Cone Pigments Research Articles

Parapinopsin, a Novel Catfish Opsin Localized to the Parapineal Organ, Defines a New Gene Family

Multiple sites of extraretinal photoreception are present in vertebrates, but the molecular basis of extraretinal phototransduction is poorly understood. This study reports the cloning of the first opsin specifically expressed in the directly photosensitive pineal and parapineal of cold-blooded vertebrates. This opsin, identified in channel catfish and termed parapinopsin, defines a new gene family of vertebrate photopigments and is expressed in a majority of parapinealocytes and a subset of pineal photoreceptor cells. Parapinopsin shows a caudal-rostral gradient of expression within the pineal organ. This study also reports the cloning of partial cDNAs encoding the channel catfish orthologues of rhodopsin and the red cone pigment-the full complement of retinal opsins in the species. In situ hybridization studies using probes derived from these retinal opsins, together with parapinopsin, reveal no expression of retinal opsins in pineal and parapineal organ and no expression of any opsin tested in the "deep brain," iris, or dermal melanophores. These data imply that phototransduction in these sites of extraretinal photoreception must be mediated by novel opsins.

Filtering effect of cone oil droplets detected in the P-111 response spectra of japanese quail

While absorption spectra of bird cone visual pigments have been well studied, physiological study of bird cone cells has been less advanced owing to their small sizes. We measured the P-III components of electroretinograms (ERG) from isolated retinas of Japanese quail. We recorded responses to monochromatic flashes of equal photon numbers, and found that the shape of the response spectrum is dependent on the incident direction of the flashes. The spectrum obtained with the flashes from the cornea side had a steeper peak around 500 nm than that with flashes from the receptor side. This is clear electrophysiological evidence of the filtering effect of the oil droplets in the cone cells, which has long been suspected. We analyzed these spectra with respect to the absorption spectra of cone pigments and transmittance spectra of oil droplets.

Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeon

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. As expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). In Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38 187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pineal gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen.

The cAMP analog 8-Cl-cAMP inhibits growth and induces differentiation and apoptosis in retinoblastoma cells.

Retinoblastomas appear to be derived from a multipotential stem cell of the retina, due to alterations of the Rb1 gene. These tumors arise only within a discrete time frame during childhood, prior to terminal differentiation of the retinal precursor cells. Treatment of retinoblastoma cells with certain agents can induce a partial differentiation of cell types resembling those of the mature retina, such as rod and cone photoreceptors, glia, conventional neurons and pigment epithelia. We have tested the effects of 8-Cl-cAMP, a synthetic analog of cAMP which preferentially binds to and activates the RII subunit of protein kinase A on the Y-79 retinoblastoma cell line in vitro. Y-79 cells treated with 8-Cl-cAMP produced short, branching processes and showed a substantial increase in staining for neuron-specific enolase, a marker for conventional neuronal differentiation. In contrast, dibutyryl-cAMP gives a strong increase in the glial marker glial acidic fibrillary protein. Y-79 cell proliferation was strongly inhibited by 8-Cl-cAMP at concentrations as low as 5-25 microM. 8-Cl-cAMP significantly increased the rate of apoptosis of Y-79 cells in a dose-dependent manner. It also modulated expression of the RI regulatory subunit of intracellular cAMP-dependent protein kinase A, which is produced in abnormal quantities by Y-79 cells. A decrease in protein production was observed, with no clear effect on the RI subunit mRNA expression, suggesting that RI regulation occurs post-transcriptionally.

Photopic transduction implicated in human circadian entrainment

Despite the preeminence of light as the synchronizer of the circadian timing system, the phototransductive machinery in mammals which transmits photic information from the retina to the hypothalamic circadian pacemaker remains largely undefined. To determine the class of photopigments which this phototransductive system uses, we exposed a group ( n=7) of human subjects to red light below the sensitivity threshold of a scotopic (i.e. rhodopsin/rod-based) system, yet of sufficient strength to activate a photopic (i.e. cone-based) system. Exposure to this light stimulus was sufficient to reset significantly the human circadian pacemaker, indicating that the cone pigments which mediate color vision can also mediate circadian vision.

