infrastructure of Remnant Photoreceptors in Advanced Hereditary Retinal Degeneration

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1 Articles infrastructure of Remnant Photoreceptors in Advanced Hereditary Retinal Degeneration John R. Cotter* and Werner K. Noellf The outer layers of the retinas of pigmented rats affected with hereditary retinal degeneration (rdy) were studied at an advanced stage in the degenerative process, ie, in 200 day old animals. At this age, most of the photoreceptors that survived the degenerative process were cones. The chromatin pattern of their nuclei clearly differentiated them from rods, displaced pigment cells and/or presumptive macrophages that also were found in the outer nuclear layer. None of the cones encountered had outer segments although structures resembling outer segment discs were found with a single cilium. Cones that had inner segments were found in regions of the retina that contained large accumulations of cellular debris. Cones that had lost both inner or outer segments, on the other hand, were found in regions that contained less debris. In such cells, the perikaryon of the cone was rich in mitochondria and other organelles; and the cilium arose directly from the cell body. The morphology of the cones and the fact that they were found in regions of the retina that contained different amounts of cellular debris suggested that cones with inner segments were in an early stage of degeneration while those that had lost inner segments were in a later stage of degeneration. All the cones encountered contained a variety of organelles including: free ribosomes, rough endoplasmic reticulum, and the Golgi apparatus. The cones that survived retinal degeneration therefore appeared to retain the cellular organelles needed for the production of photosensitive pigments. As a consequence, they may be capable of photoreceptor functions. Invest Ophthalmol Vis Sci 25: , 1984 Royal College of Surgeons (RCS) and rdy (retinal dystophic) rats are descended from the original stock of animals described by Bourne, Campbell, and Tansley in 1938.' These rats inherit a retinal disorder that gradually destroys the outer layers of the neural retina. The development of each layer is completed following birth. 1 " 5 Beginning in the third postnatal week, however, the outer segments of photoreceptor cells degenerate 2 " 5 and the byproducts produced accumulate beneath the pigment epithelium in the layer of rods and cones. 6 " 8 Subsequent and concomitant to the deposition of debris, the photoreceptor cells of the retina die. 2 ' 3 ' 5 ' 8 The degeneration of the photoreceptor cells occurs in two phases 9 : in the first phase, ie, in dayold animals, virtually all the rods are destroyed; and in the second phase, ie, in day-old animals, most but not necessarily all the remaining photoreceptors are destroyed. From the Departments of Anatomical Sciences* and Physiology,t State University of New York at Buffalo, Buffalo, New York. Supported by National Institutes of Health grant 2R01-EY Submitted for publication: May 14, Reprint requests: J. R. Cotter, PhD, The University of Kansas, Department of Ophthalmology, 39th & Rainbow Blvd., Kansas City, KS Surprisingly, despite the dramatic loss of rods that normally comprise approximately 99% of the photoreceptor cell types in the mutant rat 10 and the gradual loss of photoreceptors in the second phase, mutant rats retain visual capabilities in advanced stages of retinal degeneration. 10 " 13 The cells that survive, which are for the most part cones, 1 ' 2910 may account for the fact that vision is preserved. If this is the case, it is still remarkable since the outer segments of the cones are presumed to be destroyed in the early phase of the degenerative process. 39 La Vail and co- workers" have in fact confirmed the loss of cone outer segments in 9-month-old, 1-year-old, and 2-year-old RCS rats. In our investigation, we studied the outer retinal layers of 200-day-old pigmented rdy rats. Light microscopic examination of the eyes of the pigmented rdy strain indicated a reduction of the outer nuclear layer at the age of 200 days to one row of nuclei throughout the central and peripheral regions, of which most had a chromatin organization typical for cone cells. 6 Compared with normal retinas of the control strain (Long Evans), the number of these cone nuclei had decreased by still less than 30%. 9 On the other hand, the mass of debris, the product of rod outer segment degeneration between the neural retina and pigment epithelium had disappeared'to a small fraction of its thickness at the age of 4 months. 1366

