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S.R. Grotzner, F.A. F. Rocha, S.M. A. Lima, D.E. Hamassaki–Britto, T.S. Vihtelic, O. Hisatomi, D.F. Ventura; Topographic Density Of L, M And S Cones In The Turtle Retina Investigated By Immunocytochemistry . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5433.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:This study investigates the topographic distribution of red, green and blue single cones and double cones in the turtle retina using anti–opsin antibodies. Methods:Wholemounted turtle retinas were fixed in 4% paraformaldehyde for one hour, washed in 0.1M PB for two hours, incubated with an affinity–purified rabbit antibody against the zebrafish L and M cone pigments (1:500 for every one), revealed with CY5. The CY3 conjugated streptavidin was added to the secondary antibody to label the outer limiting membrane in which we counted the number of double cones and the total population of the photoreceptors. In order to recognize the S cones we used an affinity–purified rat antibody against the bullfrog (RcVP–MS) combined with an affinity–purified rabbit antibody against the human M/L cones (JH 492) at a dilution of 1:500 and 1:5,000 respectively, revealed with FITC and CY5 respectively. Digitalized images of all regions of the retina, at the level of the outer segments of the photoreceptors and at the outer limiting membrane, in 37,987 mm2 areas, were captured by a confocal microscope (Zeiss LSM 410). Cell counts were made with the NIH Scion Image 4.02 beta program and isodensity contours were traced with the Delta Graph 4.0 program. Results:Endogenous biotin is present in the ellipsoid, in the outer limiting membrane and in the pigment epithelium. Its presence in the outer limiting membrane allowed us to count the total number of photoreceptors and to identify the double cones. The average density and standard deviations for the different cone types were: L cones = 2,865 ± 1,992 cells/mm2, M cones = 2,020 ± 906 cells/mm2, S cones = 1,492 ± 690 cells/mm2 and double cones = 1,886 ± 1,026 cells/mm2. For the rods the average density was 728 ± 495 cells/mm2. The distribution of L and M cones showed a gradual increase from the periphery to the center with the peak of density in the visual streak. In the periphery the L cone density is between 2000–3000 cells/mm2 and to the M cone is 1000–2000 cells/mm2. In the visual streak the L cone density is about 10,000 cells/mm2 and to the M cone is about 7,000 cells/mm2. Conclusions:Photoreceptor counts of the S, L and M cones, double cones and rods, were successfully carried out using opsin labeling of the outer segments and endogenous biotin. The results matched the percentages of each receptor type made by other authors.
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