TAM receptor function in the retinal pigment epithelium

The TAM receptor tyrosine kinase Mer is expressed by cells of the retinal pigment epithelium (RPE), and genetic studies have demonstrated that Mer is essential for RPE function. RPE cells that lack Mer exhibit a severely compromised ability to phagocytose the distal ends of photoreceptor (PR) outer segments, which leads to the complete postnatal degeneration of photoreceptors and to blindness. Although in vitro experiments have implicated Gas6 as the critical TAM ligand for this process, we find that Gas6 mutant mice have a histologically intact retina with no photoreceptor degeneration. We further find that, in addition to Mer, RPE cells also express another TAM receptor--Tyro 3--and that both of these receptors are instead activated independently by the Gas6-related ligand Protein S. This protein is also expressed by RPE cells. Finally, we demonstrate that loss of Mer function is accompanied by a substantial down-regulation in Tyro 3 as well. These observations indicate that both Mer and Tyro 3 act in mouse RPE cells and suggest that their biologically relevant ligand in these cells is Protein S.     

Photoreceptors and CSF-contacting neurons in the pineal organ of a teleost fish have direct axonal connections with the brain: an HRP-electron-microscopic study

Neural signals transmitted from the pineal organ to the brain in cold-blooded vertebrates provide information about ambient illumination, information of importance for the synchronization of activity rhythms with the light-dark cycle. The ultrastructure of intrapineal projection neurons (pineal "ganglion cells") was studied after retrograde filling with HRP through their cut axons. The dominating neuronal type is a small bipolar cell. It is present in largest numbers in the pineal stalk. This cell type displays several morphological features characteristic of cerebrospinal fluid (CSF)-contacting neurons. An apical dendritelike process extends toward the central lumen of the pineal organ. This dendritic process contains numerous mitochondria, it may have several fine branches, and it may possess a ciliumlike structure. An axon emerges from the basal pole of the neuron and joins the pineal tract. This CSF-contacting neuron is postsynaptic to photoreceptor basal pedicles with ribbon-type synapses. Such synapses may occur on the neuronal soma but are mostly observed on small, basally located processes in the vicinity of the axon. There is a significant similarity between this cell type and the bipolar cells bearing a Landolt's club in the retina. In the rostral part of the pineal end-vesicle, several large photoreceptors were labeled. These photoreceptors may, consequently, have axons more than 1 mm long. An intriguing possibility is that this previously unknown vertebrate photoreceptor type conveys graded potentials over long distances.     

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.     

The structural and neurochemical development of the fetal guinea pig retina and optic nerve in experimental growth retardation.

In this study we have examined structural and neurochemical aspects of retinal and optic nerve development in experimentally growth-retarded fetal guinea pigs following maternal unilateral artery ligation. Eye weight (n = 4) and total retinal area (n = 6) at 62 days gestation (term approximately 66 days) were both relatively spared when expressed as a percentage of body weight but in absolute terms were significantly reduced by 18% (P less than 0.001) and 13% (P less than 0.05) respectively when compared with age-matched controls. The numerical density of neurons in the ganglion cell layer was significantly higher at both 52 days (n = 4) and 62 days (n = 4) in growth-retarded fetuses compared with controls. However, there was no difference between the groups in the total number of neurons in this retinal layer at either age, since retinal areas are reduced in growth retardation. The area of neuronal somata in the ganglion and inner nuclear layers was significantly reduced in growth-retarded fetuses compared with controls. There was a concomitant reduction in the width of the cellular layers in the retina and also in the plexiform (synaptic) and photoreceptor layers. The growth of the outer segments of the photoreceptor layer was particularly affected in peripheral retina. The higher packing density of cells and the reduced growth of the plexiform layers suggests a reduction in the growth of the neuropile in growth-retarded fetuses compared with controls. The radial bundling of ganglion cell axons coursing across the retina to enter the optic nerve head was poorly defined in growth retardation. In addition myelination was delayed in the optic nerve with the numerical density of myelinated axons being significantly reduced (P less than 0.005) in growth-retarded fetuses compared with controls. There was a significant reduction (P less than 0.01) in the number of amacrine cells in the inner plexiform layer expressing Substance P-like immunoreactivity in growth-retarded fetuses compared with controls. Glutamate-like immunoreactivity was most intense in the five laminae of the inner plexiform layer and in the outer plexiform layer and less pronounced in photoreceptors, ganglion cells and their axons. There was no qualitative difference in glutamate immunoreactivity between control and growth-retarded fetuses in any of these structures. Thus we have shown that intrauterine growth retardation has specific effects on the development of the fetal guinea pig retina, reducing the growth of several types of neurons and their processes and affecting the expression of the neuropeptide substance-P in amacrine cells.     