Spectral sensitivity of macaque monkeys measured with ERG flicker photometry

Macaque monkeys are widely used as a model species for investigations of the biology of human vision. Previous measurements suggest that the cone-based spectral sensitivity of these two primates is greatly similar, but perhaps not identical. We measured the photopic spectral sensitivity of 42 male macaque monkeys from two species (Macaca mulatta, M. fascicularis) using an objective index, electroretinogram flicker photometry. The variations among individuals and between the two species were very small and there was no evidence for any significant cone pigment polymorphism in this sample. There are small but systematic differences in spectral sensitivity between macaque monkeys and equivalently tested human subjects--the monkeys were slightly more sensitive to short wavelengths (< 520 nm) and slightly less sensitive to wavelengths longer than this value. The results obtained from the curve fitting of standard photopigment absorption spectra to the spectral-sensitivity functions suggest that the difference between human and macaque monkey spectral sensitivity principally reflects differences in the relative proportions of the long- and middle-wavelength cones in the retinas of the two species.

The Pax2 homolog sparkling is required for development of cone and pigment cells in the Drosophila eye.

A new Drosophila Pax gene, sparkling (spa), implicated in eye development, was isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system and in cone, primary pigment, and bristle cells of larval and pupal eye discs. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. We suggest close functional analogies between Spa and Pax2 in the development of the insect and vertebrate eye.

Mechanisms of spectral tuning in the mouse green cone pigment.

Diversification of cone pigment spectral sensitivities during evolution is a prerequisite for the development of color vision. Previous studies have identified two naturally occurring mechanisms that produce variation among vertebrate pigments by red-shifting visual pigment absorbance: addition of hydroxyl groups to the putative chromophore binding pocket and binding of chloride to a putative extracellular loop. In this paper we describe the use of two blue-shifting mechanisms during the evolution of rodent long-wave cone pigments. The mouse green pigment belongs to the long-wave subfamily of cone pigments, but its absorption maximum is 508 nm, similar to that of the rhodopsin subfamily of visual pigments, but blue-shifted 44 nm relative to the human red pigment, its closest homologue. We show that acquisition of a hydroxyl group near the retinylidene Schiff base and loss of the chloride binding site mentioned above fully account for the observed blue shift. These data indicate that the chloride binding site is not a universal attribute of long-wave cone pigments as generally supposed, and that, depending upon location, hydroxyl groups can alter the environment of the chromophore to produce either red or blue shifts.

Visual pigments and oil droplets from six classes of photoreceptor in the retinas of birds

Microspectrophotometric examination of the retinal photoreceptors of the budgerigar (shell parakeet), Melopsittacus undulatus (Psittaciformes) and the zebra finch, Taeniopygia guttata (Passeriformes), demonstrate the presence of four, spectrally distinct classes of single cone that contain visual pigments absorbing maximally at about 565, 507, 430–445 and 360–380 nm. The three longer-wave cone classes contain coloured oil droplets acting as long pass filters with cut-offs at about 570, 500–520 and 445 nm, respectively, whereas the ultraviolet-sensitive cones contain a transparent droplet. The two species possess double cones in which both members contain the long-wave-sensitive visual pigment, but only the principal member contains an oil droplet, with cut-off at about 420 nm. A survey of the cones of the pigeon, Columba livia (Columbiformes), confirms the presence of the three longer-wave classes of single cone, but also reveals the presence of a fourth class containing a visual pigment with maximum absorbance at about 409 nm, combined with a transparent droplet. No evidence was found for a fifth, ultraviolet-sensitive receptor. In the chicken, Gallus gallus (Galliformes), the cone class with a transparent droplet contains “chicken violet” with maximum absorbance at about 418 nm. The rods of all four species contain visual pigments that are spectrally similar, with maximum absorbance between about 506 and 509 nm. Noticeably, in any given species, the maximum absorbance of the rods is spectrally very similar the maximum absorbance of the middle-wavelength-sensitive cone pigments.

