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During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P5232to P5011) and then a total change in gene expression with the down regulation of a \u201cfreshwater\u201d opsin and its concomitant replacement by the expression of a typical \u201cdeep-sea\u201d opsin (P5011to P4821). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540\u2013545 nm and 435\u2013440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the \u201cgreen\u201d-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.
\n \n\n \n \nAbstractHere we reveal a population of cells that express cone photoreceptor opsins that are located in the inner retina, distant from outer retinal photoreceptors. These cells are present in rodents and human. They also express a range of key proteins critical in the cone phototransduction cascade and make contact with other retinal neurons. Their opsins are not generally confined to cellular specialized regions but are present throughout the plasma membrane, although their nuclear configurations are similar to those of outer retinal cones. This population is distinct from the ganglion cells that contain melanopsin and which are known to be inner retinal irradiance detectors regulating circadian behaviour. Surprisingly, the size of the population of short wavelength opsin positive cells in the ganglion cell layer is plastic. In normal animals their number declines with age. However, their numbers increase significantly in response to outer retinal photoreceptor loss, probably by drawing on a pool of inner retinal cells that express cone specific markers, but not opsins.
\n \n\n \n \nAbstractThe melanopsin positive, intrinsically photosensitive retinal ganglion cells (ipRGCs) of the inner retina have been shown to send wide-ranging projections throughout the brain. To investigate the response of this important cell type during retinal dystrophy, we use the Royal College of Surgeons (RCS) dystrophic rat, a major model of retinal degeneration. We find that ipRGCs exhibit a distinctive molecular profile that remains unaltered during early stages of outer retinal pathology (15 weeks of age). In particular, these cells express \u03b2III tubulin, \u03b1-acetylated tubulin, and microtubule-associated proteins (MAPs), while remaining negative for other RGC markers such as neurofilaments, calretinin, and parvalbumin. By 14 months of age, melanopsin positive fibers invade ectopic locations in the dystrophic retina and ipRGC axons/dendrites become distorted (a process that may involve vascular remodeling). The morphological abnormalities in melanopsin processes are associated with elevated immunoreactivity for MAP1b and a reduction in \u03b1-acetylated tubulin. Quantification of ipRGCs in whole mounts reveals reduced melanopsin cell number with increasing age. Focusing on the retinal periphery, we find a significant decline in melanopsin cell density contrasted by a stability of melanopsin positive processes. In addition to these findings, we describe for the first time, a distinct plexus of melanopsin processes in the far peripheral retina, a structure that is coincident with a short wavelength opsin cone-enriched rim. We conclude that some ipRGCs are lost in RCS dystrophic rats as the disease progresses and that this loss may involve vascular remodeling. However, a significant number of melanopsin positive cells survive into advanced stages of retinal degeneration and show indications of remodeling in response to pathology. Our findings underline the importance of early intervention in human retinal disease in order to preserve integrity of the inner retinal photoreceptive network.
\n \n\n \n \nAbstractMany factors probably regulate the process of natural cell death during development. It is present in both the early undifferentiated retina and later following differentiation. Melanin production plays a role in regulating retinal development and when it is absent, cell proliferation and death are enhanced. Here we examine the effects of hyperoxia on this process, as oxygen has been shown to reduce cell death among differentiated photoreceptors late in development. However, in this study we examine its effects much earlier in pigmented and albino pigmentation phenotypes, when most cells are still actively dividing and are not committed to a specific fate. Newborn mice were exposed to high oxygen levels for 24\u2003h and then returned to normal air for varying periods and their retinae examined. Hyperoxia had a dramatic effect on the number of dying cells, reducing them by almost 60% in pigmented animals and by over 80% in albinos. Following the return to normal air there was a gradual increase in their number over 360\u2003min back to normal levels in pigmented mice; however, in albinos there was a complete rebound in levels of cell death within 40\u2003min, reflecting the increased metabolic stress present in albino retinae due to their abnormal levels of proliferation. These results highlight the important role played by oxygen during early natural cell death in the retina and reveal the different developmental conditions present in the retinae of the two pigmentation phenotypes examined.
\n \n\n \n \nThe visual system is the part of the central nervous system that detects light. It is sub-served by the -photoreceptor detectors within the eye, and this information allows creatures to build a representation of the visual world as well as regulating a whole range of subconscious physiological processes. Here we describe seven techniques to probe the functionality of the visual system in rodents. They encompass direct electrical recordings and functional anatomy, as well as reflexes and behavioural response outputs of light detection. Used in concert, these techniques can enable the assessment of which photoreceptor classes are functioning as well as to indicate whether light information is processed at a sub-cortical and/or cortical level. \u00a9 2011 Springer Science+Business Media, LLC.