Novel distribution of junctional adhesion molecule-C in the neural retina and retinal pigment epithelium

Junction adhesion molecules-A, -B, and -C (Jams) are cell surface glycoproteins that have been shown to play an important role in the assembly and maintenance of tight junctions and in the establishment of epithelial cell polarity. Recent studies reported that Jam-C mRNA was increased threefold in the all-cone retina of the Nrl(-/-) mouse, suggesting that Jam-C is required for maturation and polarization of cone photoreceptors cells. We examined the expression of Jams in the mouse retina by using confocal immunofluorescence localization. Jam-C was detected in tight junctions of retinal pigment epithelium (RPE) and at the outer limiting membrane (OLM) in the specialized adherens junctions between Müller and photoreceptor cells. Additionally, Jam-C labeling was observed in the long apical processes of Müller and RPE cells that extend between the inner segments and outer segments of photoreceptors, respectively. Jam-B was also detected at the OLM. In the developing retina, Jam-B and -C were detected at the apical junctions of embryonic retinal neuroepithelia, suggesting a role for Jams in retinogenesis. In eyes from Jam-C(-/-) mice, retinal lamination, polarity, and photoreceptor morphology appeared normal. Although Jam-A was not detected at the OLM in wild-type retinas, it was present at the OLM in retinas of Jam-C(-/-) mice. These findings indicate that up-regulation of Jam-A in the retina compensates for the loss of Jam-C. The nonclassical distribution of Jam-C in the apical membranes of Müller cells and RPE suggests that Jam-C has a novel function in the retina.     

Dye-induced 'photo-degeneration' and 'photo-permeabilization' of mammalian neurons in vivo

Dyes are known to induce neuronal 'photo-degeneration' and 'photo-permeabilization' in fly photoreceptor cells in vivo. In the present study, we attempted to generalize this photodynamic damage to vertebrate neurons, using the rat retina, a brain part which is optically accessible in vivo. After intravitreal injection of the photosensitizing dye Rose Bengal (RB), irradiation of the retina of a living rat with a T-shaped microbeam was found to induce striking 'optograms' which could be observed on the excised retina. The T-shaped pattern which was to be seen in the translucent retina under transmitted light was attributed to neuronal degeneration of the neurons irradiated in the presence of RB, as attested by classical degenerative features such as a cytoplasmic darkening or a drastic swelling. The T-shaped pattern could also be observed on adding the dye Lucifer yellow to the extracellular space of the retina either in vitro or in vivo, showing that the cells irradiated in the presence of RB became permeable. These structural reactions were observed in the cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), in the processes in both plexiform layers, and in the ganglion cell axons crossing this area, whereas the photoreceptors in the outer retina appeared to be undamaged. From these reactions, due to photo-degeneration and photo-permeabilization, it was possible to identify the photodynamic damage to the nervous system histologically at the macroscopic, cellular and ultrastructural levels. In view of its accuracy and reproducibility, the photo-lesion technique holds great potential as a tool for investigating various nervous systems.     

