The identity and the functional properties of the retinal circadian clock.

视网膜生物钟的身份和功能特性。

• 批准号: 8423081
• 负责人: Ethan D Buhr
• 金额: $ 5.57万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8423081
• 关键词: Adult; Anatomy; Animals; Birth; Carbenoxolone; Cells; Circadian Rhythms; Development; Dopamine; Exhibits; Eye; Future; Gene Expression; Gene Mutation; Genes; Genetic Transcription; Glutamates; Growth; Hour; Human; Immunohistochemistry; Individual; Label; Light; Luciferases; Measures; Metabolism; Molecular; Mus; Mutation; Pathway interactions; Pattern; Periodicity; Pharmacology; Phase; Phospholipase C; Photophobia; Photoreceptors; Physiologic pulse; Physiology; Population Dynamics; Predisposition; Property; Protein Kinase C; Proteins; Regulation; Regulatory Pathway; Reporter; Research; Retina; Retinal; Role; Second Messenger Systems; Slice; Staging; Synapses; Techniques; Testing; Therapeutic; Tissues; Vertebrate Photoreceptors; Vision; Visual; cell type; circadian pacemaker; citrate carrier; gamma-Aminobutyric Acid; ganglion cell; inhibitor/antagonist; insight; intercellular communication; light entrainment; phosphodiesterase 6; photoreceptor degeneration; postnatal; pup; research study; response; second messenger; tissue culture

DESCRIPTION (provided by applicant): The retina is not a static tissue, but a dynamic population of cells whose function is modulated through the day. The circadian regulation of the retina is evident from the level of gene transcription to the connectivity of cell networks. The circadian regulation of gene transcription includes genes involved in cellular metabolism and synaptic transduction. This daily rhythmicity of retinal physiology persists in the absence of light and in many cases even when the retina is cultured in isolation. This demonstrates that the circadian rhythms of the retina are controlled by local circadian clocks. Intriguingly, these retinal circadian clocks are also able to synchronize, or entrain, to light:dark cycles when cultured in a dish. These are the only known mammalian circadian oscillators which exhibit this light sensitivity. The identity of the cell type or types which exhibits robust circadian gene expression and susceptibility to light cycles is still unknown. Using a bioluminescent reporter of the circadian clock gene, Per2, the phase, amplitude and period of the circadian clocks present in many mouse tissues can be measured in organotypic tissue culture as a representation of the animal's endogenous rhythms prior to culture of the tissue. This technique has confirmed that the cells in the mouse retina which express the circadian clock are viable in culture and offers a unique opportunity to determine their identity. Using immunohistochemistry, the retinal cells which are strongly expressing PER2 as synchronized by a light:dark cycle will be identified by co-labeling with markers for retinal cell types. Also, mutations in genes which reduce or remove the function of key retinal cell types will be tested for their influence on retinal circadian rhythms. Because the cultures are still light entrainable, this also offers an opportunity to determine the wavelength of light to which the rhythms are most sensitive, which provides insight into the photoreceptive molecule. In addition, tissue culture allows for the use of pharmacology to test the cellular pathways involved in photo transduction. Inhibitors of known visual second messenger pathways and other circadian regulatory pathways will be used in the presence of light:dark cycles to test their influence on synchronization to light. This will be done with complementary genetic mutations of known retinal cell types as described above. Finally, the ontogeny of retinal circadian clocks and their light sensitivity will be assessed in mouse pups in order to describe the onset of circadian rhythmicity in the developing retina. These experiments will provide answers to fundamental questions in regular retinal anatomy and physiology. They will also open up future avenues of research and potential therapeutic regulation of retinal function.

描述（由申请人提供）：视网膜不是静态的组织，而是动态的细胞群，其功能在一天中被调节。从基因转录水平到细胞网络的连接，视网膜的昼夜节律调节是显而易见的。基因转录的昼夜调节包括参与细胞代谢和突触转导的基因。这种视网膜生理学的每日节律性在没有光的情况下持续存在，并且在许多情况下甚至当视网膜被隔离培养时也是如此。这表明视网膜的昼夜节律是由局部生物钟控制的。有趣的是，当在培养皿中培养时，这些视网膜生物钟也能够同步或引导光：暗周期。这些是唯一已知的哺乳动物昼夜节律振荡器，表现出这种光敏感性。表现出稳健的昼夜节律基因表达和对光周期敏感性的细胞类型的身份仍然是未知的。使用生物钟基因Per2的生物发光报道基因，可以在器官型组织培养物中测量存在于许多小鼠组织中的生物钟的相位、振幅和周期，作为组织培养之前动物内源性节律的代表。这项技术已经证实，表达生物钟的小鼠视网膜细胞在培养中是可行的，并提供了一个独特的机会来确定它们的身份。使用免疫组织化学，通过与视网膜细胞类型的标志物共标记来鉴定通过光：暗循环同步的强烈表达PER 2的视网膜细胞。此外，将测试减少或消除关键视网膜细胞类型功能的基因突变对视网膜昼夜节律的影响。因为培养物仍然是光敏感的，这也提供了一个机会来确定节奏最敏感的光波长，这提供了对感光分子的洞察力。此外，组织培养允许使用药理学来测试参与光转导的细胞途径。已知的视觉第二信使途径和其他昼夜节律调节途径的抑制剂将在光：暗周期的存在下使用，以测试它们对与光同步的影响。这将用如上所述的已知视网膜细胞类型的互补基因突变来完成。最后，将在小鼠幼仔中评估视网膜昼夜节律钟的个体发育及其光敏感性，以描述发育中视网膜的昼夜节律性的开始。这些实验将为常规视网膜解剖学和生理学中的基本问题提供答案。他们还将开辟未来的研究途径和视网膜功能的潜在治疗调节。