Light-Driven Regeneration of Cone Visual Pigments through a Mechanism Involving RGR Opsin in Müller Glial Cells.

While rods in the mammalian retina regenerate rhodopsin through a well-characterized pathway in cells of the retinal pigment epithelium (RPE), cone visual pigments are thought to regenerate in part through an additional pathway in Müller cells of the neural retina. The proteins comprising this intrinsic retinal visual cycle are unknown. Here, we show that RGR opsin and retinol dehydrogenase-10 (Rdh10) convert all-trans-retinol to 11-cis-retinol during exposure to visible light. Isolated retinas from Rgr+/+ and Rgr-/- mice were exposed to continuous light, and cone photoresponses were recorded. Cones in Rgr-/- retinas lost sensitivity at a faster rate than cones in Rgr+/+ retinas. A similar effect was seen in Rgr+/+ retinas following treatment with the glial cell toxin, α-aminoadipic acid. These results show that RGR opsin is a critical component of the Müller cell visual cycle and that regeneration of cone visual pigment can be driven by light.

Retention and losses of ultraviolet-sensitive visual pigments in bats.

Ultraviolet (UV)-sensitive visual pigment and its corresponding ability for UV vision was retained in early mammals from their common ancestry with sauropsids. Subsequently, UV-sensitive pigments, encoded by the short wavelength-sensitive 1 (SWS1) opsin gene, were converted to violet sensitivity or have lost function in multiple lineages during the diversification of mammals. However, many mammalian species, including most bats, are suggested to retain a UV-sensitive pigment. Notably, some cave-dwelling fruit bats and high duty cycle echolocating bats have lost their SWS1 genes, which are proposed to be due to their roosting ecology and as a sensory trade-off between vision and echolocation, respectively. Here, we sequenced SWS1 genes from ecologically diverse bats and found that this gene is also non-functional in both common vampire bat (Desmodus rotundus) and white-winged vampire bat (Diaemus youngi). Apart from species with pesudogenes, our evolutionary and functional studies demonstrate that the SWS1 pigment of bats are UV-sensitive and well-conserved since their common ancestor, suggesting an important role across major ecological types. Given the constrained function of SWS1 pigments in these bats, why some other species, such as vampire bats, have lost this gene is even more interesting and needs further investigation.

THE EFFECT OF PHOTOPIGMENT BLEACHING ON FUNDUS AUTOFLUORESCENCE IN ACUTE CENTRAL SEROUS CHORIORETINOPATHY.

PURPOSE: To evaluate the effect of photobleaching on fundus autofluorescence (FAF) images in acute central serous chorioretinopathy. METHODS: We obtained prephotobleaching and postphotobleaching images using an Optomap 200Tx, and photobleaching was induced with a Heidelberg Retina Angiograph 2. Degrees of photobleaching were assessed as grayscale values in Optomap images. Concordances among the three kinds of images were analyzed. Hyper-AF lesions in prephotobleaching images were classified as Type 1 (changed to normal-AF after photobleaching) and Type 2 (unchanged after photobleaching). The FAF composite patterns of central serous chorioretinopathy lesions were classified as diffuse or mottled. Initial and final best-corrected visual acuity, central retinal thickness, and disease duration were compared according to fovea FAF type. RESULTS: Forty-one eyes of 41 patients were analyzed. The lesion brightness of postphotobleaching Optomap FAF showed greater concordance with Heidelberg Retina Angiograph 2 FAF (94.74%) than the prephotobleaching Optomap FAF (80.49%). Eyes with Type 1 fovea had greater initial and final best-corrected visual acuity (20/23 vs. 20/41, 20/21 vs. 20/32, P < 0.0001, P = 0.001, respectively) and shorter disease duration (19.68 ± 12.98 vs. 51.55 ± 44.98 days, P = 0.043) than those with Type 2 fovea. However, eyes with diffuse Type 2 fovea had only lower initial and final best-corrected visual acuity (20/23 vs. 20/45, 20/21 vs. 20/36, P < 0.0001, P < 0.0001, respectively) than those with Type 1 fovea. CONCLUSION: Understanding the photobleaching effect is necessary for the accurate interpretation of FAF images. Furthermore, comparing prephotobleaching and postphotobleaching FAF images may be helpful for estimation of lesion status in central serous chorioretinopathy.

The Protective Effect of Brown-, Gray-, and Blue-Tinted Lenses against Blue LED Light-Induced Cell Death in A2E-Laden Human Retinal Pigment Epithelial Cells.

