Photic and Circadian Regulation of Retinal Melatonin

描述（由申请人提供）：哺乳动物视网膜含有生物钟，其通过驱动基因表达、光感受器外节膜更新和视觉敏感性的昼夜节律来调节视网膜功能的多个方面。先前的工作已经表明，褪黑激素通过g蛋白偶联的褪黑激素受体（MTRs）起作用，在调节各种各样的视网膜昼夜节律中起关键作用，并且褪黑激素参与衰老期间视网膜细胞活力的调节。在本申请中，我们将通过使用褪黑激素熟练小鼠（C3 H-f +/+）小鼠进一步研究MTR去除对视网膜的影响，其中两种不同类型的g蛋白偶联的褪黑激素受体（MTR）已经被去除。我们实验室开发的这些新的动物模型提供了一种独特而强大的工具，用于研究褪黑激素和MTRs在视网膜中的作用。我们的初步数据表明，褪黑激素的行动内的一些视网膜细胞可能是通过形成褪黑激素受体1和2异聚体（MTRh）介导的。本申请包括三个具体目的。在具体目标1中，我们将确定褪黑激素受体1型和褪黑激素受体2型是否在视网膜细胞中形成异聚体。在具体目标2中，我们将研究褪黑激素及其相关受体在褪黑激素熟练小鼠和褪黑激素受体敲除小鼠中调节椎间盘脱落的每日节律中的作用。最后，在具体目标3中，我们将确定外源性褪黑激素的施用是否通过MTR的激活来增加视网膜对光诱导的损伤的敏感性，并且我们将剖析由褪黑激素在感光细胞和RPE细胞中激活的信号传导途径。在我们的研究中，我们将使用广泛的新技术和先进的技术，如定量真实的时间Q-RT-PCR，生物发光共振能量转移（BRET），激光捕获解剖显微镜，和微阵列分析。

DESCRIPTION (provided by applicant): The mammalian retina contains circadian clocks that regulate multiple aspects of retinal function by driving circadian rhythms of gene expression, photoreceptor outer segment membrane turnover, and visual sensitivity. Previous work has shown that melatonin acting via g protein-coupled melatonin receptors (MTRs) plays a key role in the regulation of a wide variety of retinal circadian rhythms and melatonin is involved in the modulation of retinal cell viability during aging. In the present application we will further investigate the effect of MTR removal on the retina by using melatonin proficient mice (C3H-f+/+) mouse in which the two different types of g- protein coupled melatonin receptors (MTRs) have been removed. These new animal models developed in our laboratory provide a unique and powerful tool with which to investigate the role of melatonin and MTRs in the retina. Our preliminary data suggest that melatonin action within the some retinal cells is likely to be mediated via the formation of melatonin receptor 1 and 2 heteromers (MTRh). The present application comprises three specific aims. In specific aim 1, we will determine whether melatonin receptor type 1 and melatonin receptor type 2 form heteromers in retinal cells. In Specific aim 2, we will investigate the role of melatonin and its associated receptors in the regulation of the daily rhythms in disc shedding in melatonin proficient mice and melatonin receptor knock-out mice. Finally, in specific aim 3 we will determine whether administration of exogenous melatonin increase the sensitivity of the retina to light-induced damage via activation of MTRs and we will dissect the signaling pathways activate by melatonin in the photoreceptor and RPE cells. In our research, we will use a wide array of new and technologically advanced techniques, such as quantitative real time Q-RT-PCR, Bioluminescence Resonance Energy Transfert (BRET), laser capture dissecting microscopy, and microarray analysis.