Pigment epithelial ensheathment and phagocytosis of extrafoveal cones in human retina.

The association between extrafoveal cone outer segments and pigment epithelial cells was studied by transmission electron microscopy in three human retinas; ages 5,45 and 60. The pigment epithelial apical surface from a fourth human retina, age 38,was viewed in the scanning electron microscope. Multiple villous-like apical processes protrude from the pigment epithelium into the space above each cone. Sometimes one or more of these processes is sheet-like in form and contains a wealth of intracellular organelles, including mitochondria. One or more of the villous-like procesess reaches the cone and expands to ensheath the upper one-third of the outer segment. Llike vertebrate rods, extrafoveal human cones shed their terminal disks in packets and these packets are phagocytosed by the ensheathing apical processes. The phagosomes then ascend in the processes toward the pigment epithelia soma. Digestion of phagosomes appears to begin in the apical processes.     

Relationship of cholesterol content to spatial distribution and age of disc membranes in retinal rod outer segments

The initial events of visual transduction occur on disc membranes which are sequestered within the photoreceptor outer segment. In rod cells, the discs are stacked in the outer segment. Discs are formed at the base of the rod outer segment (ROS) from evaginations of the plasma membrane. As new discs form, older discs move toward the apical tip of the rod, from which they are eventually shed and subsequently phagocytosed by the adjacent pigment epithelium. Thus, disc membranes within a given rod cell are not of uniform age. We have recently shown that disc membranes are not homogeneous with respect to cholesterol content (Boesze-Battaglia, K., Hennessey, T., and Albert, A. D. (1989) J. Biol. Chem. 264, 8151-8155). In the present study, freshly isolated bovine retinas were incubated with [3H]leucine for 4 h in order to allow sufficient time for the radiolabeled proteins to become incorporated into the basal-most (newest) discs. Osmotically intact discs were then isolated. After the addition of digitonin, the discs were fractionated based on cholesterol content, and radioactivity (indicative of newly synthesized protein) was measured. Discs which exhibited high cholesterol content also exhibited high radio-activity. These results demonstrate that the cholesterol heterogeneity of ROS disc membranes is related to the age, and thus the position, of the discs in the ROS.     

A study of photoreceptor-retinal pigment epithelium complex: age-related changes in monkeys

Retinas of 4-, 10-, and 20-year-old monkeys were studied by light microscopy, electron microscopy, and scanning electron microscopy. Sections from the midperipheral region of every retina were selected for comparison. Although no significant differences were found between 4- and 10-year-old retinas, four major changes were found in 20-year-old monkey retinas: (i) increased number of displaced photoreceptor cells (DPC), (ii) increased number of macrophages of different morphology in subretinal space, (iii) increase in pigment granules in retinal pigment epithelium (RPE) cells, and (iv) altered morphology of Muller cells. DPC included both rods and cones. Their location and morphology depended on the stage of their displacement. These cells were usually oval or rounded in shape and were found either among the outer segments of other photoreceptor cells, having stalks extending into the outer nuclear layer, or were located in the subretinal space and had no stalk. A narrow space around the DPC stalks, indicating a change in the intercellular connection between photoreceptor cells and Muller cells, was observed. Furthermore, the Muller cells related to DPC had shortened and markedly reduced microvilli. Two types of macrophages were found in the subretinal space of aged monkey retinas. One type was similar in morphology to RPE cells. Some of these cells were noticed detaching from RPE. Other types of macrophages were nonpigmented. The modifications in RPE were closely related to the changes in the associated neuroretina. The RPE cells in aged retina were devoid of microvilli or had a few thin microvilli. The pleomorphic pigment granules were dispersed throughout the cytoplasm. These cells varied in their size, shape, and surface features. These changes could significantly alter the retinal metabolic equilibrium and may be indicative of age related degenerative processes.     