Distribution of muscarinic acetylcholine receptors on processes of isolated retinal cells

Binding of propylbenzilylcholine mustard, a muscarinic acetylcholine receptor antagonist, to isolated retinal cells was examined with light microscopic autoradiography. Dissociation of the adult tiger salamander retina yielded identifiable rod, cone, horizontal, bipolar, amacrine/ganglion, and Müller cells. Preservation of fine structure was assessed with conventional electron microscopy. For all cell types, the plasmalemma was intact and free of adhering debris; in addition, presynaptic ribbon complexes were present in photoreceptor and bipolar axon terminals indicating that synaptic structures were retained. Specific binding to cell bodies and processes was analyzed separately by using morphometric and statistical techniques. The highest grain densities occurred on processes of amacrine/ganglion cells and axons and 2 degrees and 3 degrees dendrites of bipolar neurons. Bipolar cells, however, seemed to be a heterogeneous population because there was great variation in the density of binding sites on both their axons and distal dendrites. Intermediate levels of binding were found on bipolar 1 degree dendrites and horizontal cells. No specific binding was detected on Müller cells and most parts of photoreceptors. Comparisons between cells showed that grain densities were similar for bipolar axons and amacrine/ganglion cell processes but bipolar dendrites were richer in binding sites than horizontal cell dendrites. Thus, muscarinic receptors in the salamander retina are located on amacrine/ganglion, bipolar, and horizontal cells and primarily confined to the processes which compose the two synaptic layers. In the inner plexiform layer, muscarinic receptors reside on processes from all three inner retinal neurons: in the outer synaptic layer, receptors are only on second-order cells and are more numerous along bipolar than horizontal cell dendrites.     

The immunocytochemical localization of connexin 36 at rod and cone gap junctions in the guinea pig retina

Connexin 36 (Cx36) is a channel-forming protein found in the membranes of apposed cells, forming the hexameric hemichannels of intercellular gap junction channels. It localizes to certain neurons in various regions of the brain including the retina. We characterized the expression pattern of neuronal Cx36 in the guinea pig retina by immunocytochemistry using specific antisera against Cx36 and green/red cone opsin or recoverin. Strong Cx36 immunoreactivity was visible in the ON sublamina of the inner plexiform layer and in the outer plexiform layer, as punctate labelling patterns. Double-labelling experiments with antibody directed against Cx36 and green/red cone opsin or recoverin showed that strong clustered Cx36 immunoreactivity localized to the axon terminals of cone or close to rod photoreceptors. By electron microscopy, Cx36 immunoreactivity was visible in the gap junctions as well as in the cytoplasmic matrices of both sides of cone photoreceptors. In the gap junctions between cone and rod photoreceptors, Cx36 immunoreactivity was only visible in the cytoplasmic matrices of cone photoreceptors. These results clearly indicate that Cx36 forms homologous gap junctions between neighbouring cone photoreceptors, and forms heterologous gap junctions between cone and rod photoreceptors in guinea pig retina. This focal location of Cx36 at the terminals of the photoreceptor suggests that rod photoreceptors can transmit rod signals to the pedicle of a neighbouring cone photoreceptor via Cx36, and that the cone in turn signals to corresponding ganglion cells via ON and OFF cone bipolar cells.     

Differential expression of neuronal calcium sensor-1 in the developing chick retina

Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) binding protein that has been implicated in the regulation of neurotransmission and synaptogenesis. In this study we investigated the developmental expression and localization of NCS-1 in the chick retina. Single- and double-labeling experiments with three-dimensional reconstruction as well as ultrastructural data of the distribution of NCS-1 suggest that this protein is also involved in axonal process outgrowth. We found an early expression of NCS-1 in ganglion cells and their axons, in amacrine, and in horizontal cells, whereas photoreceptors were immunonegative at embryonic stages. In the early posthatching days we found strong immunostaining for NCS-1 in horizontal cells and their processes in the outer plexiform layer. In contrast, synaptic vesicle protein 2 (SV2) was prominent only in photoreceptor synaptic terminals. Ultrastructural analysis confirmed that NCS-1 was localized postsynaptically in horizontal cell processes, whereas presynaptic terminals were immunonegative. However, at late posthatching days we observed that photoreceptor ribbon synapses (from rods and/or cones) also expressed NCS-1. Thus the results support the notion that NCS-1 is involved in neuronal process outgrowth and is localized in pre- and postsynaptic compartments including mature photoreceptor synapses.     

