The 9-methyl group of retinal is essential for rapid Meta II decay and phototransduction quenching in red cones

Cone photoreceptors of the vertebrate retina terminate their response to light much faster than rod photoreceptors. However, the molecular mechanisms underlying this rapid response termination in cones are poorly understood. The experiments presented here tested two related hypotheses: first, that the rapid decay rate of metarhodopsin (Meta) II in red-sensitive cones depends on interactions between the 9-methyl group of retinal and the opsin part of the pigment molecule, and second, that rapid Meta II decay is critical for rapid recovery from saturation of red-sensitive cones after exposure to bright light. Microspectrophotometric measurements of pigment photolysis, microfluorometric measurements of retinol production, and single-cell electrophysiological recordings of flash responses of salamander cones were performed to test these hypotheses. In all cases, cones were bleached and their visual pigment was regenerated with either 11-cis retinal or with 11-cis 9-demethyl retinal, an analogue of retinal lacking the 9-methyl group. Meta II decay was four to five times slower and subsequent retinol production was three to four times slower in red-sensitive cones lacking the 9-methyl group of retinal. This was accompanied by a significant slowing of the recovery from saturation in cones lacking the 9-methyl group after exposure to bright (>0.1% visual pigment photoactivated) but not dim light. A mathematical model of the turn-off process of phototransduction revealed that the slower recovery of photoresponse can be explained by slower Meta decay of 9-demethyl visual pigment. These results demonstrate that the 9-methyl group of retinal is required for steric chromophore–opsin interactions that favor both the rapid decay of Meta II and the rapid response recovery after exposure to bright light in red-sensitive cones.     

Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase

Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.     

Aberrant metabolites in mouse models of congenital blinding diseases: formation and storage of retinyl esters

Regeneration of the visual chromophore, 11-cis-retinal, is a critical step in restoring photoreceptors to their dark-adapted conditions. This regeneration process, called the retinoid cycle, takes place in the photoreceptor outer segments and the retinal pigment epithelium (RPE). Disabling mutations in nearly all of the retinoid cycle genes are linked to human conditions that cause congenital or progressive defects in vision. Several mouse models with disrupted genes related to this cycle contain abnormal fatty acid retinyl ester levels in the RPE. To investigate the mechanisms of retinyl ester accumulation, we generated single or double knockout mice lacking retinoid cycle genes. All-trans-retinyl esters accumulated in mice lacking RPE65, but they are reduced in double knockout mice also lacking opsin, suggesting a connection between visual pigment regeneration and the retinoid cycle. Only Rdh5-deficient mice accumulate cis-retinyl esters, regardless of the simultaneous disruption of RPE65, opsin, and prRDH. 13-cis-Retinoids are produced at higher levels when the flow of retinoid through the cycle was increased, and these esters are stored in specific structures called retinosomes. Most importantly, retinylamine, a specific and effective inhibitor of the 11-cis-retinol formation, also inhibits the production of 13-cis-retinyl esters. The data presented here support the idea that 13-cis-retinyl esters are formed through an aberrant enzymatic isomerization process.     

Comparative morphology of photoreceptors in free-living plathelminths — a survey

In free-living Plathelminthes, the best-known photoreceptors are pigment-cup ocelli, eyes formed of one or several pigmented supportive cells into whose cup-shaped cavity project the light-sensitive elements of one or several sensory cells. Besides these, so-called Sehkolben, photoreceptors lacking pigment granules, are found in some species. Sensory cells in plathelminth photoreceptors most commonly use microvilli as the light-sensitive organelles, but some use cilia and combinations of microvilli and cilia. Lamellate ciliary bodies with cilia whose membranes are strongly flattened and rolled and pericerebral ciliary aggregations with interwoven cilia protruding into an intracellular cavity are likely photoreceptors in that they show amplification of membrane likely to bear photoreceptive pigments. Cells with ballooned cilia and tubular vacuoles are other differentiations to which light-sensitivity has been attributed. A variety of structures serve as lenses, all usually formed from parts of the pigment cell.     

Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.     