Membrane turnover in rod photoreceptors: ensheathment and phagocytosis of outer segment distal tips by pseudopodia of the retinal pigment epithelium

We have documented the ultrastructural changes that occur within the photoreceptor outer segment and the retinal pigment epithelium (RPE) during photosensitive membrane turnover. We employed an in vitro eyecup preparation from Xenopus laevis in which a large shedding event was induced by adding the excitatory amino acid L-aspartate (Greenberger & Besharse 1985; J. comp. Neurol. 239, 361-372). We found that during L-aspartate-induced shedding the RPE cells formed, on their apical domains, previously undescribed processes that were directly involved in disc phagocytosis. These processes are structurally similar to processes formed by macrophages during phagocytosis and are accordingly referred to as pseudopodia. Pseudopodia were distinguishable from the apical villous process normally extended from the RPE in that they were closely applied to the surface of the outer segment, had a cytoplasmic matrix of low electron density that was devoid of most cellular organelles and were enriched in thin (7 nm diameter) filaments. Filament size, specific pseudopodial staining with the actin-specific probe rhodamine phalloidin and inhibition of pseudopod formation by cytochalasin D suggested that the thin filaments were composed of actin. Pseudopodial formation also occurs during a normal light-initiated shedding event. However, the low frequency of shedding, the asynchrony of the individual shedding events and the transient appearance of the pseudopodia prevented a full appreciation of their role during normal disc shedding. Associated with massive shedding and pseudopodial formation, there was an increased adherence between retina and RPE. During L-aspartate treatment, the apical portions of the RPE cells partitioned with the distal outer segment during retinal isolation. This effect was directly related to the development of pseudopodia and may reflect alteration of surface features of the rod outer segment (ROS)-RPE interface related to phagocytosis. Our observations show that transiently forming pseudopodia are the organelles of phagocytosis and that they may play a role in disc detachment as well.     

The retinal pigment epithelium and photoreceptor cells light-and electron microscopic studies on monkey eyes

Summary In the perifoveal retina of the monkey, Cercopithecus aethiops, the melanin granules are accumulated in apical cytoplasmatic protrusions of the pigment epithelial cells, facing the end of the cones. The rods are inserted deeper into the pigment epithelium than the cones; they reach the bottom of the infoldings of the apical surface membrane of the pigment epithelial cells. No melanin granules or other inclusions are situated at the end of the rods. The outer extremity of the rods is considerably inclined and in sections often appears as groups of rod discs which are incompletely or completely separated from the main part of the outer segments. This separation is regarded as an artifact caused by the inclination of the rods, and it is therefore not considered to represent phagocytosis of the outer segments by the pigment epithelium.The inclusions of the pigment epithelial cells are classified in five categories which seem to be related to each other owing to their shared structural characteristics. It is suggested that melanin granules are produced, modified and destroyed by the pigment epithelial cells of the adult.Because of the relations between the photoreceptors and the melanin granules it is suggested that light scattered by the melanin granules may pass backwards through the outer segments of the cones, but not of the rods.This investigation was supported in part by the Danish Foundation for the Advancement of Science and by the Danish Medical Research Council.     

Primary structure of a visual pigment in bullfrog green rods

In frog retina there are special rod photoreceptor cells ('green rods') with physiological properties similar to those of typical vertebrate rods ('red rods'). A cDNA fragment encoding the putative green rod visual pigment was isolated from a retinal cDNA library of the bullfrog, Rana catesbeiana. Its deduced amino acid sequence has more than 65% identity with those of blue-sensitive cone pigments such as chicken blue and goldfish blue. Antisera raised against its C-terminal amino acid sequence recognized green rods. It is concluded that bullfrog green rods contain a visual pigment which is closely related to the blue-sensitive cone pigments of other non-mammalian vertebrates.     

Intracellular topography of rhodopsin regeneration in vertebrate rods

The vertebrate visual pigment of rods, rhodopsin, bleaches in light and regenerates in darkness. When the bleaching and regeneration are carried out in vivo, it is found that the regeneration takes place at nonuniform rates along the rod outer segment (ROS): toads and frogs regenerate rhodopsin faster in the proximal ends of the ROS than in the distal ends. Rats do the reverse. These patterns of regeneration persist whether the bleaching is done with flashes or with steady light. They are also independent of the extent to which the retinal pigment epithelium contains melanin. Furthermore, the dichotomy of patterns (proximal faster vs. distal faster) does not seem to depend upon the presence of an excess of stored retinoid in the eye. Instead, it is suggested that the villous processes of the epithelial cells may play an important role in the regeneration patterns. These processes in amphibia extend nearly to the rod inner segment but in the rat they surround only the apical end of the outer segment. If they "funnel" the retinoids back to the ROS, their location and morphology could explain the two different kinds of patterns seen.     

