VISUAL PIGMENT AND PHOTORECEPTOR ADAPTATION

描述（由申请人提供）：视杆光感受器暴露于明亮（视紫红质漂白）光触发类维生素A视觉周期的操作，并显著降低视杆闪光反应的敏感性。随后的暗适应的杆需要，除了兴奋衰减（关闭激活的转导中间体和cGMP补充），恢复从“沉默”脱敏过程，抑制转导信号，即使在接近完全恢复的循环电流。体外研究表明，沉默脱敏的幅度远远超过漂白诱导的量子捕获减少，并已确定了许多可能的贡献反应。然而，缺乏信息的操作沉默脱敏在活的眼睛是一个主要的障碍，最终了解在体内的机制和相关作用的贡献反应。该项目的一个主要重点是确定在体内漂白依赖的沉默脱敏的幅度和时间在小鼠和人类杆使用配对闪光视网膜电图（ERG）记录，最近开发的技术，允许非侵入性的确定杆弱闪光反应的全时程。在正常功能的小鼠和人类受试者的视杆细胞中，以及在由于缺乏全反式视网膜漂白产物的ABCA 4转运蛋白而表现出缓慢的兴奋衰减的abcr-/-小鼠中，我们将测试兴奋衰减速率限制沉默脱敏持续时间的特定假设。配对闪光方法在定量测定视杆闪光反应中的准确性--具体而言，ERG b波侵入偏离视杆反应振幅的可能性--将在nob小鼠（一种b波缺陷模型）的实验中进行测试。该项目的另一个重点是测试最近提出的关于视网膜色素上皮（RPE）中类维生素A代谢的假设，这一过程与RPE在暗适应杆中支持视紫红质再生的作用直接相关。这一假设--视网膜色素上皮中的11-顺式维甲酸调节视网膜色素上皮基底外侧膜上全反式视黄醇的流出--将在对表现出正常或受损视觉周期操作的小鼠进行的放射性标记实验中进行测试。该项目的结果将推进视杆细胞暗适应的基础知识，并为进一步研究人类受试者和实验动物的光感受器疾病提供基础。

DESCRIPTION (provided by applicant): Exposure of rod photoreceptors to bright (rhodopsin bleaching) light triggers operation of the retinoid visual cycle and markedly desensitizes the rod flash response. Subsequent dark adaptation of the rods requires, in addition to excitation decay (shut-off of activated transduction intermediates and cGMP replenishment), recovery from "silent" desensitization processes that depress transduction signaling even after the near-complete recovery of circulating current. In vitro studies show that the magnitude of silent desensitization far exceeds that attributable to bleach-induced reduction in quantum catch, and have identified numerous likely contributing reactions. However, the lack of information on the operation of silent desensitization in the living eye is a major current obstacle to ultimately understanding in vivo mechanisms and relative roles of the contributing reactions. A main focus of the project is to determine the in vivo bleaching-dependence of the silent desensitizations's magnitude and timing in mouse and in human rods using paired-flash electroretinographic (ERG) recording, a recently developed technique that permits noninvasive determination of the full time course of the rod weak-flash response. In normally functioning rods of mice and of human subjects, and in abcr-/- mice that exhibit sluggish excitation decay due to lack of the ABCA4 transporter of all-trans retinal bleaching product, we will test the specific hypothesis that excitation decay rate-limits the duration of silent desensitization. The accuracy of the paired-flash method in quantitatively determining the rod flash response -- specifically, the possibility that ERG b-wave intrusion skews derivation of the rod response amplitude -- will be tested in experiments on the nob mouse, a b-wave deficient model. A further focus of the project is to test a recently raised hypothesis concerning retinoid metabolism in the retinal pigment epithelium (RPE), a process directly linked with the RPE's role in supporting rhodopsin regeneration in dark adapting rods. The hypothesis -- that 11-cis retinoid in the RPE regulates the efflux of all-trans retinol at the RPE basolateral membrane -- will be tested in radiolabeling experiments on mice that exhibit normal or impaired visual cycle operation. Results of the project will advance fundamental knowledge of rod dark adaptation and provide foundation for further studies of photoreceptor disease in human subjects and experimental animals.