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

视网膜不是一个静态的组织，而是一个动态的细胞群体，其功能在一天中受到调节。从基因转录水平到细胞网络的连通性，视网膜的昼夜节律是显而易见的。基因转录的昼夜节律调控包括参与细胞代谢和突触转导的基因。这种视网膜生理的日常节律性在没有光线的情况下持续存在，在许多情况下，甚至在视网膜被隔离培养的情况下也是如此。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，这些视网膜生物钟还能够同步或缠绕到光：培养在培养皿中的黑暗周期。这是唯一已知的哺乳动物昼夜节律振荡器，表现出这种光敏感度。表现出强健的昼夜节律基因表达和对光周期的敏感性的一个或多个细胞类型的识别仍然未知。利用生物钟基因的生物发光报告基因PER2，许多小鼠组织中存在的生物钟的相位、幅度和周期可以在器官型组织培养中测量，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，小鼠视网膜中表达生物钟的细胞在培养中是活的，并提供了一个独特的机会来确定它们的身份。利用免疫组织化学技术，通过与视网膜细胞类型标记物的共同标记来识别与光：暗周期同步的表达PER2的视网膜细胞。此外，还将测试降低或移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携带的，这也提供了一个机会来确定节奏最敏感的光的波长，这提供了对光接受分子的洞察。此外，组织培养允许使用药理学来测试参与光转导的细胞通路。已知的视觉第二信使通路和其他昼夜节律调节通路的抑制剂将在光：暗周期的存在下使用，以测试它们对光同步的影响。如上所述，这将通过已知视网膜细胞类型的互补基因突变来完成。最后，将评估小鼠视网膜生物钟的个体发育和它们的光敏感度，以描述发育中的视网膜的昼夜节律的开始。这些实验将为常规视网膜解剖和生理学中的基本问题提供答案。它们还将开辟未来研究和潜在的视网膜功能治疗调节的途径。

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.