Rhythmic daily shedding of outer-segment membranes by visual cells in the goldfish

Goldfish were placed on a daily light cycle of 12 h light and 12 h darkness for 18 days or longer. The visual cells and pigment epithelium of the retina were then examined by microscopy at many intervals throughout the cycle. Goldfish rods and cones follow a rhythmic pattern in eliminating packets of photosensitive membranes from their outer segments. Rods shed membranes early in the light period. The detached membranes are ingested by pigment epithelial cells or by ameboid phagocytes, which degrade them during the remainder of the light period. Cones discard membranes from the ends of their outer segments early in the dark period. During the next several hours, this debris is digested by the pigment epithelium or by ameboid phagocytes. Thus, the disposal phase of the outer-segment renewal process is similar in rods and cones, but is displaced in time by about 12 h. There is evidence that this daily rhythm of membrane disposal in rods and cones is a general property of vertebrate visual cells.     

Evidence for melanogenesis in the retinal pigment epithelium of adult cattle and golden hamster.

1. The ultrastructure of the retinal pigment epithelium (RPE) of adult Syrian golden hamsters and cattle was examined with respect to pigment granules and phagosomes involved in degradation of disk membranes from rod outer segments. 2. In the RPE of cattle, phagosomes were found that contained an electron-dense melanin-like material that was not autofluorescent and therefore not lipofuscin. 3. Disk membranes of rods are about 4 nm thick and become enlarged (7-20 nm) and electron-dense during degradation in the RPE. 4. Additionally electron-dense vesiculo-globular bodies (10-100 nm) were found in phagosomes during disk membrane degradation and in mature melanin granules. 5. In the RPE of adult hamsters that had been exposed to intense light, premelanosomes containing unmelanised filaments with a striated periodicity were found in the cytoplasm or in association with mature melanin granules. Early and late stage melanosomes were also present. Phagosomes in the RPE contained degraded disk membranes, melanin-like material and melanofilaments. 6. Dopa oxidase was detected ultrastructurally within shed disk membranes that were in close contact with the microvilli of the RPE. 7. The possibility of melanogenesis within phagosomes during disk membrane degradation is discussed.     

Dystrophin in rod spherules; submembranous dense regions facing bipolar cell processes

 It is known that the retina contains the protein dystrophin in the ribbon synapse, but the ultrastructural analysis is not yet fully elucidated. Our previous study reported that dystrophin is localized under the rod cell membranes in rat retinas. In the present study, we have investigated the relationship between dystrophin-rich regions of rod cell membranes and other neuronal processes in mouse retinas with a monoclonal antibody raised against the human dystrophin C-terminus. Immunoblotting, immunofluorescence stainings, and immunoelectron microscopy were employed. Immunoblotting analysis indicated that mouse retinas possessed some of the dystrophin isoforms of approximately 260 kDa, 140 kDa, and 70 kDa molecular weight. Confocal images showed a punctate appearance in the outer plexiform layer, as previously described. Immunoelectron microscopy showed that dystrophin immunoreactive products were always observed at submembranous dense regions of the rod spherule abutting bipolar processes. These results suggest that retinal dystrophin may be closely involved in signal transmission from rods to bipolar cells. Accepted: 7 May 1997     

Scanning electron microscopy of the bullfrog's retina and pigment epithelium

Summary The retina and pigment epithelium of the bullfrog (Rana catesbiana) were studied with the scanning electron microscope. Fixed-dehydrated tissues were critical point dried with CO₂, then cracked in the plane of the long axis of the photoreceptors. The cellular layers of the retina and the lateral surfaces of pigment epithelial cells were visualized. The four major types of frog photoreceptor were identified: red rod, green rod, single cone, and double cone. Cone myoids were observed to be contracted in light-adapted retinas and elongated in more dark adapted retinas.This work was supported by a career development award EY-18,083 to the author and research grant EY 00468 to Dr. Kenneth T. Brown.The author gratefully acknowledges the skillful technical assistance of Ms. Maria T. Maglio.     