Scanning electron microscopy and microspectrophotometry of the photoreceptors of ictalurid catfishes

The retinal photoreceptors from larval channel catfish (Ictalurus punctatus) were studied using single cell, in situ microspectrophotometry. Rods appear at 5 days after hatch; cones are present from day one. The rods contain a visual pigment which absorbs light maximally at 540 nm. The cones contain either a green sensitive visual pigment with peak absorbance at 535 nm or a red sensitive visual pigment with peak absorbance at 608 nm. All pigments are based on vitamin A₂. Visual pigment complement does not change with age, as photoreceptors from adultI. punctatus, I. catus andI. melas contain visual pigments virtually identical to those of the larvalI. punctatus. Regardless of age, no visual pigment with peak absorbance in the short wavelength region of the spectrum was ever observed. Scanning electron microscopy of adultI. punctatus retinas showed large rods with long, cylindrical outer segments and smaller cones with short, tapered outer segments. The myoids of both rods and cones are extensable. The rods, embedded in a granular tapetal material, comprise from 50 to 60% of the photoreceptors. Only single cones are present. The data are consistent with the idea that the ictalurid catfishes spend their entire lives in an environment deficient in blue light.     

Metarhodopsin control by arrestin,light-filtering screening pigments,and visual pigment turnover in invertebrate microvillar photoreceptors

The visual pigments of most invertebrate photoreceptors have two thermostable photo-interconvertible states, the ground state rhodopsin and photo-activated metarhodopsin, which triggers the phototransduction cascade until it binds arrestin. The ratio of the two states in photoequilibrium is determined by their absorbance spectra and the effective spectral distribution of illumination. Calculations indicate that metarhodopsin levels in fly photoreceptors are maintained below ~35% in normal diurnal environments, due to the combination of a blue-green rhodopsin, an orange-absorbing metarhodopsin and red transparent screening pigments. Slow metarhodopsin degradation and rhodopsin regeneration processes further subserve visual pigment maintenance. In most insect eyes, where the majority of photoreceptors have green-absorbing rhodopsins and blue-absorbing metarhodopsins, natural illuminants are predicted to create metarhodopsin levels greater than 60% at high intensities. However, fast metarhodopsin decay and rhodopsin regeneration also play an important role in controlling metarhodopsin in green receptors, resulting in a high rhodopsin content at low light intensities and a reduced overall visual pigment content in bright light. A simple model for the visual pigment–arrestin cycle is used to illustrate the dependence of the visual pigment population states on light intensity, arrestin levels and pigment turnover.     

Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)

The spectral absorption characteristics of the retinal photoreceptors of the blue tit (Parus caeruleus) and blackbird (Turdus merula) were investigated using microspectrophotometry. The retinae of both species contained rods, double cones and four spectrally distinct types of single cone. Whilst the visual pigments and cone oil droplets in the other receptor types are very similar in both species, the wavelength of maximum sensitivity (λ_max) of long-wavelength-sensitive single and double cone visual pigment occurs at a shorter wavelength (557 nm) in the blackbird than in the blue tit (563 nm). Oil droplets located in the long-wavelength-sensitivesingle cones of both species cut off wavelengths below 570–573 nm, theoretically shifting cone peak spectral sensitivity some 40 nm towards the long-wavelength end of the spectrum. This raises the possibility that the precise λ_max of the long-wavelength-sensitive visual pigment is optimised for the visual function of the double cones. The distribution of cone photoreceptors across the retina, determined using conventional light and fluorescence microscopy, also varies between the two species and may reflect differences in their visual ecology. Accepted: 8 January 2000     

Fine structure of photoreceptors in larval trematodes

Summary The fine structure of photoreceptors is described in miracidia of Fasciola hepatica, Heronimus chelydrae, Allocreadium lobatum, and Spirorchis sp., and in a spirorchiid cercaria. All have in common eyespots consisting of pigment cells with chambers occupied by rhabdomeres consisting of retinular cell dendrites with numerous microvilli. Photoreceptors of the miracidia show a bilateral asymmetry which is most pronounced in H. chelydrae with a pair of well separated eyespots unequal in size. The smaller right one consists of a pigment cell and two rhabdomeres; the larger left eyespot has an anterior pigment cell with two rhabdomeres and a posterior cell containing one rhabdomere. Photoreceptors in the other species of miracidia also have five rhabdomeres but contain only two pigment cells which are closely apposed. Each contains a pair of lateral rhabdomeres and a fifth one occupies a posteromedian extension of the left pigment cell. In the number of rhabdomeres, their relationship to pigment cells and the resulting asymmetry, photoreceptors are more alike in the distantly related species of miracidia studied than they are in ocellate cercariae or even in the miracidium and cercaria of the same species or two closely related ones. From the asymmetry of photoreceptors in larvae of certain flatworms other than digenetic trematodes, it seems that eyespots of miracidia have retained an ancestral pattern whereas the diversity of photoreceptors in cercariae reflects the varied phototactic behavior of those larvae which complete their life cycles by all the means known for cercariae with a free-swimming period. In both miracidia and cercariae, photoreceptors show an anterior-posterior organization that would seem to be concerned with orientation of the larvae with respect to light.Supported in part by a David Ross Fellowship of the Purdue Research Foundation and in part by U.S.P.H.S. Grants 1T1 GM 1392 01 and 2T1 Al 106 07. We express thanks to Dr. Keith Dixon for aid in obtaining and processing miracidia of Fasciola hepatica; to Prof. Clark P. Read for his valuable comments and suggestions; and to Profs. Charles W. Philpott and Richard H. White for advice concerning electron microscopy.     