Basic fibroblast growth factor (bFGF) immunolocalization in the rodent outer retina demonstrated with an anti-rodent bFGF antibody

The distribution of bFGF in mouse and rat retinas was established using an antibody generated against a rat specific bFGF sequence. The patterns established with this antibody were distinctly different in comparison to the immunostaining patterns using antibodies prepared against similar regions of bovine and human bFGF sequences. Using the anti-rat antibody, at postpartum day 5, both species demonstrate bFGF-like labeling in the outer retina, primarily associated with developing photoreceptors. The anti-rat bFGF antibody in the adult mouse produces intense labeling at the level of the photoreceptor outer segment, whereas in the adult rat, Müller's cells and RPE were intensely labeled but the photoreceptors appeared to be unlabeled. In contrast, the bovine and human antibodies consistently label ganglion cells in both mouse and rat retinas, cells in the inner aspect of the inner nuclear layer and developing horizontal cells, whereas no photoreceptor labeling was observed. These results suggest that minor species differences in a short segment of the bFGF molecule used to generate these antibodies may result in major differences in apparent bFGF-like immunolocalization.     

Glycinergic contacts in the outer plexiform layer of the Xenopus laevis retina characterized by antibodies to glycine, GABA and glycine receptors

Electrophysiological experiments have predicted a direct synaptic input from glycinergic interplexiform cells (IPCs) to GABAergic horizontal cells in the Xenopus retina. However, previous ultrastructural studies failed to demonstrate this input. Here, we used three immunocytochemical approaches to investigate this issue. First, double-label postembedding immunocytochemistry with GABA- and glycine-like immunoreactivity (GABA-LI and glycine-LI) was used to study possible interactions of the glycinergic IPC with GABAergic horizontal cells. Processes postsynaptic to glycine-LI IPC terminals in the outer plexiform layer (OPL) fell into two groups, small microtubule-filled processes and larger electron-lucent processes with sparse microtubules and occasional mitochondria. In no case did we find glycine-LI synapses onto GABA-LI cells or processes. Second, pre-embedding immunocytochemistry was used to label GABA-LI cells and processes in the OPL. GABA-LI was sparse in horizontal cell axons and more intense in horizontal cell somas and in small processes. In agreement with our first set of experiments, GABA-LI profiles did not receive input from conventional synapses. Third, we localized glycine-receptor-like immunoreactivity (GlyR-LI) to several types of apparent synapses in the OPL. As expected, it was found at IPC synapses. Unexpectedly, GlyR-LI was also subsynaptic at photoreceptor synapses onto second order neurons, both at ribbon and basal junction type synapses. At least some of the GlyR-LI photoreceptor synapses were from cones. Also, GlyR-LI was apposed to photoreceptors and to unidentified small diameter processes, where no other indication of synaptic input was evident. Because glycine-LI is not found in photoreceptors, we suggest that glycine receptors at photoreceptor synapses are stimulated by glycine that diffuses from other sites, possibly from IPCs. This interpretation is consistent with available physiological studies of glycinergic effects in this retina.     