Synaptic activity of frog retinal photoreceptors. A peroxidase uptake study

The uptake of horseradish peroxidase (HRP) into membranous structures, detectable by light and electron microscopy, is used here to monitor the synaptic activity of photoreceptors of isolated frog retinas maintained in the dark or under various illumination conditions. The major findings are: (a) Neurotransmission from photoreceptor terminals seems to involve the same types of endocytic membrane-retrieval processes that occur at other nerve terminals. Presumably, the endocytic processes compensate for exocytic events associated with neurotransmission. The retrieved membrane is "recycled" to form vesicles. Some of these accumulate near the synaptic ribbons, perhaps indicating reutilization for exocytosis. On the other hand, some retrieved membrane evidently is degraded via multivesicular bodies that appear to undergo "retrograde" transport from the receptor synapses to the myoid regions. (b) Photoreceptor terminals take up much HRP in the dark. Steady illumination markedly decreases uptake by rods. Uptake by cones is notably reduced only at illumination intensities higher than those that have maximal effects on rods. (c) The decrease in rod HRP uptake with light is reversible when retinas are allowed to adapt to the dark, if the light exposures used were at intensities that bleach very little visual pigment. Such "recovery" is not observed after light exposures that bleach a considerable amount of visual pigment. The cones recover their dark levels of HRP uptake even after light exposures that bleach considerable amounts of visual pigment. The changes in HRP uptake that we observe parallel expectations for photoreceptor synaptic neurotransmission derived from indirect physiological evidence.     

THE VISUAL CELLS AND VISUAL PIGMENT OF THE MUDPUPPY, NECTURUS

Electron microscopy of the visual cells of the mudpuppy Necturus have revealed several new or hitherto neglected features of organization: (a) A system of deeply staining micelles in virtually crystalline array, probably located in the lamellae of the rod outer segments. These particles may contain the visual pigment, porphyropsin. Counts of the micelles, and microspectrophotometric measurements of porphyropsin in the retina and single rods yield the estimate that each lamellar micelle may contain about 50 molecules of porphyropsin. (b) Systems of about 30 cytoplasmic filaments (here called dendrites), continuous with the cytoplasm of the inner segment, and standing like a palisade about the outer segments of the rods and cones. In the rods, one such filament stands in the mouth of each of the approximately 30 deep fissures that carve the outer segment into a radial array of lobules. (c) A system of deeply staining particles in the membranes of the dendrites, and another in the membranes of the pigment epithelial processes. It is suggested that these may have a part in interchanges of material with the outer segments. The ciliary process is found to penetrate more deeply than is commonly supposed into the outer segments of the rods and cones. The edge of each double-membrane disc in rods forms a differentiated rim structure, both around the disc circumference and bordering the fissures. These anatomical arrangements are summarized in Figs. 13 and 14, and the relevant measurements in Table I. The dilution of visual pigment in Necturus rods and cones and a general consideration of their microstructures make it seem unlikely that such typically solid state processes as exciton migration or photoconduction can transport the effects of light far from the site of absorption. Excitation must, therefore, be conveyed to the receptor as a whole by some axial structure. Among axial structures, the plasma membrane is most likely to be the site of nervous excitation. The ciliary process probably plays its main role in the embryogenesis and regeneration of outer segments; and the dendrites and pigment epithelial processes in exchanges of material with the outer segments and perhaps with one another.     

