Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.     

绿色荧光蛋白基因修饰的人视网膜色素上皮细胞异种移植的长期观察

探讨利用视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞移植介导目的基因转移至视网膜的可行性。人的RPE细胞在体外经携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒感染及G4 1 8筛选后 ,手术显微镜直视下经睫状体平坦部注射到兔眼视网膜下间隙。术后通过活体荧光眼底照相 ,荧光显微镜、共聚焦显微镜及透射电镜等观察眼球铺片及切片 ,发现经 gfp基因修饰的人RPE细胞在兔眼视网膜下间隙可存活一年以上。移植的RPE gfp细胞不仅仅局限于移植部位 ,大多扩散到超过 2～ 3个象限的眼底 ,镶嵌于宿主RPE细胞之间或呈单层排列在宿主色素上皮与神经视网膜之间 ,并持续高水平表达GFP。移植后早期玻璃体内每周注射免疫抑制剂普乐可复 (FK5 0 6 )可明显改善移植细胞的存活状态     

Evidence of decreased adhesion between the neural retina and retinal pigmented epithelium of the Mitf vit (vitiligo) mutant mouse

In order for the retina to function properly, photoreceptor cell outer segments must be in contact with the adjacent retinal pigmented epithelium (RPE). A mouse model homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene manifests disruption of the outer segment/RPE interdigitation and demonstrates progressive loss of the photoreceptor cells. The mouse nevertheless has near normal levels of rhodopsin for many weeks and it is not known whether there is an in vivo loss of adhesion or whether the disruption is visible following tissue processing for histology. To assess this, a mechanical separation experiment was performed in which neural retinas were peeled free from the RPE and examined for the amount of pigment adherent to them. The peeling experiment indicated that control neural retinas retained significant amounts of adherent pigment at all ages examined. Neural retinas of mutant mice at age 2 weeks demonstrated adherent pigment, but older animals retained minimal pigment. Scanning electron microscopy indicated that the RPE cells of control mice were markedly damaged upon peeling and displayed different planes of cleavage, whereas those of mutants showed minimal cellular damage upon peeling, suggestive of decreased adhesion. A recombination experiment revealed that the mutant RPE/eyecup could reappose mutant and control retinas under in vitro conditions, suggesting that RPE fluid transport abilities were intact. The data provide the first direct experimental evidence that the Mitf^vit mutant mouse has a naturally occurring retinal detachment and hence support its value as a model for studies of retina/RPE adhesion.     

The localization of glutathione peroxidase in the photoreceptor cells and the retinal pigment epithelial cells of Wistar and Royal College of Surgeons dystrophic rats

INTRODUCTION: If degenerating photoreceptor outer segments not phagocytized by RPE cells in the retina of Royal College Surgeons (RCS) rats were to undergo peroxidation, the distribution of glutathione peroxidase (GSH-PO) in the mitochondria or cytoplasm of the retina might be altered. We evaluated the immunocytochemical localization of GSH-PO to identify subcellular organelles in sections of the retinas of RCS rats. METHODS: Immunoblot analysis confirmed the presence of GSH-PO molecules in the retinas of RCS and Wistar rats aged 3 weeks. Sections were reacted with the F(ab) fragment of anti-rat alphaGSH-PO and then examined by laser scanning microscopy (LSM) and transmission electron microscopy (TEM). RESULTS: The size of the GSH-PO molecule in the retina was about 21 KD in the mitochondria and 23 KD in the cytosol in both strains of rats. LSM revealed fluorescent granules in the photoreceptor inner segments of the Wistar rats, and immunohistochemical TEM revealed GSH-PO in the mitochondria of their photoreceptor inner segments and retinal pigment epithelial (RPE) cells. In the RCS rats, the degenerating photoreceptor outer segments were clearly seen to be positive for anti-GSH-PO by conventional light microscopy (CLM). However, the photoreceptor inner segments of the RCS rats were negative for staining with anti-GSH-PO by LSM, and no GSH-PO could be detected in the mitochondria of the photoreceptor inner segments or RPE cells by immuno-TEM. CONCLUSION: Degeneration of the photoreceptor outer segments induced mitochondrial damage in the photoreceptor inner segments, and as a result GSH-PO shifted from the photoreceptor inner segments to the degenerating outer segments.     

