Spectral and Metabolic Basis of Visual Responses

我们将研究脊椎动物视网膜视杆细胞和视锥细胞在强光期间和之后的敏感性调节机制。为此，我们将在漂白前后和外源掺入视网膜及其类似物之前和之后，在黑暗中对膜电流进行电生理记录，显微分光光度法测量视觉色素，并在孤立视杆和视锥细胞中进行视网膜和视黄醇的显微荧光测量。将检验以下假设：(1)全反式视黄醇还原为全反式视黄醇在调节脊椎动物光感受器的反应动力学和敏感性方面起着重要的作用。我们将对孤立的火蜥蜴视杆和视锥以及小鼠视杆进行显微荧光实验，以确定和比较漂白和视网膜类似物处理后视网膜还原为视黄醇的动力学、空间分布和代谢依赖性。我们将确定细胞外试剂(如IRBP)从漂白细胞中去除视网膜的动力学。我们将利用细胞外电流记录在相同的条件下同时测定细胞的生理状态，以确定减少漂白的光色素对敏感度恢复的影响。最后，我们将同时进行荧光和电生理测量，以确定视网膜减少是在视网膜从视蛋白结合部位释放之前、同时还是之后发生的。(1)视蛋白中非共价结合部位的占据状态对光感受器的适应状态起着至关重要的作用。我们将测量经过漂白和视网膜类似物处理的火蜥蜴光感受器的PDE和鸟苷酸环化酶速度，以确定在暗适应过程中色素再生和敏感度恢复所需的视蛋白/视网膜相互作用的基本结构特征。

We will investigate the mechanisms that regulate the sensitivity of vertebrate retinal rods and cones during and following bright light. To do this, we will make electrophysiological recordings of membrane current microspectrophotometric measurements of visual pigment, and microfluorometric measurements of retinal and retinol in solitary rods and cones in darkness before and following bleaching and following exogenous incorporation of retinal and its analogs. The following hypotheses will be tested: (1) The reduction of all-trans retinal to all- trans retinol plays an important rote in regulation of response kinetics and sensitivity in vertebrate photoreceptors. We will perform microfluorometric experiments on solitary salamander rods and cones as well as mouse rods to determine and compare the kinetics, spatial distribution and metabolic dependence of reduction of retinal to retinol following bleaching and treatment with retinal analogs. We shall determine the kinetics of removal of retinal from bleached cells by extracellular agents (e.g. IRBP). We will make simultaneous determinations of the physiological state of cells under these same conditions using extracellular current recordings to determine the effect that reduction of bleached photopigment has on sensitivity recovery. Finally, we shall make simultaneous fluorometric and electrophysiological measurements to determine if retinal reduction occurs before, at the same time as, or after retinal is released from the opsin binding site. (1) The state of occupation of the non-covalent binding site in opsin plays a crucial role in determining the adaptational state of photoreceptors. We shall measure PDE and guanylyl cyclase velocities in salamander photoreceptors that have been bleached and treated with analogs of retinal to determine the essential structural features of the opsin/retinal interaction required for pigment regeneration and recovery of sensitivity during dark-adaptation.