Developmental disturbances in the hamster retina caused by a mutant of mumps virus

Newborn hamsters were inoculated intracerebrally with either the neurovirulent Kilham strain of mumps virus or a mutant (M13) strain of this virus. The M13 strain has an alteration in the haemagglutinin-neuraminidase protein of its envelope and causes a low-grade infection of the brain. Both strains spread consistently to the retina where the Kilham strain caused an extensive necrotizing infection. In contrast, the M13 strain predominantly caused an infection of the retinal pigment epithelium (RPE) with the involvement of scattered neurons in the retina. Only minimal degenerative or inflammatory changes were seen, but at 12 days of age developmental alterations were seen in all eyes. These included stretches with failure of photoreceptor segment development and the formation of folds in the outer nuclear layer. The former changes occurred in areas with loss of RPE cells and the latter generally in connection with displaced pigment-loaded cells from the RPE layer. It is suggested that these retinal alterations are mainly secondary to the RPE infection with the M13 strain.     

Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the functional reconstruction of retinas lacking photoreceptors.

We have investigated the possibility of using transplantation of immature or mature rodent photoreceptors as well as mature human photoreceptors to reconstruct retinas in which photoreceptor degeneration is either inherited or environmentally induced. To this end, we have devised methods for isolating and transplanting the outer nuclear layer (ONL) (e.g., the photoreceptor layer) to the subretinal space of mature rodents. In addition we found that if portions of the inner retina are transplanted along with the intact photoreceptor sheet, photoreceptor organization is better maintained. In ultrastructural studies of the reconstructed retina an outer plexiform-like layer (OPL) is visible at the interface of the transplanted ONL and the host inner nuclear layer, with invaginating ribbon synapses characteristic of those formed by rod photoreceptors evident within this OPL. Ribbon synapses are found only rarely in unreconstructed retina. These results suggest that synaptic connections between transplanted photoreceptors and host cells may be made. Evidence for the potential recovery of function following photoreceptor transplantation is found in visually evoked cortical responses and behavioral responses (pupillary reflex) to light stimulation of the reconstructed eye. These findings suggest the possibility that neural transplantation can reconstruct a sensory end organ--in this case the retina--to restore evoked activity and an appropriate behavioral response to sensory stimulation.     

The entry of taurine into the neural retina and pigment epithelium of the frog.

Autoradiographs of frog retinas incubated with radioactively labelled taurine show heavy grain density over (i) cells in the position of amacrine interneurones, (ii) the inner plexiform (synaptic) regions, giving a striated appearance, (iii) photoreceptor cells and their synaptic terminals, and (iv) the pigment epithelium. The inner retina, the photoreceptor cell layer and the pigment epithelium all have uptake systems for taurine with apparent Km's in the high affinity range (Km, 20-50 micron; Vmax, 13-33 nmoles/g/min). In vivo subcutaneously injected taurine was taken up by the pigment epithelium and then passed to the photoreceptors and inner retina over about 10 days. The total level of nucleide in the retina remained constant for at least 6 weeks. There was little or no metabolism of the labelled taurine during this time.     

In vitro transdifferentiation of embryonic rat retinal pigment epithelium to neural retina

Divergence of neural retinal and retinal pigment epithelial (RPE) lineages from the optic vesicle neuroepithelium starts at a very early stage of eye development. Partially or even fully differentiated RPEs of some vertebrate species are capable of transforming into neural retina. In the present study, we have shown that mammalian RPE possesses the ability to transdifferentiate into neural retina at early embryonic stages. If cultured in serum-free medium, presumptive rat RPE became pigmented and expressed a molecular marker of mature RPE. In the presence of basic fibroblast growth factor (bFGF), cultured early embryonic rat RPE did not acquire pigment and grew to form retina-like multilayer structure containing neuronal cells and cells that express markers of retinal ganglion, amacrine and rod photoreceptor cells. The effects of bFGF occurred independently of effects on cell division and became irreversible after periods that varied with tissue age. This study has demonstrated that already differentiated embryonic rat RPE still retain the ability to become neural retina up to certain stage.     