THE RENEWAL OF ROD AND CONE OUTER SEGMENTS IN THE RHESUS MONKEY

The renewal of retinal rod and cone outer segments has been studied by radioautography in rhesus monkeys examined 2 and 4 days after injection of leucine-³H. The cell outer segment consists of a stack of photosensitive, membranous discs. In both rods and cones some of the newly formed (radioactive) protein became distributed throughout the outer segment. Furthermore, in rods (but not in cones), there was a transverse band of concentrated radioactive protein slightly above the outer segment base 2 days after injection. This was due to the formation of new discs, into which labeled protein had been incorporated. At 4 days, these radioactive discs were located farther from the outer segment base. Repeated assembly of new discs had displaced them away from the basal assembly site and along the outer segment. Measurements of the displacement rate indicated that each retinal rod produces 80–90 discs per day, and that the entire complement of outer segment discs is replaced every 9–13 days. To compensate for the continual formation of new discs, groups of old discs are intermittently shed from the apical end of the cell and phagocytized by the pigment epithelium. Each pigment epithelial cell engulfs and destroys about 2000–4000 rod outer segment discs daily. The similarity between visual cells in the rhesus monkey and those in man suggests that the same renewal processes occur in the human retina.     

Dopamine Induces Light-Adaptive Retinomotor Movements in Bullfrog Cones via D2 Receptors and in Retinal Pigment Epithelium via D1 Receptors

In the eyes of lower vertebrates, retinal photoreceptors and melanin pigment granules of the retinal pigment epithelium (RPE) exhibit characteristic retinomotor movements in response to changes in ambient illumination and to signals from an endogenous circadian clock. We previously reported that 3,4-dihydroxyphenylethylamine (dopamine) mimicked the effect of light on these movements in photo-receptors and RPE cells of green sunfish, Lepomis cyanellus, by interacting with D2 dopaminergic receptors. Here, we report that dopamine also mimics the effect of light on cone and RPE retinomotor movements in bullfrogs, Rana catesbeiana, i.e., dopamine induces cone contraction and RPE pigment dispersion. Dopamine induced cone contraction in isolated dark-adapted bullfrog retinas incubated in constant darkness in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). This effect of dopamine was inhibited by a D2 but not a D1 antagonist and mimicked by a D2 but not a D1 agonist. These results suggest that induction of cone contraction by dopamine is mediated by D2 dopaminergic receptors and that cone adenylate cyclase activity is inhibited. Thus, dopamine acts via the same type of receptor in both bullfrog and green sunfish retinas to induce cone contraction. In contrast, dopamine influences RPE retinomotor movement via different receptors in fish and bullfrog. Dopamine induced light-adaptive pigment dispersion in isolated dark-adapted bullfrog RPE-eyecups incubated in constant darkness in normal Ringer's solution. Because the retina was not present, these experiments demonstrate a direct effect of dopamine on bullfrog RPE. This effect of dopamine on bullfrog RPE was inhibited by a D1 but not a D2 antagonist and mimicked by a D1 but not a D2 agonist. Furthermore, agents that increase the concentration of intracellular cyclic AMP also induced pigment dispersion in dark-adapted bullfrog RPE-eyecups incubated in the dark. These results suggest that dopamine induces pigment dispersion in bullfrog RPE via D1 dopaminergic receptors. Thus, dopamine acts via different receptors on bullfrog (D1) versus green sunfish (D2) RPE to induce pigment dispersion. In addition, inhibitor studies indicate that pigment dispersion is actin dependent in teleost but not in bullfrog RPE. Dopamine-induced pigment dispersion was inhibited by cytochalasin D in isolated RPE sheets of green sunfish but not in RPE-eyecups of bullfrogs. Together, these observations indicate that dopamine mimics the effect of light on cone and RPE retinomotor movements in both fish and bullfrogs. However, in the RPE, different receptors mediate the effect of dopamine, and different cytoskeletal mechanisms are used to affect pigment transport.(ABSTRACT TRUNCATED AT 400 WORDS)     