\n \n\n \n \nAbstractNon\u2010rod, non\u2010cone ocular photoreceptors have been shown to mediate a range of irradiance detection tasks. The strongest candidates for these receptors are melanopsin\u2010positive retinal ganglion cells (RGCs). To provide a more complete understanding of these receptors in vivo, we have utilized a mouse that lacks rod and cone photoreceptors (rd/rd cl) and compared these animals to congenic wild\u2010types. Using real\u2010time polymerase chain reaction and immunohistochemistry, we address the following. (1) Is Fos expression within these RGCs driven by an input from the rods/cones or is it the product of the intrinsic photosensitivity of these neurons? We demonstrate that most Fos expression across the entire retina is due to the rods/cones, but in the absence of these photoreceptors, light will induce Fos within melanopsin RGCs. (2) Could the reported age\u2010related decline in circadian photosensitivity of rodents be linked to changes in the population of melanopsin RGCs? We show that old mice experience an \u223c\u200a40% reduction in melanopsin RGCs. (3) Does the loss of inner retinal neurons affect the responses of melanopsin RGCs? Aged (\u223c\u2003700\u2003days) rd/rd cl mice lose most of their inner retina but retain the retinal ganglion cell layer. In these mice, the proportion of melanopsin RGCs that express Fos in response to light is significantly reduced. Collectively, our data suggest that melanopsin RGCs form a heterogeneous population of neurons, and that most of the light\u2010induced c\u2010fos expression within these cells is associated with the endogenous photosensitivity of these neurons.
\n \n\n \n \nPURPOSE: In several species the retinal pigment epithelium (RPE) has the potential to transdifferentiate into retinal cells to regenerate functional retinal tissue after injury. However, this capacity for regeneration is lost in mammals. The synthetic retinoic acid derivative, fenretinide [N(4-hydroxyphenyl) retinamide], induces a neuronal-like phenotype in the human adult retinal pigment epithelial cell line (ARPE-19). These changes are characterized by the appearance of neural-like processes and the expression of neuronal markers not normally associated with RPE cells. Here we assess whether fenretinide can induce a neuroretinal cell phenotype in ARPE-19 cells, by examining retinal cell marker expression. METHODS: ARPE-19 cells were treated daily with culture medium containing either 3 \u03bcM fenretinide or dimethyl sulfoxide as a control for 7 days. Cells were processed for immunocytochemistry, western blotting, and for analysis by PCR to examine the expression of a panel of RPE, neural, and retinal-associated cellular markers, including classical and non-canonical opsins. RESULTS: Treatment with fenretinide for 7 days induced the formation of neuronal-like processes in ARPE-19 cells. Fenretinide induced the expression of the cone long wavelength sensitive opsin (OPN1lw) but not rhodopsin (RHO), while decreasing the expression of RPE cell markers. Many of the neuronal and retinal specific markers examined were expressed in both control and fenretinide treated cells, including those involved in photoreceptor cell development and the multipotency of neural retinal progenitor cells. Interestingly, ARPE-19 cells also expressed both photoreceptor specific and non-specific canonical opsins. CONCLUSIONS: The expression of retinal-associated markers and loss of RPE cell markers in control ARPE-19 cells suggests that these cells might have dedifferentiated from an RPE cell phenotype under standard culture conditions. The expression of molecules, such as the transcription factors paired box 6 gene (PAX6), sex determining region Y-box 2 (SOX2), cone-rod homeobox (CRX), and neural retina leucine zipper (NRL), further implies that in culture these cells are predisposed toward a retinal progenitor-like state. The fenretinide-induced increase in photoreceptor cell markers, accompanied by a decrease in RPE cell markers, suggests that retinoids may play a role in the transdifferentiation of RPE cells. Importantly, our data show for the first time the expression of a vertebrate ciliary opsin (OPN1lw) and rhabdomeric-like opsin, opsin 4 (OPN4 also known as melanopsin) in a clonal cell line. Together these data suggest that ARPE-19 cells are primed for and possess the capacity to differentiate toward a retinal cell-like lineage.
\n \n\n \n \nPURPOSE: To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells. METHODS: HESC-RPE cells were derived from a super-confluent preparation of the Shef1 HESC line. Pigmented colonies were isolated and expanded into pigmented monolayers on Matrigel matrix-coated dishes or filters. Cells were exposed to fluorescently labeled outer segments isolated from the porcine eye and assessed for phagocytic activity at regular intervals. Expression of molecules associated with RPE phagocytosis was analyzed by RT-PCR, immunocytochemistry, and western blot. The role of Mer Tyrosine Kinase (MERTK) in the phagocytosis of outer segments was investigated using antibodies directed against MERTK to block function. In a novel approach, cells were also exposed to fresh human neural retina tissue then examined by electron microscopy for evidence of phagocytosis and changes in cell morphology. RESULTS: HESC-derived RPE cells are capable of phagocytosing isolated porcine outer segments and express molecules associated with RPE-specific phagocytosis, including MERTK. Pre-incubation with antibodies against MERTK blocked phagocytosis of photoreceptor outer segments, but not polystyrene beads. HESC-RPE cells also phagocytosed outer segments in a novel human retinal explant system. Furthermore co-culture adjacent to human retina tissue in this preparation resulted in the appearance of features in HESC-derived RPE cells normally observed only as the RPE matures. CONCLUSIONS: The ingestion of photoreceptor outer segments from an isolated population and an artificial ex vivo human retina system demonstrates HESC-derived RPE cells are functional. HESC-derived RPE possess the relevant molecules required for phagocytosis, including MERTK, which is essential for the phagocytosis of outer segments but not latex beads. Furthermore, some changes observed in cell morphology after co-culture with human retina may have implications for understanding the full development and differentiation of RPE cells.
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