Photic Environment, Visual Pigments, and the Limits of the Visible Spectrum

The limits of the visible spectrum are set by the light available for vision, and by the visual pigment absorbance. The hundreds of visual pigments studied to the present day have absorbance maxima spread within the range from 350 to 620 nm. Yet this diversity is used for vision quite nonuniformly: rod and cone visual pigments are tightly clustered around a few preferred positions in the spectrum, eg near 500 nm in the rods of land animals. The so-called ‘sensitivity hypothesis’ assumes that the clustering is to maximise the number of absorbed photons available in the animals' light environment. In most cases, however, visual pigments are substantially more short-wave (blue-shifted) than is necessary for maximum quantal absorption. Examples of the ‘blue shift’ are the Purkinje shift during cone - rod transition in dark adaptation, the hypsochromic shift of rod visual pigments in deep-water fish, and a similar shift in the cone pigments of geckos and some snakes as a result of evolutionary adaptation to nocturnal habits. It is argued that an important limiting factor in vision is the dark noise produced by thermal isomerisation of the chromophore. Measurements of the dark noise in rods with different visual pigments show that the noise increases steeply when the absorbance maximum is shifted to longer wavelengths, thus precluding the use of long-wave pigments for vision at low intensities. The optimum spectral position of a pigment may be that which ensures a maximum light-to-noise ratio in a particular photic environment.

Colour Perception 1978–1997

In the past twenty years, the spectral sensitivities of the three types of cone have been established with some certainty: direct measurements by microspectrophotometry and electrophysiology are in fair agreement with psychophysical estimates. Particularly significant was the publication of DNA sequences for the four opsins of the human eye, by Jeremy Nathans and colleagues in 1986. This work was soon to transform the understanding of retinitis pigmentosa and other retinal dystrophies, and it has given many insights into the evolution of colour vision; but, curiously, the explanations of dichromacy and anomalous trichromacy have not proved as straightforward as we all expected in 1986. What is clear, however, is that normal colour vision exhibits a genetic polymorphism: much of the intersubject variance in colour matches can be traced to differences in the amino-acid sequence of the opsins for the long-wave and middle-wave cone pigments. The last two decades have seen a major change in the status of opponent processes. In the 1970s it was still common for professors to tell undergraduates that the Young - Helmholtz theory of colour vision held at the receptor level and the Hering theory at the level of the retinal ganglion cells. It is now clear that the chromatically antagonistic processes revealed electrophysiologically and psychophysically in the early visual system do not correspond to the red - green and yellow - blue processes that Hering postulated on the basis of phenomenological observations. The existence of four unique hues is today one of the unexplained mysteries of colour science. In one salient respect, research in colour vision has been changed by instrumental advances. Computer-controlled monitors (though offering splendid pitfalls to the unwary) have allowed the study of spatially and temporally complex chromatic displays, notably in the field of colour constancy. Most recently there has been interest in the chromatic statistics of natural scenes: how well is the visual system matched to the statistics of the world and can it adapt to the gamut of chromaticities present in a given scene?

Modifying a Rodenstock scanning laser ophthalmoscope for imaging densitometry

The necessary modifications and technical requirements are described for using a commercially available scanning laser ophthalmoscope (Rodenstock Model 101 SLO) as an imaging densitometer to assess human photopigment distribution. The main requirements are a linear detector amplifier, fast shutters for the laser beams, and a trigger unit. Images must be compensated for varying laser intensity. Both rod and cone photopigments are measured with the 514-nm argon laser of the SLO. Discrimination is possible owing to the different spatial distribution. The cone pigment density peaks in the foveal center (D = 0.40) with a steep decrease with increasing eccentricity E (full width at half-maximum, 2.5 degrees ). Rod photopigment increases with increasing eccentricity (D = 0.23 for E = 11 degrees ). These values are in agreement with previous reported results obtained with scanning laser ophthalmoscopes specially designed for retinal densitometry and high stability.

Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group.

