Central Regulation of the Peripheral Visual System

周边视觉系统的中央调节

• 批准号: 6664764
• 负责人: Robert Nelson Jinks
• 金额: $ 13.59万
• 依托单位: FRANKLIN AND MARSHALL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6664764
• 关键词: SDS polyacrylamide gel electrophoresis; biological clocks; circadian rhythms; electroretinography; enzyme activity; enzyme substrate; horseshoe crabs; immunocytochemistry; immunoelectron microscopy; membrane biogenesis; octopamine; phosphoprotein phosphatase; photobiology; photostimulus; protein biosynthesis; protein kinase A; protein kinase C; retina degeneration; transmission electron microscopy; two dimensional gel electrophoresis; visual photoreceptor; visual photosensitivity; visual phototransduction; western blottings

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.

描述（由申请人提供）：每天，我们视网膜中的感光细胞，我们的光感受器，脱落并更新其感光膜的一部分。然而，这个过程发生的原因还不完全清楚;它在迄今为止研究的所有动物中普遍存在。它对正常的视觉功能至关重要。事实上，视网膜变性发生在不能脱落感光膜的突变大鼠身上。在大多数动物中，光敏膜的脱落是由生物钟（昼夜节律）调节的，并由生物钟和/或光触发。然而，人们对生物钟和光如何相互作用以触发和调节光敏膜的日常脱落和更新知之甚少。鲎的侧眼长期以来一直是研究光敏感膜脱落的重要系统。鲎提供了相对独特的优势，迄今为止，没有证据表明眼睛中存在生物钟;相反，昼夜节律从大脑中的中央时钟沿着从大脑到眼睛的视神经中的神经纤维传递到眼睛。因此，光和昼夜节律对光感受器结构和功能的影响可以通过切断视神经而在该系统中容易地解耦。在目前资助的提案（EY 13196 -01）中，开发并测试了光和生物钟在日常瞬态光敏膜脱落调节中相互作用的模型。这一竞争性延续申请提议通过以下两个具体目标继续评估该模型。 1.研究在鲎光感受器的传出启动过程中，蛋白质合成是否受到长期cAMP依赖性蛋白激酶激活的刺激，以进行短暂的横纹肌脱落。蛋白质合成抑制、免疫组织化学/免疫印迹（CREB活性）和二维凝胶电泳将用于确定侧眼的昼夜节律启动是否需要合成新蛋白质以进行短暂脱落。如果大脑需要蛋白质合成来启动眼睛进行短暂脱落，则将对新蛋白质（或来自该蛋白质的肽）进行微测序（外包）以进行推定鉴定。 2.确定在昼夜传出引发后响应于光触发瞬时横纹肌脱落的信号级联中蛋白激酶C的靶底物。丝氨酸和苏氨酸磷酸化的免疫组织化学测定将与蛋白激酶C（PKC）和cAMP依赖性蛋白激酶的药理学激活剂和抑制剂联合使用，以分离启动瞬时脱落的PKC底物。