A2E-laden ARPE-19 cells were exposed to a blue light to induce cytotoxicity, in order to investigate the protective effects of various tinted ophthalmic lenses against photo-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laden with A2E, known to be among the etiologies of age-related macular degeneration (AMD). Different-colored tinted lenses with varying levels of tint and different filtering characteristics, such as polarized, blue-cut, and photochromatic lenses, were placed over the cells, and the protective efficacies thereof were evaluated by lactate dehydrogenase assay. When tinted lenses were placed over ARPE-19 cells, there were different reductions in cytotoxicity according to the colors and tint levels. The level of protection afforded by brown-tinted lenses was 6.9, 36.1, and 49% with a tint level of 15, 50, and 80%, respectively. For gray-tinted lenses, the protective effect was 16.3, 35, and 43.4% for the corresponding degree of tint, respectively. In the case of blue-tinted lenses, a protective effect of 20% was observed with 80% tinted lenses, but 15 and 50% tinted lenses provided no significant protection. In addition, photochromic lenses showed a protective effect but blue-cut lenses and polarized lenses provided no significant protection. Tinted lenses significantly reduced cytotoxicity in RPE cells irradiated with blue light. The protection was more efficient in lenses with a brown or gray tint than in blue-tinted lenses. Tinted glasses may provide significant protection against potential blue-light-induced photochemical and photo-oxidative damage in RPE cells.

Vitamin A and Vision.

Visual systems detect light by monitoring the effect of photoisomerization of a chromophore on the release of a neurotransmitter from sensory neurons, known as rod and cone photoreceptor cells in vertebrate retina. In all known visual systems, the chromophore is 11-cis-retinal complexed with a protein, called opsin, and photoisomerization produces all-trans-retinal. In mammals, regeneration of 11-cis-retinal following photoisomerization occurs by a thermally driven isomerization reaction. Additional reactions are required during regeneration to protect cells from the toxicity of aldehyde forms of vitamin A that are essential to the visual process. Photochemical and phototransduction reactions in rods and cones are identical; however, reactions of the rod and cone visual pigment regeneration cycles differ, and perplexingly, rod and cone regeneration cycles appear to use different mechanisms to overcome the energy barrier involved in converting all-trans- to 11-cis-retinoid. Abnormal processing of all-trans-retinal in the rod regeneration cycle leads to retinal degeneration, suggesting that excessive amounts of the retinoid itself or its derivatives are toxic. This line of reasoning led to the development of various approaches to modifying the activity of the rod visual cycle as a possible therapeutic approach to delay or prevent retinal degeneration in inherited retinal diseases and perhaps in the dry form of macular degeneration (geographic atrophy). In spite of great progress in understanding the functioning of rod and cone regeneration cycles at a molecular level, resolution of a number of remaining puzzling issues will offer insight into the amelioration of several blinding retinal diseases.

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins.

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.

Functional characterisation of the chromatically antagonistic photosensitive mechanism of erythrophores in the tilapia Oreochromis niloticus.

Non-visual photoreceptors with diverse photopigments allow organisms to adapt to changing light conditions. Whereas visual photoreceptors are involved in image formation, non-visual photoreceptors mainly undertake various non-image-forming tasks. They form specialised photosensory systems that measure the quality and quantity of light and enable appropriate behavioural and physiological responses. Chromatophores are dermal non-visual photoreceptors directly exposed to light and they not only receive ambient photic input but also respond to it. These specialised photosensitive pigment cells enable animals to adjust body coloration to fit environments, and play an important role in mate choice, camouflage and ultraviolet (UV) protection. However, the signalling pathway underlying chromatophore photoresponses and the physiological importance of chromatophore colour change remain under-investigated. Here, we characterised the intrinsic photosensitive system of red chromatophores (erythrophores) in tilapia. Like some non-visual photoreceptors, tilapia erythrophores showed wavelength-dependent photoresponses in two spectral regions: aggregations of inner pigment granules under UV and short-wavelengths and dispersions under middle- and long-wavelengths. The action spectra curve suggested that two primary photopigments exert opposite effects on these light-driven processes: SWS1 (short-wavelength sensitive 1) for aggregations and RH2b (rhodopsin-like) for dispersions. Both western blot and immunohistochemistry showed SWS1 expression in integumentary tissues and erythrophores. The membrane potential of erythrophores depolarised under UV illumination, suggesting that changes in membrane potential are required for photoresponses. These results suggest that SWS1 and RH2b play key roles in mediating intrinsic erythrophore photoresponses in different spectral ranges and this chromatically dependent antagonistic photosensitive mechanism may provide an advantage to detect subtle environmental photic change.

