Spectral and Metabolic Basis of Visual Responses

我们将研究调节脊椎动物视网膜视杆细胞和视锥细胞在强光下的敏感性的机制。要做到这一点，我们将使电生理记录膜电流显微分光光度测量的视觉色素，和显微荧光测量的视网膜和视黄醇在孤独的杆和锥在黑暗中之前和之后漂白和外源性纳入视网膜及其类似物。将检验以下假设：（1）全反式视黄醇还原为全反式视黄醇在脊椎动物光感受器的反应动力学和敏感性的调节中起重要作用。我们将进行显微荧光实验孤立蝾螈杆和锥以及小鼠杆，以确定和比较动力学，空间分布和代谢依赖性的减少视网膜到视黄醇漂白和治疗视网膜类似物。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。我们将使用细胞外电流记录在这些相同条件下同时测定细胞的生理状态，以确定漂白色素的减少对敏感性恢复的影响。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否发生在视网膜从视蛋白结合位点释放之前、同时或之后。(1)视蛋白中非共价结合位点的占据状态在决定光感受器的适应状态中起着至关重要的作用。我们将测量已漂白并用类似物处理的蝾螈光感受器中的PDE和鸟苷酸环化酶速度，以确定暗适应期间色素再生和敏感性恢复所需的视蛋白/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.