Central Regulation of the Peripheral Visual System

描述(申请人提供)：每天，我们视网膜中的光敏感细胞，我们的光感受器，脱落并更新一部分光敏膜。然而，这一过程发生的原因并不完全清楚；迄今为止，所有被研究的动物都知道这一过程。对于正常的视觉功能来说，这是必不可少的。事实上，视网膜退化发生在无法摆脱感光膜的突变大鼠身上。在大多数动物中，光敏膜的脱落受生物钟(昼夜节律)的调节，并由生物钟和/或光触发。然而，关于生物钟和光如何相互作用来触发和调节光敏膜的日常脱落和更新，人们知之甚少。长期以来，河蟹的侧眼一直是研究光敏膜脱落的重要系统。到目前为止，没有证据表明眼睛中存在生物钟；相反，昼夜节律通过视神经中从大脑到眼睛的神经纤维从大脑的中央时钟传递给眼睛。因此，在这个系统中，光和昼夜节律对光感受器结构和功能的影响可以很容易地通过切断视神经来分离。在目前资助的提案(EY13196-01)中，开发了一个光与生物钟相互作用调节每日瞬时光敏膜脱落的模型，并进行了药理学测试。这个竞争性的延续应用程序建议通过以下两个具体目标继续评估该模型。 1.研究在传出预激条件下，蛋白质合成是否受到长期依赖cAMP的蛋白激酶激活的刺激。蛋白质合成抑制、免疫组织化学/免疫印迹(CREB活性)和双向凝胶电泳法将用于确定短暂性脱落的侧眼昼夜启动是否需要新蛋白质的合成(S)。如果大脑需要蛋白质合成来为眼睛的瞬间脱落做好准备，那么新的蛋白质(或来自该蛋白质的多肽)将被微测序(外包)以进行假定的鉴定。 2.确定蛋白激酶C的靶底物(S)在触发昼夜传出启动后的光反应中的瞬时条带脱落的信号级联中。丝氨酸和苏氨酸磷酸化的免疫组织化学分析将与蛋白激酶C和cAMP依赖的蛋白激酶的药理激活剂和抑制剂一起使用，以分离启动瞬时脱落的蛋白激酶C底物(S)。

DESCRIPTION (provided by applicant): Everyday, the light-sensitive cells in our retinas, our photoreceptors, shed and renew a portion of their light-sensitive membrane. Why this process occurs is not completely understood, however; it is ubiquitous to all animals studied to date. It is essential for proper visual function. In fact, retinal degeneration occurs in mutant rats unable to shed their photosensitive membrane. In most animals, shedding of the photosensitive membrane is regulated by a biological clock (circadian rhythm) and triggered by that clock and/or by light. Relatively little is known, however, about how the biological clock and light interact to trigger and regulate daily shedding and renewal of photosensitive membrane. The lateral eye of the horseshoe crab (Limulus) has long served as an important system for the study of light-sensitive membrane shedding. Limulus provides the relatively unique advantage that, to date, there is no evidence of a biological clock in the eye; instead, circadian rhythms are communicated to the eye from a central clock in the brain along nerve fibers that run from the brain to the eye in the optic nerve. As a result, the effects of light and circadian rhythms on photoreceptor structure and function can be decoupled easily in this system by severing the optic nerve. In the currently funded proposal (EY13196-01), a model for the interaction of light and the biological clock in the regulation of daily transient photosensitive membrane shedding was developed and tested pharmacologically. This competing continuation application proposes to continue to evaluate the model through the following two specific aims. 1. Investigate whether protein synthesis is stimulated by long-term cAMP-dependent protein kinase activation during efferent priming of Limulus photoreceptors for transient rhabdom shedding. Protein synthesis inhibition, immunohistochemistry/immunoblotting (for CREB activity), and two-dimensional gel electrophoresis will be used to determine whether circadian priming of the lateral eye for transient shedding requires the synthesis of a new protein(s). If protein synthesis is required for the brain to prime the eye for transient shedding, the novel protein (or peptides from that protein) will be microsequenced (outsourced) for putative identification. 2. Identify the target substrate(s) for protein kinase C in the signaling cascade that triggers transient rhabdom shedding in response to light following circadian efferent priming. Immunohistochemical assays for serine and threonine phosphorylation will be used in conjunction with pharmacological activators and inhibitors of protein kinase C (PKC) and cAMP-dependent protein kinase to isolate the PKC substrate(s) that initiate transient shedding.