2 No. 12 CONE CELL ULTRASTRUCTURE IN RDY RATS / Correr ond Noell 1367 Retinas of the pigmented rdy strain at the age of 200 days or older were, therefore, chosen for electrophysiologic and behavioral studies of preserved visual functions. Indeed, visual-evoked potentials can be recorded from the striate cortex, the animals learn to discriminate between horizontal and vertical bars, and concentric antagonistic receptive fields can be recorded from the optic tract. 913 Our study was designed to provide ultrastructural information on the outer layers of the retina for the interpretation of the visual function studies. 14 Special attention was given to the fine structure of the cones since their presence may explain the continued function of the retina at this age. The results of this study indicate the cones in 200-day-old mutant rats are indeed "cone-less" but that they are in other respects structurally preserved. Materials and Methods Animal Strain and Housing The animals were selected from an in-house colony of pigmented mutant (rdy) rats maintained since with the autosomal recessive photoreceptor degeneration. Normal controls were of the Long Evans strain originating from Charles River. The rats were reared from birth in a dark environment and all preparatory procedures prior to eye fixation were carried out under dim red light or after eye lids had been covered by light-proof tape. Visual-Evoked Cortical Potentials Prior to the preparation for electron microscopy, the approximately 200-day-old rats were tested for the state of their visual capacity, measuring the singleflash ERG (Grass photostimulator) and the computersummed flash-evoked potential of the visual cortex obtained by recording from stainless steel screws inserted in the skull over the central retino-topic region. 14 These measurements were performed in urethane anesthesia (1.2 gm/kg) and after conjunctival applications of 2% xylocaine. The single-flash ERG was not recordable at a cathode ray sensitivity of 50 /uv/cm while the normal response exceeded 1 mv. Cortical responses, however, were preserved as for the average of all rdy rats at this age, showing an increased latency compared with the normal and the typical N,, P 2, and N 2 components of reduced amplitude. 14 Within 1 hour after the electrical measurements, the rats were prepared for histology. Electron Microscopy The animals were perfused by transcardiac administration of Hanks' solution 15 to which 1% sodium nitrate was added just prior to the perfusion. Following Hanks' solution, the animals were perfused with a mixture of 2% glutaraldehyde and 2% formalin in 0.1 M cacodylate buffer. The retinas were removed and cut into small strips. These were postfixed in ferrocyanide-reduced 1% osmium tetroxide 16 and block stained for 24 hr in the dark with uranyl acetate in 0.05 M acetate buffer. The tissue was then dehydrated, embedded in Spurr's resin, and sectioned on a Sorvall MT2-B ultramicrotome. Semithin sections that were stained with toluidine blue were used for orientation of the tissue blocks. Thin sections that were stained with uranyl acetate and lead citrate were examined with a JEM- 100B transmission electron microscope (JEOL Ltd.; Tokyo, Japan). All procedures involving animals were performed according to policies set forth in the ARVO Resolution on the Use of Animals in Research. Pigment Epithelium Results Although the outer layers of the retina were each identified in 200 day old rats (Fig. 1), the neural part of the outer layers, ie, the layer of rods and cones and the outer nuclear layer, obviously was altered. On the choroidal side of the pigment epithelium, the pigment epithelial cell had numerous microvilli that ended near a basal lamina and on the apical surface, the cell membrane of the pigment epithelial cell was thrown into elaborate apical processes that extended into the neural retina (Fig. 2). The nucleus of the pigment epithelial cell was large, round, and the heterochromatin of its nucleus was located along the interior of the nuclear envelope. The cytoplasm of the cell contained premelanosomes, numerous melanosomes, mitochondria, lipid droplets, smooth endoplasmic reticulum, the Golgi apparatus and rough endoplasmic reticulum. Phagosomes containing outer segment discs were not seen. Debris In some sections there was debris between the apical processes of the pigment epithelium and the outer nuclear membrane (Fig. 3). The region of debris was in some places wide and filled with degenerating material. Lamellar whorls of degenerating material and extracellular membranes that resembled outer segment discs were found in the debris. The region of cell debris also contained intact structures including the processes of Miiller cells, inner segments and cilia. Normal outer segments were not observed though structures that vaguely resembled intact outer segment discs were found in the blind end of one cilium (Fig. 4). These were short, sac-like but not

