Circadian Clock Function in the Mammalian Retina

哺乳动物视网膜的昼夜节律时钟功能

• 批准号: 7941847
• 负责人: Christophe P. Ribelayga
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-02 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7941847
• 关键词: Affect; Amacrine Cells; Biochemistry; Biological; Cell Death; Cells; Cellular Morphology; Circadian Rhythms; Complex; Cues; Dark Adaptation; Darkness; Data; Development; Dopamine; Dopaminergic Cell; Elements; Functional disorder; Gene Expression; Gene Proteins; Goals; Grant; High Pressure Liquid Chromatography; Hypothalamic structure; Impairment; In Situ Hybridization; In Vitro; Knowledge; Light; Link; Location; Mammals; Measurement; Measures; Melatonin; Messenger RNA; Molecular; Mus; Nature; Neural Retina; Neuromodulator; Pattern; Periodicity; Phase; Phenotype; Photoreceptors; Physiological; Physiology; Process; Regulation; Research; Research Project Grants; Retina; Retinal; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Running; Structure; System; Techniques; Testing; Time; Tissues; Variant; base; circadian pacemaker; immunocytochemistry; insight; light effects; light entrainment; light intensity; protein expression; public health relevance; research study; response; suprachiasmatic nucleus

DESCRIPTION (provided by applicant): The daily organization of retinal function relies on passive responses to changes in ambient light intensity but also on a complex endogenous circadian clock system. The primary hallmark of clocks is that they continue to run in constant environmental conditions (e.g., total darkness) with periods of approximately 24 h (circadian clocks), and are synchronized to environmental rhythms through external cues, such as the light/dark cycle. By interacting with the dopamine and melatonin neuromodulator systems that are involved in light/dark adaptation, the retinal clock provides a mechanism to anticipate the change in light intensity that occurs at dawn and dusk, thus helping the retina to adapt in a time- and energy-efficient manner at the transition times. Accumulative indirect evidence indicates that impairment of the circadian regulation of retinal physiology may contribute to photoreceptor cell death in some degenerative retinal diseases. Therefore, establishing the basis of circadian rhythmicity in the retina is essential for our understanding of retinal physiology and pathophysiology. To this end, determining the nature and precise location of the circadian clock that drives daily rhythmicity in the retina is crucial. Recent developments in the field indicate that the clock machinery is a cell-based mechanism that relies on a set of genes and proteins (the clock components). Although the clock components are expressed in retinal tissue, the molecular mechanism and the exact location of the mammalian retinal clock are still elusive in part because clock gene expression is widespread among retinal layers. These observations suggest that more than one clock may be located in the mammalian retina. A circadian clock is likely located in the photoreceptor cells where it drives the rhythmic synthesis and release of melatonin. However, it is still unclear whether the rhythmic activity of the dopaminergic amacrine cells is under the control of a clock located within the dopaminergic cells or whether it is driven by the melatonin rhythm. Because melatonin and dopamine strongly interact with each other, it is also unclear whether light entrains retinal rhythms via its effects on dopamine or on melatonin. Our central hypothesis is that a fully functional circadian clock is located in the dopaminergic amacrine cells. The proposed experiments will be conducted on isolated mouse neural retinas maintained in vitro for several days in constant environmental conditions. Using an HPLC technique to measure dopamine levels and in situ hybridization and immunocytochemistry to analyze clock component expression, we will determine 1) whether a clock in the mouse retina controls dopamine levels and whether this rhythm is dependent on the melatonin rhythm and/or the presence of the photoreceptors; 2) which clock components display self-sustained expression in vitro and are required for the dopamine rhythm to occur; and 3) whether light entrainment primarily affects the dopamine and/or the melatonin rhythm and/or the expression of specific clock elements. Retinas from melatonin-deficient, melatonin-proficient and genetically-modified mice will be used in each of the specific aims. PUBLIC HEALTH RELEVANCE: Completion of this research project will provide a better understanding of the cellular and molecular basis of the circadian clock in the mammalian retina and thus increase our knowledge of how the circadian clock controls day/night differences in retinal function. Impairment of circadian rhythmicity in the retina has been linked to photoreceptor cell death and therefore this study will provide fundamental insights into the mechanisms that underlie retinal diseases such as retinitis pigmentosa.

描述（由申请人提供）：视网膜功能的日常组织依赖于对环境光强度变化的被动响应，但也依赖于复杂的内源性生物钟系统。时钟的主要标志是它们在恒定的环境条件下继续运行（例如，完全黑暗），周期大约为24小时（生物钟），并且通过外部线索（例如光/暗周期）与环境节律同步。通过与参与明/暗适应的多巴胺和褪黑激素神经调节系统相互作用，视网膜时钟提供了一种机制来预测黎明和黄昏时发生的光强度变化，从而帮助视网膜在过渡时间以时间和能量有效的方式进行适应。累积的间接证据表明，视网膜生理的昼夜节律调节的损害可能有助于在一些退行性视网膜疾病中的感光细胞死亡。因此，建立视网膜昼夜节律的基础对于我们理解视网膜生理学和病理生理学至关重要。为此，确定驱动视网膜中每日节律性的生物钟的性质和精确位置至关重要。该领域的最新进展表明，时钟机制是一种基于细胞的机制，依赖于一组基因和蛋白质（时钟组件）。虽然时钟成分在视网膜组织中表达，但哺乳动物视网膜时钟的分子机制和确切位置仍然难以捉摸，部分原因是时钟基因表达在视网膜层中广泛存在。这些观察结果表明，在哺乳动物的视网膜上可能有不止一个生物钟。生物钟可能位于感光细胞中，在那里它驱动褪黑激素的节律性合成和释放。然而，目前尚不清楚多巴胺能无长突细胞的节律性活动是否在多巴胺能细胞内的时钟控制下，或者是否由褪黑激素节律驱动。由于褪黑激素和多巴胺彼此强烈相互作用，因此还不清楚光是否通过其对多巴胺或褪黑激素的影响来影响视网膜节律。我们的中心假设是，一个功能齐全的生物钟位于多巴胺能无长突细胞。将在恒定环境条件下在体外维持数天的分离的小鼠神经视网膜上进行所提议的实验。使用HPLC技术测量多巴胺水平和原位杂交和免疫细胞化学分析时钟成分表达，我们将确定1）小鼠视网膜中的时钟是否控制多巴胺水平，以及这种节律是否依赖于褪黑激素节律和/或光感受器的存在; 2）哪些时钟成分在体外显示自我维持的表达，并且是多巴胺节律发生所需的;以及3）光夹带是否主要影响多巴胺和/或褪黑激素节律和/或特定时钟元件的表达。将在每个特定目标中使用来自褪黑激素缺乏、褪黑激素熟练和遗传修饰小鼠的视网膜。公共卫生相关性：该研究项目的完成将更好地了解哺乳动物视网膜生物钟的细胞和分子基础，从而增加我们对生物钟如何控制视网膜功能的昼夜差异的了解。视网膜昼夜节律的损害与感光细胞死亡有关，因此这项研究将为视网膜色素变性等视网膜疾病的机制提供基本见解。