This application proposes experiments to study the processes of light and dark adaptation as well as oxidative metabolism in isolated vertebrate photoreceptors. Electrical responses of these cells under background and bleach-adapted conditions will be obtained using suction microelectrodes. Changes in visual pigment content will be measured using microspectrophotometry. Oxidative metabolism of isolated cells will be measured using miniature oxygen-sensitive electrodes. Psychophysics has shown that background and bleaching adaptation are """"""""equivalent"""""""". Exceptions to this rule involve principally measures of desensitization using time varying stimuli, that could be due to non- equivalence in photoreceptor response kinetics. We propose to compare response kinetics of rods and cones at equivalent levels of background and bleaching adaptation. The internal calcium level [Ca2+]i in rod and cone outer segments has recently been shown to regulate background adaptation. We propose to investigate whether [Ca2+]i is also a mediator of bleaching adaptation. Recovery from bleaching adaptation requires regeneration of visual pigment, but using 11-cis locked retinal analogs, we have found that it does not require formation of a transducing visual pigment. We proposed using other retinal analogues to investigate whether recovery requires formation of an opsin-chromophore Schiff base, or simply occupation of the chromophore binding site. Maintenance and restoration of visual pigment levels during light and dark adaptation requires movement of retinoid between retina and pigment epithelium and likely involves IRBP (interphotoreceptor retinoid-binding protein). We have been able to transfer retinoid from IRBP to isolated photoreceptors (promoting dark adaptation). We propose to investigate further this transfer, in particular the transfer of both retinol and retinal to photoreceptors, and also to investigate the role of IRBP in the transfer of retinoid during light adaptation, i.e. transfer from photoreceptors. Previous interpretations of the O2 consumption of photoreceptors have been hampered by the measurements not having been made in isolation. Investigation of the O2 consumption of single photoreceptor cells should remove this problem. WE will investigate whether the O2 consumption is triggered by light, and whether it is controlled by changes in the levels of [Na+]i or [Ca2+]i.
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