Retinal phototransduction.

Vision is perhaps the most important of all our senses, and gives us an immense amount of information regarding the outside world. The initial format in which this information reaches the retina are photons; particles of energy radiation of a given wavelength emitted or reflected from our surroundings. The brain itself however, perceives information in electrical signals via action potentials and changes in electrochemical gradients. The processes involved in the transduction of photons into electrical potentials will be the focus of this article. This review article summarizes the recent advances in understanding these complex pathways and provides an overview of the main molecules involved in the neurobiology of vision.

Comparative studies on the late bleaching processes of four kinds of cone visual pigments and rod visual pigment.

Visual pigments in rod and cone photoreceptor cells of vertebrate retinas are highly diversified photoreceptive proteins that consist of a protein moiety opsin and a light-absorbing chromophore 11-cis-retinal. There are four types of cone visual pigments and a single type of rod visual pigment. The reaction process of the rod visual pigment, rhodopsin, has been extensively investigated, whereas there have been few studies of cone visual pigments. Here we comprehensively investigated the reaction processes of cone visual pigments on a time scale of milliseconds to minutes, using flash photolysis equipment optimized for cone visual pigment photochemistry. We used chicken violet (L-group), chicken blue (M1-group), chicken green (M2-group), and monkey green (L-group) visual pigments as representatives of the respective groups of the phylogenetic tree of cone pigments. The S, M1, and M2 pigments showed the formation of a pH-dependent mixture of meta intermediates, similar to that formed from rhodopsin. Although monkey green (L-group) also formed a mixture of meta intermediates, pH dependency of meta intermediates was not observed. However, meta intermediates of monkey green became pH dependent when the chloride ion bound to the monkey green was replaced with a nitrate ion. These results strongly suggest that rhodopsin and S, M1, and M2 cone visual pigments share a molecular mechanism for activation, whereas the L-group pigment may have a special reaction mechanism involving the chloride-binding site.

Activation of visual pigments by light and heat.

Vision begins with photoisomerization of visual pigments. Thermal energy can complement photon energy to drive photoisomerization, but it also triggers spontaneous pigment activation as noise that interferes with light detection. For half a century, the mechanism underlying this dark noise has remained controversial. We report here a quantitative relation between a pigment's photoactivation energy and its peak-absorption wavelength, λ(max). Using this relation and assuming that pigment activations by light and heat go through the same ground-state isomerization energy barrier, we can predict the relative noise of diverse pigments with multi-vibrational-mode thermal statistics. The agreement between predictions and our measurements strongly suggests that pigment noise arises from canonical isomerization. The predicted high noise for pigments with λ(max) in the infrared presumably explains why they apparently do not exist in nature.

Nitric oxide-dependent pigment migration induced by ultraviolet radiation in retinal pigment cells of the crab Neohelice granulata.

The purpose of this study was to verify the occurrence of pigment dispersion in retinal pigment cells exposed to UVA and UVB radiation, and to investigate the possible participation of a nitric oxide (NO) pathway. Retinal pigment cells from Neohelice granulata were obtained by cellular dissociation. Cells were analyzed for 30 min in the dark (control) and then exposed to 1.1 and 3.3 J cm(-2) UVA, 0.07 and 0.9 J cm(-2) UVB, 20 nmß-PDH (pigment dispersing hormone) or 10 µm SIN-1 (NO donor). Histological analyses were performed to verify the UV effect in vivo. Cultured cells were exposed to 250 µm L-NAME (NO synthase blocker) and afterwards were treated with UVA, UVB or ß-PDH. The retinal cells in culture displayed significant pigment dispersion in response to UVA, UVB and ß-PDH. The same responses to UVA and UVB were observed in vivo. SIN-1 did not induce pigment dispersion in the cell cultures. L-NAME significantly decreased the pigment dispersion induced by UVA and UVB but not by ß-PDH. All retinal cells showed an immunopositive reaction against neuronal nitric oxide synthases. Therefore, UVA and UVB radiation are capable of inducing pigment dispersion in retinal pigment cells of Neohelice granulata and this dispersion may be nitric oxide synthase dependent.

Novel snapshot imaging of photoreceptor bleaching in macaque and human retinas.