Ultrastructural study of the retinal pigment epithelium during metamorphosis in the sea lamprey (Petromyzon marinus L.)

Summary The sequence of morphological changes in the retinal pigment epithelium during the metamorphic period of the sea lamprey Petromyzon marinus L. has been investigated using electron microscopy. At early metamorphic stages (stages I and II), photoreceptors are present in a small zone of the retina. During these stages, the lateral surface of the epithelial cells shows zonulae occludentes and adhaerentes. The degree of cell differentiation varies throughout the retinal pigment epithelium. Cells covering the differentiated photoreceptors in the central retina have phagosomes, whereas pigment granules appear only in the retinal pigment epithelium dorsal to the optic nerve head. Most epithelial cells have myeloid bodies; their morphology is more complex around the optic nerve head. At stage III, when photoreceptors develop over the whole retina, the distribution of cytoplasmic organelles is almost homogeneous in the retinal pigment epithelium. Subsequently, the basal plasma membrane of the epithelial cells becomes progressively folded and their apical processes enlarged. In addition, extensive gap junctions develop between retinal pigment cells. In late metamorphic stages, noticeable growth of myeloid bodies occurs and consequently the retinal pigment epithelium resembles that of the adult. This study also describes, for the first time, the presence of wandering phagocytes in the retinal pigment epithelium of lampreys; their role in melanosome degradation is discussed.     

Statistics and quantum bumps in arthropod photoreceptors

Discrete waves of depolarizing membrane potential in arthropod photoreceptors, called quantum bumps, appear to result from single-photon absorptions of the visual pigment. Statistical analysis of bump records suggest a model for bump occurrence in dark-adapted receptors at low levels of illumination. This model assumes that a photon that isomerizes a visual pigment molecule can trigger a stochastic process that can produce no more than one bump under normal conditions, and that the stochastic processes triggered by different isomerized visual pigment molecules are independent of each other.     

Interphotoreceptor retinoid-binding protein (IRBP)

The regeneration of visual pigment in rod photoreceptors of the vertebrate retina requires an exchange of retinoids between the neural retina and the retinal pigment epithelium (RPE). It has been hypothesized that interphotoreceptor retinoid-binding protein (IRBP) functions as a two-way carrier of retinoid through the aqueous compartment (interphotoreceptor matrix) that separates the RPE and the photoreceptors. The first part of this review summarizes the cellular and molecular biology of IRBP. Work on the IRBP gene indicates that the protein contains a four-fold repeat structure that may be involved in binding multiple retinoid and fatty acid ligands. These repeats and other aspects of the gene structure indicate that the gene has had an active and complex evolutionary history. IRBP mRNA is detected only in retinal photoreceptors and in the pineal gland; expression is thus restricted to the two photosensitive tissues of vertebrate organisms. In the second part of this review, we consider the results obtained in experiments that have examined the activity of IRBP in the process of visual pigment regeneration. We also consider the results obtained on the bleaching and regeneration of rhodopsin in the acutely detached retina, as well as in experiments testing the ability of IRBP to protect its retinoid ligand from isomerization and oxidation. Taken together, the findings provide evidence that, in vivo, IRBP facilitates both the delivery of all-trans retinol to the RPE and the transfer of 11-cis retinal from the RPE to bleached rod photoreceptors, and thereby directly supports the regeneration of rhodopsin in the visual cycle.     

