Pacemaker Properties of the Avian Pineal Gland

鸟类松果体是研究鸟类松果体细胞和分子基础的重要模型系统。 生物钟和睡眠。然而，尽管主要产量的生物合成机制 这个腺体，褪黑激素，很大程度上被理解为节律产生机制的分子基础 一直难以捉摸，其起搏器特性的细胞/分子基础完全未知。 在最初的P01赠款中确定的转录图谱和新细胞培养方法的开发 几个可能在节律产生中很重要的候选基因，并表明褪黑素 广泛影响代谢和离子动态平衡，而不影响时钟基因节律。现在 一项提案旨在验证松果体起搏器活动调节中枢神经系统和外周代谢的假设 通过独立于或至少不同于输出调节的机制来调节节律 通过雏鸟的时钟基因节律。(1)我们将确定外科手术是否扰乱了昼夜节律 松果体切除和视网膜切除对糖代谢和时钟基因的影响 活体中枢神经系统和周围组织的节律(mRNA和蛋白质)。我们还会问，政府是否 外源褪黑素对小鼠糖代谢和时钟基因(mRNA和蛋白质)的不同影响 应用定量聚合酶链式反应和DNA微阵列技术对中枢神经系统和外周组织进行检测。(2)我们有 建立了一种原代培养松果体细胞与中枢神经系统共培养的新实验系统 外周靶细胞。我们将通过监测褪黑素来确定起搏器特性的动力学 介质中的节律，以及起搏器和靶细胞中的代谢活动和时钟基因节律 同时。最后，(4)我们将确定时钟基因和其他被识别的基因所起的作用 通过上调和下调这些基因和其他基因的表达，我们的转录图谱 慢病毒转导的起搏器和靶细胞。Lay综述：分子机制 鸟类松果体产生褪黑素的节律，外周组织通过这种节律对褪黑素作出反应 被研究。这一结果将为松果体起搏器活性的首次分子分析提供依据。这些研究 将在睡眠-觉醒周期中的新陈代谢和时钟基因调控之间建立一座桥梁 与夜间活动的啮齿动物相比，昼夜活动的人类的代谢需求更相似。

The avian pineal gland is an important model systemfor the study of cellular and molecular bases of circadian clocks and sleep. However, although the mechanisms of the biosynthesis of the major output of this gland, melatonin, are largely understood, the molecular bases for the rhythm generating mechanism has been elusive, and the cellular/molecular basis for its pacemaker properties is completely unknown. Transcriptional profiling and the development of new cell culture methods in the original P01 grant identified several candidate genes that were likely to be important in rhythm generation and showed that melatonin broadly affected metabolic and ionic homeostasis without affecting clock gene rhythms. The present proposal seeks to test the hypothesis that pineal pacemaker activity regulates CNS and peripheral metabolic rhythms via mechanisms that are independent of or at least differentially regulated from output regulation through clock gene rhythms in chicks. (1) We will determine whether surgical disruption of circadian organization by pinealectomy and retinectomy differentially affects glucose metabolism and clock gene rhythms (mRNA and protein) in CNS and peripheral tissues in vivo. We will also ask whether administration of exogenous melatonin differentially affects glucose metabolism and clock genes (mRNA and protein) in CNS and peripheral tissues in vivo by employing both quantitative PCR and DNA microarrays. (2) We have developed a new experimental system in which primary cultured pinealocytes are co-cultured with CNS and peripheral target cells. We will determine the dynamics of pacemaker properties by monitoring melatonin rhythms in the media, and both metabolic activity and clock gene rhythms in pacemaker and target cells simultaneously. Finally, (4) we will determine the roles played by clock genes and other genes identified in our transcriptional profiling by up-regulating and knocking down expression of these and other genes in pacemaker and target cells by lentiviral transduction. Lay Summary: The molecular mechanisms by which the avian pineal generates rhythms of melatonin and by which peripheral tissues respond to melatonin will be studied. The results will provide the first molecular analysis of pineal pacemaker activity. These studies will build a bridge between metabolic and clock gene regulation in sleep-wake cycles in a species whose metabolic demands are more similar to diurnal humans than are those of nocturnal rodents.