Both Rod and Cone Disc Shedding ore Related to Light Onset in the Cat Sreven K. Fisher,* Bruce A. Pfeffer.f and Don H. Anderson* Nineteen domestic cats were entrained to a 12-hr light/12-hr dark lighting cycle and killed at different times of the cycle. The numbers of rod and cone phagosomes in the retinal pigment epithelium (RPE) were quantified by light microscopy. The number of both rod and cone phagosomes shows a large increase within 2 hrs after light onset. Thereafter, the phagosome count remains low until the latter part of the dark period when the number of rod and cone phagosomes increases slightly. In a few cases, substantial variability in phagosome numbers was found between animals killed at the same times in the lighting cycle. Small (0.4-0.8 Mm), dense, membrane-bound granules, that have the ultrastructural features of lysosomes, occur in large numbers in the tapetal RPE cells. The granules may contain only a homogeneous matrix or matrix plus membrane fragments or melanin. Variations in the number of such granules in different animals shows no clear diurnal pattern. Phagosome number, size, and length measurements of outer segments are used to provide estimates of cone outer segment turnover time in the cat. Invest Ophthalmol Vis Sci 24:844-856, 1983 In a wide variety of vertebrate species, rod outer within the processes of the cone sheath for some time segments shed discs in response to light onset or at before reaching the RPE cell bodies.1213 In the cat the beginning of the light part of the daily light-dark retina, our results clearly show that both rods and cycle.1"7 In some species cone disc shedding occurs cones shed discs predominately during the first 2 hr predominantly at the beginning of the dark cycle,58 after light onset. while in others, it reaches a maximum somewhat later in the photoperiod.4'6 It has been suggested that the Materials and Methods temporal relationship between cone disc shedding and the onset of darkness is analogous to the link Nineteen domestic cats were kept on a 12-hr light/ between rod disc shedding and the onset of light.9 12-hr dark cycle for 2 weeks to several months. Tem- We investigated the daily cycle of photoreceptor perature and humidity were also kept constant. disc shedding in domestic cats entrained to a 12-hr Room lighting (270 lux) was provided by overhead light/12-hr dark cycle. Cats have a duplex retina dom- fluorescent lamps. Water and cat chow were available inated by rod photoreceptors, but with a moderate at all times. Animals were killed at the following times population of cones10 that are distinguished easily (0 hr = lights on, 12 hr = lights off): 0, 1 (two cats), from rods because they are shorter and do not reach 2 (two cats), 3, 6, 9, 13, 14, 18 (three cats), 19, 20, the apical border of the RPE.11'12 The cones are con- 21, 22 (two cats), 23 hr. tacted by specialized sheet-like RPE processes that fill the space between the tips of the cones and the apical Fixation surface of the RPE.1112 Thus, disc packets shed by Cats were anesthetized by an initial intramuscular cones in this species are particularly easy to distin- dose of ketamine HC1 (Bristol); once sedation oc- guish from those shed by rods, since they remain curred (within a few minutes) they were given a lethal dose of sodium pentobarbital (Nembutal®, Abbott). For animals fixed during the dark period, anesthesia From the Department of Biological Sciences,* University of was done with illumination from a flashlight equipped California, Santa Barbara, Santa Barbara, California, and the Na- tional Eye Institute,! Bethesda, Maryland. with a red-transmitting filter or a safelight equipped Supported by National Eye Institute research grants EY-00888 with a Wratten No. 25 filter. For the animals fixed and EY-02082 and NEI Research Career Development Award EY- at 13, 14, 18 (two of three cats), 19, 20, 22 hr (one 00174 to SKF. of two cats), enucleation and dissection of the eyes Submitted for publication July 8, 1982. were done under red light. For those cats killed at 18 Reprint requests: Steven K. Fisher, Department of Biological Sciences, University of California, Santa Barbara, Santa Barbara, (one of three cats), 21, 22 (one of two cats), and 23 CA 93106. hr, anesthesia was administered under red light and, 0146-0404/83/0700/844/$ 1.40 © Association for Research in Vision and Ophthalmology 844 Downloaded from iovs.arvojournals.org on 10/01/2021 No. 7 ROD AND DISC CONE SHEDDING/Fisher er ol. 845 when respiration had stopped, the lights were turned This involved counting 10 to 20 sections for each on and enucleation of the eyes had begun. In all cases, specimen. The