Uptake dynamics and retinal tolerance of phosphorothioate oligonucleotide and its direct delivery into the site of choroidal neovascularization through subretinal administration in the rat

This study aimed to investigate uptake dynamics and retinal tolerance of phosphorothioate oligonucleotides (PS-oligos) following subretinal injection. A fluorescent-labeled PS-oligo (FL-oligo) with random sequence was administered into the subretinal space of rat by transsclera-choroid-retinal pigment epithelium (RPE) injection at doses of 0.129, 1.29, and 12.9 microg in 2.0 microl solution. The uptake dynamics were evaluated by fundus fluorescent photography in real time and by fluorescence microscopy using flat mounts and cryosections. Immunophenotyping for CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages was performed to assess cellular infiltration in the retina. In addition, the FL-oligo was injected subretinally in a rat model of choroidal neovascularization (CNV) for direct delivery into the site of CNV. Subretinal administration of FL-oligo resulted in both dose-dependent and time-dependent distribution in the retina, where it accessed the RPE and all layers of the neuroretina. CD4+, CD8+ cytotoxic lymphocytes, and CD68+ macrophages were observed at the site of needle penetration. However, in areas far from the injection site where the FL-oligo appeared strongly, cellular infiltration was absent, and the retinal morphology was preserved very well. The FL-oligo was successfully delivered into the site of intense laser photocoagulation. It was predominantly localized to the RPE, macrophages, and some choroid cells and remained detectable for at least 56 days after injection. Our results demonstrate for the first time that subretinal injection efficiently introduced PS-oligo into the RPE and neuroretina with an acceptable level of safety. Subretinal administration of antiangiogenic oligonucleotides may hold great potential for the treatment of CNV.     

In vivo adenovirus-mediated gene transfer to newborn rat retinal pigment epithelial cells

A successful surgical access to the subretinal space is critical for achieving adenovirus-mediated gene transfer to the retinal pigment epithelial (RPE) cells or photoreceptor cells. We report a novel surgical approach allowing an efficient delivery of recombinant replication-deficient adenoviral vectors into the subretinal space of newborn rats. Our data suggest that this method may be useful for infecting reproducibly large areas of the RPE cell layer of normal newborn rats and should be applicable to RCS pups. We also show the feasibility of infecting ex vivo RPE cells in culture using the same recombinant adenoviral vector.     

Biocompatibility of subretinal parylene-based Ti/Pt microelectrode array in rabbit for further artificial vision studies

To evaluate the biocompatibility of subretinal implanted parylene-based Ti/Pt microelectrode arrays (MEA). Eyes were enucleated 3 months after MEAs were implanted into the subretinal space of rabbits. Morphological changes of the retinas were investigated by H&;E staining. Immunohistochemical staining for glial fibrillary acidic protein and opsin were performed to evaluate changes in Muller cells and photoreceptors in the retinas. Retina tissue around the array remained intact. Photoreceptor degeneration and glial cell activation were observed in the retina overlaying the MEA implant. However, the cells in the inner retinal layers were preserved. Photoreceptor degeneration and glial cell activation at the MEA–retina interface are expected to be a normal reaction to implantation. Material used in this experiment has good biocompatibility within the subretinal environment and is expected to be promising in the further retinal prosthesis studies.     

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.     

Ultrastructural localization of blood-retinal barrier breakdown in diabetic and galactosemic rats

Breakdown of the blood-retinal barrier (BRB) is an early event in diabetic and galactosemic rats, but the location and nature of the specific defect(s) are controversial. Using an electron microscopic immunocytochemical technique, the retinas of normal, diabetic, and galactosemic rats were immunostained for endogenous albumin. Normal rats showed little evidence of BRB breakdown at either the inner barrier (retinal vasculature) or the outer barrier (retinal pigment epithelium) (RPE). In diabetic and galactosemic rats, as was true in human diabetics, BRB breakdown occurred predominantly at the inner BRB, but in some cases at the outer barrier as well. Treatment with the aldose reductase inhibitor sorbinil largely prevented BRB failure in galactosemic rats. In the inner retina of diabetic and galactosemic rats, albumin was frequently demonstrated on the abluminal side of the retinal capillary endothelium (RCE) in intercellular spaces, basal laminae, pericytes, ganglion cells, astrocytes, and the perinuclear cytoplasm of cells in the inner nuclear layer. Albumin did not appear to cross RCE cell junctions; however, it was occasionally seen in RCE cytoplasm of galactosemic rats. In the outer retina, albumin was frequently detected in the subretinal space, in the intercellular space between photoreceptors, and in the perinuclear cytoplasm of photoreceptor cells, but was only infrequently found in the RPE cells constituting the barrier. Albumin derived from the choroidal vasculature did not appear to cross the tight junctions of the RPE. These findings suggest that specific sites of BRB compromise are infrequent but that once albumin has crossed the RCE or RPE it freely permeates the retinal tissue by filling intercellular spaces and permeating the membranes of cells not implicated in BRB formation. The diffuse cytoplasmic staining of some RCE and RPE cells suggests that the predominant means of BRB breakdown in diabetes and galactosemia involves increased focal permeability of the surface membranes of the RCE and RPE cells rather than defective tight junctions or vesicular transport.     

