Pacemaker Properties of the Avian Pineal Gland

鸟类松果体是研究鸟类免疫功能的细胞和分子基础的重要模型系统 生物钟和睡眠然而，虽然主要产物的生物合成机制 这个腺体，褪黑激素，在很大程度上被理解，节奏产生机制的分子基础 一直难以捉摸，其起搏器特性的细胞/分子基础是完全未知的。 转录谱分析和新的细胞培养方法的发展在原来的P01赠款确定 几个候选基因，可能是重要的节奏产生，并表明褪黑激素， 广泛影响代谢和离子稳态，而不影响时钟基因节律。本 一项提案试图检验松果体起搏器活动调节中枢神经系统和外周代谢的假设 通过独立于或至少与输出调节不同的机制调节的节律 通过小鸡体内的时钟基因节律。(1)我们将确定手术是否能扰乱昼夜节律 松果体切除和视网膜切除对葡萄糖代谢和时钟基因的影响 体内CNS和外周组织中的节律（mRNA和蛋白质）。我们也会问， 外源性褪黑激素的差异影响葡萄糖代谢和时钟基因（mRNA和蛋白质）， 应用定量PCR和DNA微阵列技术对中枢神经系统和外周组织进行了研究。(2)我们有 开发了一种新的实验系统，其中原代培养的松果体细胞与CNS共培养， 外周靶细胞我们将通过监测褪黑激素来确定起搏器特性的动态变化 在媒体的节奏，以及代谢活动和时钟基因的节奏在起搏器和靶细胞 同步最后，（4）我们将确定时钟基因和其他基因在基因组中所起的作用。 我们通过上调和敲低这些基因和其他基因的表达， 起搏器和靶细胞通过慢病毒转导。概述：分子机制， 鸟类松果体产生褪黑激素的节律 被研究。这一结果将首次提供松果体起搏活性的分子分析。这些研究 将在一个物种的睡眠-觉醒周期中建立代谢和时钟基因调控之间的桥梁， 与夜行啮齿动物相比，白天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.