视网膜生物钟研究进展

昼夜节律生物钟是以24h为周期的自主维持的振荡器。在高等的多细胞生物中,生物钟可以分为母钟和子钟。研究表明哺乳动物的母钟位于下丘脑视交叉上核(suprachiasmatic nucleus,SCN),由此发出信息控制全身的节律活动;子钟位于组织细胞内,调控效应器的节律。在分子水平上,生物钟的振荡由自身调控反馈环路的转录和翻译组成,并接受外界环境因素的影响,通过下丘脑视叉上核(Suprachiasmatic Nucleus,SCN)中枢震荡器的同步整和而产生作用。视网膜是一种十分节律性的组织,许多生化的、细胞的和生理的过程都是以节律的方式来进行的,如视觉灵敏度、视网膜杆细胞外片层脱落和视网膜色素上皮细胞的吞噬作用、光受体中的视觉色素基因的快速表达等。生物钟存在于很多脊椎动物的视网膜中,被认为是一种外周生物钟。本文综述了视网膜生物钟,生物钟信号传输以及生物钟网络等的最新研究进展。     

Light-Induced Dopamine Release from Teleost Retinas Acts as a Light-Adaptive Signal to the Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules migrate in and out of the cells' long apical projections in response to changes in light condition. When the RPE is in its normal association with the retina, light onset induces pigment granules to disperse into the apical projections; dark onset induces pigment granules to aggregate into the cell bodies. However, when the RPE is separated from the retina, pigment granule movement in the isolated RPE is insensitive to light onset. It thus seems likely that a signal from the retina communicates light onset to the RPE to initiate pigment dispersion. We have examined the nature of this retina-to-RPE signal in green sunfish, Lepomis cyanellus. In isolated retinas with adherent RPE, light-induced pigment dispersion in the RPE is blocked by treatments known to block Ca2+-dependent transmitter release in the retina. In addition, the medium obtained from incubating previously dark-adapted retinas in the light induces light-adaptive pigment dispersion when added to isolated RPE. In contrast, the medium obtained from incubating dark-adapted retinas in constant darkness does not affect pigment distribution when added to isolated RPE. These results are consistent with the idea that RPE pigment dispersion is triggered by a substance that diffuses from the retina at light onset. The capacity of the conditioned medium from light-incubated retinas to induce pigment dispersion in isolated RPE is inhibited by a D2 dopamine antagonist, but not by D1 or alpha-adrenergic antagonists. Light-induced pigment dispersion in whole RPE-retinas is also blocked by a D2 dopamine antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultimate fate of rod outer segments in the retinal pigment epithelium.

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Ultimate Fate of Rod Outer Segments in the Retinal Pigment Epithelium

To investigate the degradation pathway of rod outer segments (ROS) in vivo, we injected gold-labeled ROS into the subretinal space of rabbits using a pars plana approach. Histology and electron microscopy performed on the specimens 72 hr after ROS injection revealed that the retina over the injection site was reattached, the retinal pigment epithelial (RPE) cells were intact, and gold granules were localized inside melanin granules and melanosomes. These results indicate that, in RPE, in vivo degradation of ROS is associated with melanosomes.     

Pigment cells trigger the onset of gastrulation in tropical sea urchin Echinometra mathaei

In the tropical sea urchin Echinometra mathaei, pigment cells are just detectable before the onset of gastrulation, owing to an early accumulation of red pigment granules. Taking advantage of this feature, behavior of pigment cells was studied in relation to the processes of gastrulation. Before the initiation of primary invagination, pigment cells were arranged in a hemi-circle in the dorsal half of the vegetal plate. Inward bending of the vegetal plate first occurred at the position occupied by pigment cells, while the bending was not conspicuous in the ventral half of the blastopore. Rhodamine-phalloidin staining showed that actin filaments were abundant at the apical corticies of pigment cells. It was also found that the onset of gastrulation was considerably delayed in the NiCl2-treated embryos, in which pigment cells were drastically reduced in number. It is notable that the NiCl2-treated embryos began to gastrulate on schedule if they contained a number of pigment cells in spite of treatment. This shows that pigment cells are the bottle cells that trigger the onset of gastrulation. In the embryos devoid of pigment cells, a short stub-like gut rudiment formed in a delayed fashion, and several secondary mesenchyme cells (SMC) appeared at the tip of the rudiment and elongated gradually until its tip reached the apical plate. This observation suggests that the SMC that pull the gut rudiment upward are not pigment cells but blastocoelar cells, because pigment cells change their fate to blastocoelar cells upon NiCl2-treatment.