The lozenge locus is genetically complex, containing two functionally distinct units, cistrons A and B, that influence the structure of the compound eye. Extreme mutations of either cistron produce adult phenotypes that share similarities and that have striking differences. We have analyzed the expression of several developmentally important eye genes including boss, scabrous, rhomboid, seven-up, and Bar in lozenge mutant backgrounds representing both cistrons. This analysis follows the progressive recruitment of photoreceptor neurons during eye development and has confirmed that the initial development of photoreceptors is normal up to the five cell precluster stage (R8, R2/5 and R3/4). However, when lozenge is mutant, further eye development is perturbed. As cells R1, R6 and R7 are recruited, patterns of gene expression for seven-up and Bar become abnormal. We have also characterized the expression of two different enhancer trap alleles of lozenge. The lozenge product(s) appear to be first expressed in the eye disc in undifferentiated cells shortly after the five cell precluster forms. Then, as distinct cells are recruited to a fate, lozenge expression persists and is refined in those cells. Our data suggests that lozenge functions in cone cells and pigment cells as well as in specific glia. With respect to photoreceptor neurons, lozenge biases the developmental potential of cells R1, R6 and R7, by directly influencing the expression of genes important for establishing cell fate.

The Visual Photopigments of Simple Deuteranomalous Trichromats Inferred from Color Matching

Deuteranomalous trichromacy is the most common form of inherited color-vision deficiency. A modern description of its cause is a single abnormality: the normal middle-wave cone photopigment (M) is replaced by a shifted middle-wave pigment (M) that is shared by all deuteranomalous trichromats. This explanation, however, fails to account for the individual differences in color vision observed even within the sub-group of deuteranomals with good chromatic discrimination. An ensemble of color matches is used here to test whether these individual differences reflect differences in the wavelength of peak sensitivity (lambda max) of individual deuteranomals' cone photopigments. The results show variation in both the lambda max and the effective optical density of their cone pigments. The individual differences found in lambda max are in accord with recent molecular biological research that shows individual differences in the genes thought to encode deuteranomalous photopigments.

Chromatic and monochromatic optical resolution in the rainbow trout

The modulation transfer function due to measured longitudinal chromatic aberration was calculated for the otherwise unaberrated eye of the adult rainbow trout ( Oncorhynchus mykiss) under daylight conditions assuming light absorption by single retinal cone pigments, and by photopic mechanisms involving interaction between cones. The adult trout eye, with its large immobile pupil, is limited by chromatic aberration to resolution much lower than the diffraction limit, consistent with the low acuity reported for fish. This low resolution can be considered a design trade-off cost of a bright image. The measured monochromatic modulation transfer function is similar to that calculated due to chromatic aberration alone, showing that these independent aberrations are approximately balanced in the fish eye. The effect of changes in receptor length, pigment density, water depth, and pupil size upon the chromatic resolution was calculated. The calculated chromatic modulation transfer function will hold approximately for other teleost eyes with lens larger than about 1 mm.

Single amino acid residue as a functional determinant of rod and cone visual pigments.

The visual transduction processes in rod and cone photoreceptor cells begin with photon absorption by the different types of visual pigments. Cone visual pigments exhibit faster regeneration from 11-cis-retinal and opsin and faster decay of physiologically active intermediate (meta II) than does the rod visual pigment, rhodopsin, as expected, due to the functional difference between rod and cone photoreceptor cells. To identify the amino acid residue(s) responsible for the difference in molecular properties between rod and cone visual pigments, we selected three amino acid positions (64, 122, and 150), where cone visual pigments have amino acid residues electrically different from those of rhodopsin, and prepared mutants of rhodopsin and chicken green-sensitive cone visual pigment. The results showed that the replacement of Glu-122 of rhodopsin by the residue containing green- or red-sensitive cone pigment converted rhodopsin's rates of regeneration and meta II decay into those of the respective cone pigments, whereas the introduction of Glu-122 into green-sensitive cone visual pigment changed the rates of these processes into rates similar to those of rhodopsin. Furthermore, exchange of the residue at position 122 between rhodopsin and chicken green-sensitive cone pigment interchanges their efficiencies in activating retinal G protein transducin. Thus, the amino acid residue at position 122 is a functional determinant of rod and cone visual pigments.