3 PE Rd ONL OPL INL Fig. 1. The outer retinal layers of a 200-day-old animal are shown in this low-power electron micrograph. They include, beginning near the top: the pigment epithelium (PE), a region of cellular debris (Rd), which in normal retinas contains light-sensitive outer segments and the inner segments of photoreceptor cells; the outer nuclear layer (ONL), which contains cell bodies of surviving rods and cones; and the outer plexiform layer (OPL), which contains the axons of photoreceptor cells and the synaptic contacts that they make with bipolar and horizontal cells of the underlying inner nuclear layer (INL). Both rod (R) and cone (C) nuclei were identified in the outer nuclear layer (X2.8OO).

4 No. 12 CONE CELL ULTRASTRUCTURE IN RDY RATS / Correr ond Noell 1069 Fig. 2. An electron micrograph of the pigment epithelium. The choroidal side of the pigment epithelium is located in the upper left-hand portion of the field (X9,18O). numerous and they resembled the discs seen at the proximal end of normal outer segments or developing outer segments. In some sections there was little debris between the apical processes of pigment epithelial cells and nuclei of the outer nuclear layers so that the processes of pigment epithelial cells were closely opposed to the processes of Miiller cells. In other sections, the space between the two was occupied by cell processes (Fig. 5) that contained vesicle-like structures. These measured approximately 40 nm. The origin of the fibers could not be determined, but they may represent the axonal processes of surviving photoreceptors. Miiller Cells In areas of the retina that contained considerable amounts of debris, cell junctions between adjacent Miiller cells formed an obvious outer limiting membrane. In this area (Fig. 3) microvilli arose from the Miiller cells and projected into the region that contained debris. When inner segments were present, the microvilli surrounded them. Beneath the outer limiting membrane some cell bodies were invested by several layers of cytoplasmic processes. These have been shown to be the processes of Miiller cells." In areas of the retina where there was no distinct outer limiting membrane, the cell processes of the pigment epithelium were separated from the outer nuclear layer by layers of Miiller cell processes. Cells of the Outer Nuclear Layer The outer nuclear layer, which was reduced to a single row of nuclei, contained three distinct cell types. The nucleus of one cell type had a single clump of chromatin material surrounded by less electron dense euchromatin (Fig. 1). Cells with this chromatin pattern in the nucleus were classified as rods.

5 1070 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1984 Vol '*> " _ - - * Fig. 3. The outer limiting membrane (OLM) is formed by membrane junctions of adjacent Muller cell processes. Numerous microvilli (MP) arise from the apical ends of Muller cells. The region above the outer limiting membrane is filled with debris and lamellar whorls of degenerating debris (RD). The nucleus of a cone (CN) is located below. The cytoplasm of the cone can be traced beyond the OLM into the region of the debris where the inner segment abruptly ends (XI 5,960). Cones were the most frequently encountered cell type. They were identified by the chromatin pattern of the nucleus which consistently formed two or more clumps of heterochromatin (Fig. 1). Occasionally, synaptic ribbons were found in the perikaryon, and this helped to identify the cones as true photoreceptor cells. In addition a cilium, if present, identified these as photoreceptors. Sixty-four cones were identified in this manner. Their cell bodies were present as isolated structures or they were clustered into small groups. In patches of the retina where the outer limiting membrane was