Tryptophan hydroxylase Is Required for Eye Melanogenesis in the Planarian Schmidtea mediterranea

Melanins are ubiquitous and biologically important pigments, yet the molecular mechanisms that regulate their synthesis and biochemical composition are not fully understood. Here we present a study that supports a role for serotonin in melanin synthesis in the planarian Schmidtea mediterranea. We characterize the tryptophan hydroxylase (tph) gene, which encodes the rate-limiting enzyme in serotonin synthesis, and demonstrate by RNA interference that tph is essential for melanin production in the pigment cups of the planarian photoreceptors. We exploit this phenotype to investigate the biological function of pigment cups using a quantitative light-avoidance behavioral assay. Planarians lacking eye pigment remain phototactic, indicating that eye pigmentation is not essential for light avoidance in S. mediterranea, though it improves the efficiency of the photophobic response. Finally, we show that the eye pigmentation defect observed in tph knockdown animals can be rescued by injection of either the product of TPH, 5-hydroxytryptophan (5-HTP), or serotonin. Together, these results highlight a role for serotonin in melanogenesis, perhaps as a regulatory signal or as a pigment substrate. To our knowledge, this is the first example of this relationship to be reported outside of mammalian systems.     

Ultrastructure of phaosomous photoreceptors in Stylaria lacustris (Naididae, ‘Oligochaeta’, Clitellata) and their importance for the position of the Clitellata in the phylogenetic system of the Annelida

Many species of Naididae possess a pair of pigmented eyes. Within Clitellata, eyes are generally present in Hirudinea, whereas Naididae are the only oligochaete taxon having these sense organs. The eyes of Naididae are epidermal structures and consist of a multicellular pigment cup in which a single row of five to six photoreceptor cells is embedded. The sensory cells are typical phaosomes: the photoreceptive structures (microvilli) project into a cavity formed by the sensory cell itself. In Stylaria lacustris this cavity opens to the exterior, clearly documenting that it represents an invagination of the apical cell membrane. The density of sensory microvilli is comparatively low and a central vitreous body is lacking. Similar phaosomous photoreceptors, not associated with either pigmented or unpigmented supporting cells, occur in the epidermis of the anterior end. These photoreceptors correspond to those found in other Clitellata, confirming that phaosomes are the only known type of photoreceptor cell occurring in this taxon. As a result of their simple structure they have been regarded as plesiomorphic for Annelida. However, an out‐group comparison with eyes and photoreceptors occurring in polychaetes and other spiralians reveals that they, in fact, are a rather specialized type of photoreceptor. Despite the simple structure, they most likely represent an autapomorphy of Clitellata. It follows that in all probability, these phaosomes are a secondarily evolved type of photoreceptor, which arose within the oligochaete clade after the primary photoreceptors present in the out‐groups had been lost. This loss might have occurred during evolution of a burrowing life style within the sediment and subsequent invasion of the terrestrial environment.     

The Polarity of the Retinal Pigment Epithelium

The diversity of epithelia in the body permits a multitude of organ-specific functions. One of the foremost examples of this is the retinal pigment epithelium. Located between the photoreceptors of the retina and their principal blood supply, the choriocapillaris, the retinal pigment epithelium is critical for the survival and function of retinal photoreceptors. To serve this purpose, the retinal pigment epithelium cell has adapted the classic Golgi-to-cell-surface targeting pathways first described in such prototypic epithelial cell models as the Madin-Darby canine kidney cell, to arrive at a unique distribution of membrane and secreted proteins. More recent data suggest that the retinal pigment epithelium also takes advantage of its inherent asymmetry to augment the classical pathways of Golgi-to-cell-surface traffic. As retinal pigment epithelium transplants and gene therapy represent potential cures for retinal degenerative diseases, understanding the basis of the unique polarity properties of retinal pigment epithelium cells will be a critical issue for the development of future therapies.     

Heme-mediated reactive oxygen species toxicity to retinal pigment epithelial cells is reduced by hemopexin