PURPOSE: Various methods have been used to obtain a topographic map of bleached photopigments in human retinas in the past. The purpose of this study was to determine whether the bleaching topography of the photoreceptors could be obtained by snapshot imaging reflectometry. METHODS: Four to five fundus photographs of one rhesus monkey and three healthy human subjects were taken by white flashes at intervals of 4 s, with a commercial fundus camera with minimal modifications. The flash-induced reflectance increases (bleaching) were calculated by dividing the reflectance of the first image into the subsequent images, pixel by pixel. RESULTS: The topography of the bleached macula corresponded well with the anatomical distribution of the cones. The ratio of reflectance changes in the center to that in the surrounding tissue was high for red and low for green and blue images. These results indicate that the reflectivity changes were not artifacts but were derived from changes in the photopigment density in the cones and rods. CONCLUSIONS: The topography of bleached photoreceptors obtained with a commercial fundus camera from one monkey and three healthy human subjects showed that this technique has potential as a new clinical method for examining photoreceptor function in both normal and diseased retinas.

Photopigment quenching is Ca2+ dependent and controls response duration in salamander L-cone photoreceptors.

The time scale of the photoresponse in photoreceptor cells is set by the slowest of the steps that quench the light-induced activity of the phototransduction cascade. In vertebrate photoreceptor cells, this rate-limiting reaction is thought to be either shutoff of catalytic activity in the photopigment or shutoff of the pigment's effector, the transducin-GTP-phosphodiesterase complex. In suction pipette recordings from isolated salamander L-cones, we found that preventing changes in internal [Ca(2+)] delayed the recovery of the light response and prolonged the dominant time constant for recovery. Evidence that the Ca(2+)-sensitive step involved the pigment itself was provided by the observation that removal of Cl(-) from the pigment's anion-binding site accelerated the dominant time constant for response recovery. Collectively, these observations indicate that in L-cones, unlike amphibian rods where the dominant time constant is insensitive to [Ca(2+)], pigment quenching rate limits recovery and provides an additional mechanism for modulating the cone response during light adaptation.

Photoreceptor responses of fruitflies with normal and reduced arrestin content studied by simultaneous measurements of visual pigment fluorescence and ERG.

We have simultaneously measured the electroretinogram (ERG) and the metarhodopsin content via fluorescence in white-eyed, wild-type Drosophila and the arrestin2 hypomorphic mutant (w(-);arr2 (3)) at a range of stimulus wavelengths and intensities. Photoreceptor response amplitude and termination (transition between full repolarization and prolonged depolarizing afterpotential, PDA) were related to visual pigment conversions and arrestin concentration. The data were implemented in a kinetic model of the rhodopsin-arrestin cycle, allowing us to estimate the active metarhodopsin concentration as a function of effective light intensity and arrestin concentration. Arrestin reduction in the mutant modestly increased the light sensitivity and decreased the photoreceptor dynamic range. Compared to the wild type, in the mutant the transition between full repolarization and PDA occurred at a lower metarhodopsin fraction and was more abrupt. We developed a steady-state stochastic model to interpret the dependence of the PDA on effective light intensity and arrestin content and to help deduce the arrestin to rhodopsin ratio from the sensitivity and PDA data. The feasibility of different experimental methods for the estimation of arrestin content from ERG and PDA is discussed.

Straylight in the human eye: testing objectivity and optical character of the psychophysical measurement.

The point spread function or PSF of the human eye encompasses hugely different domains: a small-angle, high-intensity domain, called the 'PSF core', and a large-angle, low-intensity domain, usually referred to as 'straylight'. The first domain can be assessed by available double-pass or other optical techniques. For the second domain psychophysical techniques have been developed, in particular the Compensation Comparison or CC technique, recently made available for clinical application in the C-Quant instrument. We address the question of whether the psychophysical technique gives measures of straylight that are compatible with those made by optical methods. With a small adaptation the CC method can be used to assess straylight from physical light scattering samples, instead of straylight in the eye, using the same psychophysics, but without interference from the ocular straylight. The light scattered by each of seven light-scattering samples, encompassing the range of straylight values observed in human eyes, was measured by two optical methods and by the psychophysical technique. The results showed that the optical and psychophysical measurements for the seven samples were almost identical.

Effects of 400 nm, 420 nm, and 435.8 nm radiations on cultured human retinal pigment epithelial cells.