6 No. 12 CONE CELL ULTRASTRUCTURE IN f\dy RATS / Correr and Noell 1371 r Fig. 4. An inner segment and a cilium are illustrated in this electron micrograph. Small structures that resemble outer segment discs (arrow) were found in the cytoplasm of this cilium (X45,760). easily identified, the cells were twisted and the cytoplasm surrounding the nucleus extended through the outer limiting membrane beyond the processes of the Miiller cells into the region of debris and into structures that correspond to the ellipsoid and myoid regions of inner segments (Fig. 3). Cilia were associated with inner segments (Fig. 4), but in no instance was a cilium continuous with an outer segment. Though these cilia did not connect with outer segments they were near to lamellar whorls. The cilia originating from still preserved inner segments were found in regions that contained considerable amounts of cellular debris. In regions where there was little debris, the cytoplasm of the cone was restricted to the cell body (Fig. 5). Some of the cone cells had a cilium that arose directly from the cell body in the region of the nucleus (Fig. 6). Because of distortion of the tissue, the cilium was disoriented with respect to its normal radial position. Within the cytoplasm of the cone there were free and fixed ribosomes, mitochondria, the Golgi apparatus, microtubules, basal bodies, and in some cells autophagic vacuoles. A third cell type contained rough endoplasmic reticulum, which was abundant, and the Golgi apparatus. The heterochromatin of the nucleus was located along the inner aspect of the nuclear envelope. Some cells of this type also contained large, electron dense, irregularly shaped structures that resembled melanosomes or dense bodies (Fig. 7) and in some, the cytoplasm of the cell was branched. Although the cells were not identified definitively, they may be macrophages 17 or displaced pigment cells." Outer Plexiform Layer Cell processes that resembled those found in the space between the pigment epithelium and the outer nuclear layer were found in the outer plexiform layer. The processes contained synaptic vesicles and synaptic ribbons that were surrounded by a halo of synaptic vesicles. A few structures resembled cone pedicles. In

7 1372 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1984 Vol. 25 Fig. 5. Although two lamellar whorls of degenerated material are present, there is very little debris between the apical processes of the pigment epithelium and the two cone nuclei that are seen in this section. Notice that the region between the nuclei of the outer nuclear layer and the apical processes of the pigment epithelium is filled by numerous processes many of which contain vesicle-like structures (XI 2,320). these the presynaptic component of the pedicle contained mitochondria, synaptic vesicles, and synaptic ribbons. Some of the postsynaptic process were invaginating the presynaptic element. Discussion Of the three or four cell types (cones, rods, macrophages, and/or displaced pigment cells) found in the outer nuclear layer of 200-day-old rdy rats, cones were the one type most frequently encountered. This is consistent with the observation that the retina of rdy rats has degenerated at this age to one which has lost almost all rods. 910 Cones of normal retina are tall columnar cells that can be subdivided into several subdivisions including outer segment, inner segment, and perikaryon. 18 In contrast, the morphology of the cone in the 200-dayold rdy rat was altered. In particular, none appeared to have outer segments. Some cells did have inner segments but the shape of the cell was usually distorted. The fact that these cones were found in regions that contained heavy debris suggested that they were in an early stage of degeneration. In areas of the retina where there was little debris, the inner segments were missing, and the cell consisted solely of a perikaryon and a modified outer fiber. A cilium that arose directly from the cell body was observed in most cells. In other cones of the same class, the cilium was probably not seen because of the plane of section. Basal bodies, presumably those associated with the cilia of the cones also were identified in the perikaryon and not the inner segment. The position of the cilium was very interesting. The cilium normally connects inner and outer segments and is located in the layer of rods and cones. Finding the cilium so close to the cone nucleus therefore suggested that either the origin of the cilium had shifted from the inner segment layer to the outer nuclei or that a new one had formed. The absence of outer segments in all cones and the absence of inner segments in some suggests that there is progressive alteration of cone cell morphology and even though the cones encountered are from animals

8 No. 12 CONE CELL ULTRASTRUCTURE IN RDY RATS / Correr ond Noell 1370 Fig. 6. In this electron micrograph, a cilium (arrow) can be seen originating from a cone. Notice that the origin of the cilium is very close to the nucleus, Normally the cilium arises from the apex of the inner segment (X27,000). of the same age, a pattern emerges that suggests that differences in cone morphology are related to stages of cone degeneration (Fig. 8). The first stage involves the destruction of outer segments and formation of a "cone-less" cone, that is, a cone that lacks a cone-shaped outer segment. This presumably occurs before the animals are 200 days old since nothing even vaguely resembling outer segments was encountered in animals of this age. Next there is a retraction of the inner segment and

9 1374 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / December 1984 Vol. 25 Fig. 7. Cells other than photoreceptors were found in the outer nuclear layer. These cells always contained large, irregularly shaped electron dense structures (arrows) {XI 6,000). Fig. 8. Hypothetical stages of cone degeneration in rdy rats. In the first stage (1) outer segments degenerate.-next (2-3) inner segments are destroyed, and what remains of the inner segments and their organelles are retracted along with the cilium toward the outer limiting membrane (double linear line). Finally (4) the cilium of the cone and organelles are sequestered in the cell body of the cone.