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.     

Purinergic signalling in the subretinal space: a role in the communication between the retina and the RPE

The retinal pigment epithelium (RPE) is separated from the photoreceptor outer segments by the subretinal space. While the actual volume of this space is minimal, the communication that occurs across this microenvironment is important to the visual process, and accumulating evidence suggests the purines ATP and adenosine contribute to this communication. P1 and P2 receptors are localized to membranes on both the photoreceptor outer segments and on the apical membrane of the RPE which border subretinal space. ATP is released across the apical membrane of the RPE into this space in response to various triggers including glutamate and chemical ischemia. This ATP is dephosphorylated into adenosine by a series of ectoenzymes on the RPE apical membrane. Regulation of release and ectoenzyme activity in response to light-sensitive signals can alter the balance of purines in subretinal space, and thus coordinate communication across subretinal space with the visual process.     

Age-dependent accumulation of lipofuscin in perivascular and subretinal microglia in experimental mice

Fundus autofluorescence (AF) imaging by confocal scanning laser ophthalmoscopy has been widely used by ophthalmologists in the diagnosis/monitoring of various retinal disorders. It is believed that fundus AF is derived from lipofuscin in retinal pigment epithelial (RPE) cells; however, direct clinicopathological correlation has not been possible in humans. We examined fundus AF by confocal scanning laser ophthalmoscopy and confocal microscopy in normal C57BL/6 mice of different ages. Increasingly strong AF signals were observed with age in the neuroretina and subretinal/RPE layer by confocal scanning laser ophthalmoscopy. Unlike fundus AF detected in normal human subjects, mouse fundus AF appeared as discrete foci distributed throughout the retina. Most of the AF signals in the neuroretina were distributed around retinal vessels. Confocal microscopy of retinal and choroid/RPE flat mounts demonstrated that most of the AF signals were derived from Iba-1+ perivascular and subretinal microglia. An age-dependent accumulation of Iba-1+ microglia at the subretinal space was observed. Lipofuscin granules were detected in large numbers in subretinal microglia by electron microscopy. The number of AF+ microglia and the amount of AF granules/cell increased with age. AF granules/lipofuscin were also observed in RPE cells in mice older than 12 months, but the number of AF+ RPE cells was very low (1.48 mm(-2) and 5.02 mm(-2) for 12 and 24 months, respectively) compared to the number of AF+ microglial cells (20.63 mm(-2) and 76.36 mm(-2) for 6 and 24 months, respectively). The fluorescence emission fingerprints of AF granules in subretinal microglia were the same as those in RPE cells. Our observation suggests that perivascular and subretinal microglia are the main cells producing lipofuscin in normal aged mouse retina and are responsible for in vivo fundus AF. Microglia may play an important role in retinal aging and age-related retinal diseases.     

A Highlights from MBoC Selection: Chloride Intracellular Channel 4 Is Critical for the Epithelial Morphogenesis of RPE Cells and Retinal Attachment

Retinal detachment is a sight-threatening condition. The molecular mechanism underlying the adhesion between the RPE and photoreceptors is poorly understood because the intimate interactions between these two cell types are impossible to model and study in vitro. In this article, we show that chloride intracellular channel 4 (CLIC4) is enriched at apical RPE microvilli, which are interdigitated with the photoreceptor outer segment. We used a novel plasmid-based transfection method to cell-autonomously suppress CLIC4 in RPE in situ. CLIC4 silenced RPE cells exhibited a significant loss of apical microvilli and basal infoldings, reduced retinal adhesion, and epithelial-mesenchymal transition. Ectopically expressing ezrin failed to rescue the morphological changes exerted by CLIC4 silencing. Neural retinas adjacent to the CLIC4-suppressed RPE cells display severe dysplasia. Finally, a high level of aquaporin 1 unexpectedly appeared at the apical surfaces of CLIC4-suppressed RPE cells, together with a concomitant loss of basal surface expression of monocarboxylate transporter MCT3. Our results suggested that CLIC4 plays an important role in RPE-photoreceptor adhesion, perhaps by modulating the activity of cell surface channels/transporters. We propose that these changes may be attributable to subretinal fluid accumulation in our novel retinal detachment animal model.     

Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size

Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.     

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.     

Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses

Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.     

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.     

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)