mean number of phagosomes/mm is enucleation and dissection of the eyes took less than derived from counts of each of several sections, and 10 min. the standard error was used as a measure of variability After enucleation, the cornea, lens, and as much between the different sections. Each section used for of the vitreous as possible were dissected away, and counting was separated from its nearest neighbor by the eye was immersed in a fixative composed of glu- at least 5 ixra to avoid duplication of phagosome taraldehyde and formaldehyde (1% each) in 0.086 M counts. Phagosomes within the cone sheaths were not sodium phosphate buffer (pH 7.2) with 20 mM CaCl2 counted in the "RPE phagosome" category but were added.14 They were kept in fixative for 12 to 24 hrs counted separately as cone phagosomes. Because of at 4 C and then washed several times in phosphate the relatively low number of cones, the cone phago- buffer with 4.5% sucrose and 20 mM CaCl2 at pH somes were tabulated as number of phagosomes/100 7.2. During the buffer wash, a notch was cut in the well-aligned cone sheaths. Since phagosomes tend to superior pole of the eye cup to allow orientation after stay in the center of the cone sheath as they ascend osmication. The eyes were post-fixed in 2% osmium to the RPE, a 1-jtm-thick section through a well- tetroxide (in veronal acetate buffer with 4.5% sucrose aligned cone outer segment and sheath will almost and 20 mM CaCl2 at pH 7.1) for 2 to 2V2 hr at 4 C, certainly reveal any phagosomes within that sheath. rinsed in distilled H2O, and stained overnight in 0.5% All slides were coded so that the two individuals doing aqueous uranyl acetate. After a brief wash in distilled counts did not know the time of fixation. H2O, the tissue was dehydrated in a cold ethanol-H20 The diameter of RPE phagosomes was measured, series, passed through propylene oxide, and embed- using an optical reticule, for cats killed at 1,2, and ded in Araldite 6005® (Cargille). While in absolute 6 hr after light onset and for phagosomes in the cone ethanol, the eyecup was divided into superior and sheaths of cats killed at 1 and 2 hr after light onset. inferior halves and the superior half further divided Small (0.4-0.8 nm diameter), homogeneous granules into two quadrants with the more temporal portion (distinct from phagosomes) were also counted in 1 including the area centralis. Only this quadrant was mm of RPE for each animal. used in our study. Measurements of outer segment length from pho- tomicrographs and of the areas under the curves con- Microscopy structed from phagosome frequency data were made using a Zeiss digital image processor (MOP-3). One micrometer thick sections were taken from the superior-temporal quadrant of one eye from each Results cat; the tapetal region just peripheral to the area cen- tralis was used because in the area centralis itself, Cat Tapetal Retinal Pigment Epithelium there is little or no space between cone outer segment tips and the apical border of the RPE (unpublished The RPE cells of the posterior tapetal region are observations). One micrometer thick sections for irregularly shaped because large choroidal capillaries phagosome counts were taken with a Sorvall MT2-B indent their basal surfaces (see Fig. 5A for example). ultramicrotome, placed on glass slides, and stained In longitudinal sections, the RPE cell depth, as mea- either with a solution of 0.1% toluidine blue and 0.1% sured from the apical to basal surfaces, varies from azure II in 0.5% aqueous sodium tetraborate or with 2 to 9 fxm; their width from one lateral border to the saturated, aqueous p-phenylenediamine. next is between 14 and 17 /xm when the section plane passes through the center of the nucleus. Thin sections (50-70 nm thick) were stained with Three major cytoplasmic inclusions can be rec- uranyl acetate and lead citrate and examined by elec- ognized by light microscopy in these cells: phago- tron microscopy. somes (phagocytosed packets of discs shed from un- derlying photoreceptors), melanin granules, and a Quantitative Data large population of homogeneously staining dense Phagosomes were identified by light microscopy on granules measuring 0.4 to 0.8 nm in diameter. Mature the basis of their size and staining characteristics (see melanin granules are found in all of our samples, but results). Phagosome counts were made using a Zeiss their frequency is low as would be expected from Universal Research Microscope fitted with a 63X retina overlying a tapetum. They are distinguished planapochromat objective at a total magnification of easily from phagosomes by their staining properties, 787.5 times.