Abstract
Abstract: :
Purpose: The subcellular localization of Arrestin (Arr) and α–Transducin (αT) in rod photoreceptors has been shown to be transient and dependent on the lighting environment to which mice are exposed. Semi–quantitative analysis of the light/dark induced temporal changes in the concentrations of Arr in the frog and αT in the rat by other labs have produced very different kinetics of translocation between the ROS and the RIS. The simultaneous evaluation of the amounts of these proteins in the same sections of mouse retina was undertaken to provide a more detailed analysis of the kinetics of the temporal changes in their subcellular compartmentation. On a comparative basis, cone αT was also studied. Methods: Albino Balb/cJ mice were exposed to light/dark environments for increasing periods of time, killed and the eyes enucleated, fixed, paraffin embedded, sectioned and subjected to immunofluorescent staining using double labeling with antibodies against Arr and rod αT or cone αT. Fluorescent images were acquired with a Nikon Eclipse 800 microscope coupled to a digital camera and fluorescence intensity was quantitated using Metamorph software. The concentration of these proteins was determined at selected times following either the onset of light or darkness and the results presented in graphic format. Results: The magnitude of the effects of the translocation are dramatically contrasted in images of the retinas of animals that have been maximally light adapted verses retinas that have been maximally dark adapted. After the onset of light, almost all of rod αT has translocated in less than two minutes whereas the translocation of the majority of Arr requires at least six minutes. Translocation in the opposite direction, from light to dark, occurs more slowly for both proteins with Arr requiring almost 30 minutes and αT needing more than 200 minutes to complete its journey. Unlike the frog, prolonged exposure of mice to light does not send Arrestin back to the RIS. Cone αTdoes not translocate under the same conditions which completely translocate rod Arr and rod αT. Conclusions: Collectively, the data support the conclusions that rod Arr and rod αT translocate in each direction by independent mechanisms and suggest that the functional increase in sensitivity of rods for the detection of photons in dim light is secondary to the "shut down" of phototransduction in bright light. Cones apparently do not need the benefits of αT translocation.
Keywords: photoreceptors • microscopy: light/fluorescence/immunohistochemistry • cytology