10 No. 12 CONE CELL ULTRASTRUCTUPvE IN RDY RATS / Correr and Noell 1375 the cilium toward the outer limiting membrane. Finally the cilium and organelles of the cells are sequestered in the cytoplasm surrounding the nucleus. The fact that cones are present at 200 days is itself significant because any one or all of remnant cones could account for the continued function of the retina at this age. Though they are modified morphologically, cones retain organelles needed to synthesize photosensitive pigments, and hence they may function without outer segments. However, the mechanisms by which cones are activated by light in the absence of outer segments remains to be determined. Photosensitive pigments are probably inserted in the plasma membrane, 1014 but whether or not this insertion and the preservation of the visual machinery require the presence of the basal bodies of the cilium is an open question. Key words: inherited retinal degeneration, rdy rats, cone ultrastructure Acknowledgments The authors are indebted to Ms. Elizabeth Stachiewicz for sectioning material, to Dr. Thaddeus Szczesny for printing the micrographs, and to John Nyquist for making the line drawing. References 1. Bourne MC, Campbell DA, and Tansley K: Hereditary degeneration of the rat retina. Br J Ophthalmol 22:613, Lucus DR, Attfield M, and Davey JB: Retinal dystrophy in the rat. J Path Bact 70:469, Dowling JE and Sidman RL: Inherited retinal dystrophy in the rat. J Cell Biol 14:73, Noell WK: Cellular physiology of the retina. J Opt Soc Am 53:36, Bok D and Hall MO: The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J Cell Biol 49:664, Noell WK: Aspects of experimental and hereditary retinal degeneration. In Biochemistry of the Retina, Graymore CN, editor. New York, Academic Press, 1965, pp La Vail MM, Sidman RL, and O'Neil D: Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J Cell Biol 53:185, La Vail MM and Battelle B-A: Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat. ExpEye Res 21:167, Noell WK, Stockton RA, Spongr V, and Braniecki MA: Visual capabilities in advanced retinal degeneration. I: In the rdy (RCS) rat. ARVO Abstracts. Invest Ophthalmol Vis Sci 20(Suppl):238, Cicerone CM, Green DG, and Fisher LJ: Cone inputs to ganglion cells in hereditary retinal degeneration. Science 203:1113, La Vail MM, Sidman M, Rausin R, and Sidman RL: Discrimination of light intensity by rats with inherited retinal degeneration. Vision Res 14:693, Kaitz M: The effect of light on brightness perception in rats with retinal dystrophy. Vision Res 16:141, Schnitzer SB, Noell WK, and Zezelic M: Discrimination of rectangular orientation by the rdy (RCS) rat at an advanced stage of visual cell degeneration. ARVO Abstracts. Invest Ophthalmol Vis Sci 20(Suppl):238, Noell WK, Salinsky MC, Stockton RA, Schnitzer SB, and Kan V: Electrophysiological studies of the visual capacities at advanced stages of photoreceptor degeneration in the rat. Doc Ophthalmol Proc Ser 27:175, Hanks JH and Wallace RE: Relation of oxygen and temperature in the preservation of tissues by refrigeration. Proc Soc Exp Biol Med 71:196, Karnovsky MJ: Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J Cell Biol 51:146A, La Vail MM: The retinal pigment epithelium in mice and rats with inherited retinal degeneration. In The Retinal Pigment Epithelium, Zinn KM and Marmor MF, editors. Cambridge, Harvard University Press, 1979, pp Rodieck RW: The Vertebrate Retina. San Francisco, Freeman and Company, 1973.