起搏器对明暗周期的夹带，以及更多 具体地，SCN中Fos表达可以是 夹带机制。提出了研究四个问题的协议 指向这一假设。 (1)光照和昼夜节律对基因表达有什么影响 关于Fos的，关于它与Jun的相互作用，以及它们与DNA的结合？ Fos/Jun的表达模式将通过免疫组织化学进行评估 和原位杂交，免疫反应和mRNA的鉴定 通过Western和Northern杂交分析物种，并检测 通过迁移率漂移(凝胶延迟)分析进行序列特异性DNA结合。 (2)FOS本地化对 视网膜受体腹外侧型SCN和IGL？可能的共址 Fos对合成已知神经肽的神经元的作用及其依赖性 对来自IGL的视觉输入的SCN中光诱导的Fos激活的影响 将会被评估。 (3)影响起搏器夹带的刺激物是否能改变Fos的表达 南华早报？注射后观察Fos表达的变化 在每日注射N-甲基-D-天冬氨酸拮抗剂MK-801后 褪黑激素，在胎儿的SCN中。 (4)实验性降低SCN细胞内Fos水平 抑制光的夹带作用？一种反义寡核苷酸 与大鼠c-fos基因5‘端互补的基因将通过 微量渗透压泵向SCN内立体定向灌注及其效果 关于光诱导的显性行为节律性的相移 量过了。

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.