Spectral and Metabolic Basis of Visual Responses

描述(申请人提供)：我们将研究调节锥体和视杆细胞在暴露于强光期间和之后的灵敏度和响应性恢复的机制，这些机制允许这些细胞对巨大强度范围的光做出反应。为了做到这一点，我们将进行感受器电流的电生理记录，以及视觉色素的显微分光光度测量和视网膜到视黄醇还原的显微荧光测量。测量将在黑暗中进行，在漂白之前和之后，以及在外源合并视网膜及其类似物之后。将检验以下假设：(1)视网膜假体基团与不同视锥类型的视蛋白之间的临界非共价相互作用通过不同的机制调节光激活视锥视蛋白(Meta II)的衰变。这些机制有助于影响在强光照射期间和之后发生的暗电流和闪光反应的快速恢复。我们将在漂白和随后将各种天然和类似的维甲酸掺入完整细胞的视觉色素后对Meta II腐烂、视网膜产生和受体反应恢复的影响进行关联，以确定调节反应恢复的视网膜上特定的相互作用部位。(2)在色素再生过程中，锥体结合部位的非共价占据有助于非常迅速地恢复暗适应过程中的敏感性。这种占有率在燃料棒中具有相反的效果。我们将确定视锥细胞和视杆细胞中相对于色素再生的敏感度恢复的时间进程，这些视锥细胞和视杆细胞暴露在视黄素类化合物中，这些视黄素要么简单地占据视蛋白结合部位，要么与视蛋白结合并随后形成视觉色素。我们将使用不同的维甲酸类似物来确定在这些细胞的暗适应过程中，维甲酸必须具备的加速/阻碍敏感度恢复的基本特征。(3)NADPH将全反式视黄醇还原为全反式视黄醇，随后视黄醇从视蛋白中释放，限制了脊椎动物视杆细胞视色素的再生和视觉敏感性的恢复。我们将在使用含有NADPH或其代谢前体(葡萄糖-6磷酸、三磷酸腺苷等)的溶液透析前后，测量漂白棒外段的局部敏感性恢复。以确定是否必须发生视网膜到视黄醇的减少，以允许暗电流和闪光敏感性的恢复，并了解这种反应恢复是否会受到操作系统中NADPH供应的限制。Layer摘要：在强光照射期间和之后，视杆细胞和视锥细胞的灵敏度和响应性的恢复对于我们感知光环境变化的能力非常重要。我们建议进行生理和生化实验，以增加我们对这种能力背后的细胞机制的理解。我们的最终目标是了解正常和疾病相关条件下的视网膜功能。

DESCRIPTION (provided by applicant): We will investigate the mechanisms that regulate recovery of sensitivity and responsiveness of cones and rods during and following exposure to bright light which allow these cells to respond to light over an immense intensity range. To do this we will make electrophysiological recordings of receptor current together with microspectrophotometric measurements of visual pigment and microfluorometric measurements of retinal to retinol reduction in solitary rods and cones. Measurements will be made in darkness before and following bleaching and following exogenous incorporation of retinal and its analogs. The following hypotheses will be tested: (1) Critical non-covalent interactions between prosthetic groups on retinal and opsin in different cone types regulate the decay of light-activated cone opsin (Meta II) by different mechanisms. These mechanisms assist to affect the rapid recovery of dark current and flash responses that occur during and following bright light exposure. We shall correlate effects on Meta II decay, retinal production, and receptor response recovery following bleaching and subsequent incorporation of a variety of native and analog retinoids into the visual pigment of intact cells to determine specific sites of interaction that on retinal that regulate response recovery. (2) Non-covalent occupancy of the cone binding site during pigment regeneration assists in the very rapid recovery of sensitivity that occurs during dark-adaptation. Such occupancy has the opposite effects in rods. We shall determine the time course of sensitivity recovery relative to that of pigment regeneration in cones and rods that have been exposed to retinoids that either simply occupy the opsin binding site or bind to opsin and subsequently form a visual pigment. We will use different retinoid analogs to determine the essential features that a retinoid must have to accelerate/impede sensitivity recovery during dark adaptation in these cells. (3) The reduction of all-trans retinal to all-trans retinol by NADPH, and subsequent retinol release from opsin limit the regeneration of visual pigment and recovery of visual sensitivity in vertebrate rods. We shall measure the recovery of sensitivity locally within outer segments of bleached rods before and following dialysis with solutions containing NADPH or its metabolic precursors (glucose-6 phosphate, ATP, etc.) to determine whether the reduction of retinal to retinol must occur to allow recovery of dark current and flash sensitivity, and to see if this response recovery can be limited by the supply of NADPH in the OS. Lay Summary: Recovery of sensitivity and responsiveness of rods and cones during and following bright light exposure is important for our ability to sense changes in our light environment. We propose physiological and biochemical experiments designed to increase our understanding of the cellular mechanisms that underlie this ability. Our ultimate goal is to understand retinal function under normal and disease related conditions.