Catalysis of the formation of reactive oxygen species (RO₂S) by low molecular weight complexes of iron has been implicated in several pathological conditions in the retina since photoreceptors and retinal pigment epithelial cells are likely to be especially sensitive to RO₂S. Since protective proteins cannot cross the blood-retinal barrier, it is likely that the retina performs its own protective functions by synthesizing proteins that bind iron and nonprotein iron complexes, the major catalysts of RO₂S generation. Investigations were carried out to determine whether pigment epithelial cells are themselves sensitive to iron-generated RO₂S and whether apo-transferrin and apo-hemopexin, known to be made locally in the retina, can perform a protective function. In ⁵¹Cr release assays, the toxicity of exogenous RO₂S including hydrogen peroxide or superoxide (generated by xanthine oxidase/hypoxanthine) to human retinal pigment epithelial cells was inhibited by the iron chelators, desferrioxamine and apo-transferrin. Free but not protein-bound ferric iron and heme exacerbated the toxic effect. The toxic effect of heme was abolished by the heme-scavenging, extracellular antioxidant, apo-hemopexin, and also by exogenous bovine serum albumin. In addition, heme toxicity was inhibited by a 3 h preincubation of cells with either heme, apo-hemopexin, or heme-hemopexin 24 h prior to the toxicity assay. It is concluded, first, that toxic effects of iron and heme can be prevented by apo-transferrin or apo-hemopexin and, second, that exposure of RPE cells to free heme or hemopexin sets in motion a series of biochemical events resulting in protection against oxidative stress. It is probable that these include heme oxygenase induction. © 1996 Wiley-Liss, Inc.     

Pigment migration and illumination of single photoreceptors in a moth

Summary The relation between intensity of illumination of single photoreceptors and position of the screening pigment was studied in the compound eye of the moth Deilephila elpenor. The amplitude of the response to test flashes of white light was measured in 7 dark adapted photoreceptor cells with the screening pigment in the extreme distal and in a proximal position. The experiments demonstrate that the response amplitude of individual photoreceptors varies with the pigment position. The associated variation in screening effect was 1.0 to 1.9 log units.The research reported in this study was supported by Swedish Medical Research Council, Sällskapet för Medicinsk Forskning and Stifteisen Gustaf och Tyra Svenssons Minne.     

Light exposure during embryonic and yolk-sac alevin development of Chinook salmon Oncorhynchus tshawytscha does not alter the spectral phenotype of photoreceptors

Colour vision is mediated by the expression of different visual pigments in photoreceptors of the vertebrate retina. Each visual pigment is a complex of a protein (opsin) and a vitamin A chromophore; alterations to either component affects visual pigment absorbance and, potentially, the visual capabilities of an animal. Many species of fish undergo changes in opsin expression during retinal development. In the case of salmonid fishes the single cone photoreceptors undergo a switch in opsin expression from SWS1 (ultraviolet sensitive) to SWS2 (blue-light sensitive) starting at the yolk-sac alevin stage, around the time when they first experience light. Whether light may initiate this event or produce a plastic response in the various photoreceptors is unknown. In this study, Chinook salmon Oncorhynchus tshawytscha were exposed to light from the embryonic (5 days prior to hatching) into the yolk sac alevin (25 days post hatching) stage and the spectral phenotype of photoreceptors assessed with respect to that of unexposed controls by in situ hybridization with opsin riboprobes. Light exposure did not change the spectral phenotype of photoreceptors, their overall morphology or spatial arrangement. These results concur with those from a variety of fish species and suggest that plasticity in photoreceptor spectral phenotype via changes in opsin expression may not be a widespread occurrence among teleosts.     

Sunscreen for Fish: Co-Option of UV Light Protection for Camouflage

Many animals change their body pigmentation according to illumination of their environment. In aquatic vertebrates, this reaction is mediated through aggregation or dispersion of melanin-filled vesicles (melanosomes) in dermal pigment cells (melanophores). The adaptive value of this behavior is usually seen in camouflage by allowing the animal to visually blend into the background. When exposed to visible light from below, however, dark-adapted zebrafish embryos at the age of 2 days post fertilization (dpf) surprisingly display dispersal instead of aggregation of melanosomes, i.e. their body coloration becomes dark on a bright background. Melanosomes of older embryos and early larvae (3–5 dpf) on the other hand aggregate as expected under these conditions. Here we provide an explanation to this puzzling finding: Melanosome dispersion in larvae 3 dpf and older is efficiently triggered by ultraviolet (UV) light, irrespective of the visual background, suggesting that the extent of pigmentation is a trade-off between threats from predation and UV irradiation. The UV light-induced dispersion of melanosomes thereby is dependent on input from retinal short wavelength-sensitive (SWS) cone photoreceptors. In young embryos still lacking a functional retina, protection from UV light predominates, and light triggers a dispersal of melanosomes via photoreceptors intrinsic to the melanophores, regardless of the actual UV content. In older embryos and early larvae with functional retinal photoreceptors in contrast, this light-induced dispersion is counteracted by a delayed aggregation in the absence of UV light. These data suggest that the primary function of melanosome dispersal has evolved as a protective adaption to prevent UV damage, which was only later co-opted for camouflage.