The present study demonstrates narrowband short-wavelengths radiation- (400, 420, and 435.8 nm) induced cellular damage of cultured human retinal pigment epithelial cells using in vitro biological assays to determine wavelengths that are responsible for photochemical lesions of the retina. This work involved the exposure of retinal pigment epithelial (RPE) cells (ARPE-19) to narrowband light of three different wavelengths (400, 420, and 435.8 nm) using a xenon arc lamp and interference filters. Cellular viability, mitochondrial distribution, and nucleic acid (both DNA and RNA) damage were quantified after various energy levels of exposure, using the Alamar blue assay, and confocal laser scanning microscopy with two fluorescent stains (Rhodamine 123 and Acridine Orange). The results clearly show that 400 nm light radiation can cause significant dose-dependent decreases in RPE cell viability as well as degradations of DNA/RNA and mitochondria in RPE cells, while 420 and 435.8 nm light radiation cause no cellular damage. While further evaluations may be needed to assess specificity and confounding factors of these assessment tools, the results may be a matter for consideration in future IOL design efforts.

Evolution of dim-light and color vision pigments.

A striking level of diversity of visual systems in different species reflects their adaptive responses to various light environments. To study the adaptive evolution of visual systems, we need to understand how visual pigments, the light-sensitive molecules, have tuned their wavelengths of light absorption. The molecular basis of spectral tuning in visual pigments, a central unsolved problem in phototransduction, can be understood only by studying how different species have adapted to various light environments. Certain amino acid replacements at 30 residues explain some dim-light and color vision in vertebrates. To better understand the molecular and functional adaptations of visual pigments, we must identify all critical amino acid replacements that are involved in the spectral tuning and elucidate the effects of their interactions on the spectral shifts.

Thermal recovery of iodopsin from photobleaching intermediates.

The chloride effect on the photobleaching process of iodopsin, a chicken red-sensitive cone visual pigment, was studied in detail by time-resolved low-temperature spectroscopy at -40 degrees C to -10 degrees C. Decay-associated difference spectra obtained by kinetic analysis using the singular value decomposition method were composed of spectra of BL-iodopsin, lumiiodopsin, metaiodopsin I, metaiodopsin II and metaiodopsin III, essentially identical to those at room temperature. In each conversion step however, iodopsin was partially regenerated, which is not observed in the bleaching process for other visual pigments or iodopsin at room temperature. Moreover, iodopsin was slowly regenerated from the bleached species. The reverse reactions were completely suppressed by substitution of lyotropic NO(3)(-) for Cl(-), suggesting that Cl(-) binding to iodopsin interferes with light-induced cis-trans isomerization of the chromophore. It is likely that the water molecule hydrating Cl(-) forms the additional hydrogen bond(s), by which the protein conformational change necessary to release this steric hindrance becomes enthalpic. As progress of the bleaching process is a consequence of protein conformational change, it is suppressed at low temperatures, resulting in thermal back-isomerization.

Melanopsin: an exciting photopigment.

The discovery that mice lacking rods and cones are capable of regulating their circadian rhythms by light provided the conceptual framework for the discovery of an entirely new photoreceptor system within the mammalian eye. We now know that a small subset of retinal ganglion cells are directly photosensitive and utilize an opsin/vitamin A-based photopigment called melanopsin maximally sensitive in the blue part of the spectrum. We also know that these photosensitive retinal ganglion cells mediate a broad range of physiological responses to light, ranging from the regulation of circadian rhythms to pupil constriction. Most recently, it has become clear that the melanopsins are only distantly related to visual pigments and in terms of their biochemistry share more in common with invertebrate photopigments. Here we outline the discovery of this remarkable new photoreceptor system, review the structure of melanopsin and conclude with a working model of melanopsin phototransduction.

Spectral tuning of shortwave-sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows some of the largest shifts in lambda(max), with values ranging in different species from 390-435 nm in the violet region of the spectrum to < 360 nm in the ultraviolet. Phylogenetic evidence indicates that the ancestral pigment most probably had a lambda(max) in the UV and that shifts between violet and UV have occurred many times during evolution. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UV-sensitive (UVS) pigments, it is almost certainly unprotonated. The generation of VS pigments in amphibia, birds and mammals from ancestral UVS pigments must involve therefore the stabilization of protonation. Similarly, stabilization must be lost in the evolution of avian UVS pigments from a VS ancestral pigment. The key residues in the opsin protein for these shifts are at sites 86 and 90, both adjacent to the Schiff base and the counterion at Glu113. In this review, the various molecular mechanisms for the UV and violet shifts in the different vertebrate groups are presented and the changes in the opsin protein that are responsible for the spectral shifts are discussed in the context of the structural model of bovine rhodopsin.