Density of Rods, S– and UV– Cones in the Retina of the Turtle Investigated by Immunocytochemical Techniques

Abstract

Abstract: : Purpose: To investigate the topographic distribution of rods, S– and UV single cones in the turtle retina using anti–opsin antibodies. Methods:Wholemounted turtle retinas (n=3) were fixed in 4% paraformaldehyde in 0.1M phosphate buffer during one hour, incubated with an affinity–purified rat antibody against the bullfrog (RcVP–MS, 1:500) combined with an affinity–purified rabbit antibody against the human M/L cones (JH 492, 1:5,000) and revealed with FITC and CY5, respectively. The streptavidin conjugated–CY3 was added to the secondary IgG to label the outer limiting membrane. We counted the number of double cones and estimate the total population of the photoreceptors using digitalized images (area/field =14,100 µm²) from all regions of the retina that were captured using a confocal microscope (LSM 410, Zeiss). Cell counts were made with the NIH Scion Image 4.02 beta program and isodensity contours were traced using Graph 4.0 software. Results: The total numbers of photoreceptors and double cones, counted at the outer limiting membrane level, were 13,961 ± 10,699 and 2,359 ± 1,053, respectively. The mean density and standard deviations for the S–, UV cones, and rods was: 1,458 ± 1,064 S–cones; 722 ± 536 UV–cones and 845 ± 411 rods). The total number of photoreceptors in the visual streak was about 54,000 cells/mm², with about 3,000 UV–cones/mm², 5,600 S–cones/mm² and close to 10,000 double cones/mm². The distributions of S–cones, double cones and total photoreceptors showed a gradual reduction of density ventral to the visual streak (in the temporal direction) than dorsal. Rod density varied between 1,700–1,900 cells/mm², with the peak density at extreme ventral periphery. These value represents 22–25% of the total of the photoreceptors. Conclusions: Rods, S–cones, UV–cones and double cones were successfully identified using opsin labeling of the outer segments. Our results matched the general percentages of each receptor type found by other authors using other methods.

Keywords: retina • immunohistochemistry • opsins