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KJ Strissel, M Sokolov, IB Leskov, VI Govardovskii, VY Arshavsky; An In Situ Model for Investigating Mechanisms of Light-Dependent Transducin Movement in Rod Photoreceptors of the Rodent Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1387.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Light stimulates both the alpha and beta/gamma subunits of the photoreceptor-specific G protein, transducin, to redistribute from the rod outer segment (ROS) to other parts of the photoreceptor cell in living rats, leading to a reduction in photoresponse sensitivity and contributing to light adaptation. As a first step towards determining the cellular mechanisms that control this light-stimulated redistribution, we needed to develop a model that would allow us to uniformly and reliably deliver agents to the living retina to test their potential effects upon light-stimulated transducin movement. Two models, an eyecup and isolated retina, were considered. Methods: Rats were dark-adapted 15 hours prior to all experiments. The animals were euthanized and the eyes extracted under dim red light. For eyecups, the extra ocular tissue, central cornea and the lens were carefully removed. For isolated retina, the entire anterior portion of the eye was cut away and the retina dissected intact. Dissections were performed in supplemented Ringer's maintained at 37 C with constant aeration (5% CO2/95% O2). Retinas and eyecups were also maintained under these conditions for the in situ cultures. For experiments, retinas and eyecups were then subjected to various time periods of illumination using a light source measured by a calibrated light meter at a light intensity level known to stimulate maximal transducin movement in vivo. At the end of the illumination period the isolated retinas or retinas freshly dissected from the illuminated eyecups were flat-mounted and immediately frozen for serial tangential frozen sectioning and Western blot analysis for protein content determination. Results: Retinas maintained in either the eyecup or isolated retina using these culture conditions exhibited light-stimulated transducin redistribution when compared to cultured retinas left in the dark. The greatest extent of light-stimulated movement observed under the in situ conditions for the transducin alpha subunit was 60% of the total alpha subunit pool, somewhat less than the maximum of 90% observed in vivo. For the transducin beta subunit, 80% of the total pool redistributed from ROS upon illumination in situ, identical to the maximum observed in vivo. Conclusion: We succeeded in developing an in situ retina model in which light-stimulated transducin movement takes place very close to the extent observed in the rodent retina in vivo. We may now use a pharmacological approach to begin testing specific hypotheses about essential requirements and mechanisms of this phenomenon.
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