IGF-1 produced by cone photoreceptors regulates rod progenitor proliferation in the teleost retina

Teleost eyes grow throughout life by adding neurons and stretching extant tissue. New retinal neurons of all types are added at the ciliary margin and new rod photoreceptors are inserted throughout retina in the outer nuclear layer (ONL). New rod photoreceptors result from the division of progenitor cells located in the ONL amidst functioning rod photoreceptor cell nuclei, but it is not known how new rod addition is regulated. Previous experiments using an organotypic retinal slice preparation revealed that insulin-like growth factor 1 (IGF-1) up-regulates the division of the rod progenitor cells [Dev. Brain Res. 76 (1993) 183], but the site of IGF-1 action was unknown. Here, we show where in the retina IGF-1 is made, where IGF receptors are located, and we identify the role of IGF-1 in adult retinal rod neurogenesis with both gain-and loss-of-function experiments. We found that IGF-1 is expressed by cone photoreceptor cells and its abundance varies with a daily rhythm, being significantly higher at night. In vivo application of exogenous IGF-1 increases rod progenitor cell division, an effect that is greater at night than during the day. We also show that inhibiting the function of IGF receptors decreases proliferation of rod progenitor cells. Finally, we show that IGF receptors are located on rod progenitor cells as well as on cone and rod photoreceptors. Taken together, these data suggest that the rhythmic production and release of IGF-1 plays a role in regulating the insertion of new rod photoreceptors into the retina. The diurnal change in IGF-1 abundance and effects of exogenous IGF-1 are consistent with the previous demonstration that rod progenitor cell division is threefold greater at night than in the day [Brain Res. 673 (1995) 119; Brain Res. 712 (1996) 40]. We also show that the insertion of new rod photoreceptors at the central edge of the ciliary neurogenic zone very likely also depends on IGF-1 production by cone photoreceptors. We propose that addition of new rod photoreceptors into the functioning retina is regulated through a feedback mechanism mediated at least in part via the IGF-1 produced in the cone photoreceptors.     

Activin A promotes progenitor differentiation into photoreceptors in rodent retina

Activins are TGF beta-like proteins that were first discovered for their actions on the reproductive system, but have subsequently been shown to play a role in a variety of developmental processes. Previous studies have demonstrated that activins and their receptors are present in the developing retina, as well as other regions of the embryonic nervous system. We used both in vitro and in vivo approaches to test for functions of activin during retinal development. We found that activin A treatment of embryonic day 18 rat retinal cultures causes the progenitor cells in the cultures to exit the cell cycle and differentiate into rod photoreceptors. This effect is dose-dependent and the promotion of rod photoreceptor differentiation is specific, since the other primary retinal neurons generated in these cultures, the C1+ amacrine cells, are not affected by activin A treatment. Mice with homozygous deletion of the activin betaA gene show a specific decrease in the number of rod photoreceptors compared to wild-type or heterozygous littermates. These data demonstrate that activin A is an important regulator of photoreceptor differentiation in the developing retina.     

Localization of zinc in the outer retina of carp: a light- and electron-microscopic study

Many lines of evidence suggest that zinc may play an important neuromodulatory role in the central nervous system, including the retina. In this work, localization of zinc in the outer retina of carp was studied, using the silver amplification method, by light and electron microscopy. Reaction products (silver grains) were widely distributed throughout the retina, including photoreceptors, the outer and inner nuclear layers (ONL and INL), the ganglion cell layer (GCL), as well as in both outer and inner plexiform layers (OPL and IPL). Generally, staining for zinc was stronger in the outer retina than in the inner retina, and grains were aggregated with the greatest density in the OPL and the outer limiting membrane (OLM). Silver precipitates were also detected in the inner segments, axons, but not outer segments of photoreceptors. At the ultrastructural level, zinc was localized to myoid regions, mitochondria in the inner segments, internuclear space and nuclei of photoreceptors. In addition, silver grains were found in the terminals of photoreceptors, cone pedicles, and rod spherules, as well as in some processes in the OPL, which might be dendrites of horizontal cells. The presence of zinc in the terminals of photoreceptors suggests that zinc might be released from photoreceptor terminals and play modulatory roles in the outer retina.