Visual Pigment Assignments in Regenerated Retina

Retinas of adult teleost fish can regenerate after injury. Two important issues regarding this phenomenon are the assembly of the regenerated retina and the neuronal images of the visual scene that the regenerated retina produces. Here we report experiments in which the visual pigment content of photoreceptors derived from native and regenerated sunfish retinas was determined by microspectrophotometry. In native retina, there is an apparently perfect correspondence between cone morphology and visual pigment content; all rods contain a middle-wavelength pigment, all single cones contain a different middle-wavelength pigment, and all double cone members contain a long-wavelength pigment. The visual pigments in regenerated rods and double cones were the same as in native retina; however, triple cones, a morphology never observed in native retina, contained the long-wavelength pigment. Moreover, although approximately 60% of regenerated single cones contained the expected middle-wavelength pigment, all other single cones contained the long-wavelength pigment. This mismatch between morphology of regenerated single cones and their visual pigment assignment indicated the following: (1) There is a degree of independence between the mechanisms that establish cone morphology and pigment content during regeneration, which suggests that cone photoreceptor regeneration is not a straightforward recapitulation of the normal cone photoreceptor developmental plan. (2) Although anomalous, the long-wavelength single cones may enable regenerated retina to restore the native spectral sampling of the visual scene.

Rhodopsin: A Prototypical G Protein-Coupled Receptor

A variety of spectroscopic and biochemical studies of recombinant site-directed mutants of rhodopsin and related visual pigments have been reported over the past 9 years. These studies have elucidated key structural elements common to visual pigments. In addition, systematic analysis of the chromophore-binding pocket in rhodopsin and cone pigments has led to an improved understanding of the mechanism of the opsin shift, and of particular molecular determinants underlying color vision in humans. Identification of the conformational changes that occur on rhodopsin photoactivation has been of particular recent concern. Assignments of light-dependent molecular alterations to specific regions of the chromophore have also been attempted by studying native opsins regenerated with synthetic retinal analogs. Site-directed mutagenesis of rhodopsin has also provided useful information about the retinal-binding pocket and the molecular mechanism of rhodopsin photoactivation. Individual molecular groups have been identified to undergo structural alterations or environmental changes during photoactivation. Analysis of particular mutant pigments in which specific groups are locked into their respective "off" or "on" states has provided a framework to identify determinants of the active conformation, as well as the minimal number of intramolecular transitions required to switch between inactive and active conformations. A simple model for the active state of rhodopsin can be compared to structural models of its ground state to localize chromophore-protein interactions that may be important in the photoactivation mechanism. This review focuses on the recent functional characterization of site-directed mutants of bovine rhodopsin and some cone pigments. In addition, an attempt is made to reconcile previous key findings and existing structural models with information gained from the analysis of site-directed mutant pigments.

Bilirubin, Rem Sleep, and Phototransduction of Environmental Time Cues. A Hypothesis

The prevailing hypothesis for phototransduction is that visual (rod or cone) pigments mediate light's primary effects on biological clock systems. Common light-responsive chronobiological behavioral properties of plants and animals and some common molecular structures of plants and animals suggest the possibility that heme moieties and bile pigments in animals mediate some nonvisual influences of light on neuroactive gases and biological rhythms. As plant phytochrome resets the plant biological clock, the similar chromophore in bile pigments is proposed to transduce environmental light zeitgeber signals to endogenous biological clocks. The temporal association of plasma bilirubin and rapid-eye-movement (REM) sleep in populations, the correlation of secretion of biliary bilirubin with REM sleep among 10 different species (Spearman r = 0.89, p < 0.002), and the known responses of bilirubin to light lead to the hypothesis that bilirubin, in particular, plays an evolutionary role in the regulation of REM sleep and in mediating some of light's antidepressant effects. (Chronobiology International, 14(3), 319–329, 1997)

Functional expression of human cone pigments using recombinant baculovirus: compatibility with histidine tagging and evidence for N-glycosylation

Mammalian color vision is mediated by light-sensitive pigments in retinal cone cells. Biochemical studies on native mammalian cone visual pigments are seriously hampered by their low levels and instability. We describe a novel approach for their functional expression, employing the baculovirus system in combination with histidine tagging to allow future purification and structural analysis. The human red and green cone pigments are produced in relatively large amounts and can be detected by immunocytochemistry as well as by immunoblotting. Histidine tagging has no significant effect on the absorbance maxima. The first evidence is presented that these pigments are N-glycosylation.