Rod Photopigment Research Articles

The Impact of Photopigment Bleaching on the Human Rod Photoreceptor Subretinal Space Measured Via Optical Coherence Tomography.

The purpose of this study was to investigate rod photopigment bleaching-driven intrinsic optical signals (IOS) in the human outer retina and its measurement repeatability based on a commercial optical coherence tomography (OCT) platform. The optical path length of the rod photoreceptor subretinal space (SRS), that is, the distance between signal bands of rod outer segment tips and retinal pigment epithelium, was measured in 15 healthy subjects in ambient light and during a long-duration bleaching white-light exposure. On 2 identical study days (day 1 and day 2 [D1 and D2]), light stimulation resulted in a significant decrease in rod SRS by 21.3 ± 7.6% and 19.8 ± 8.5% (both P<0.001), respectively. The test-retest reliability of the SRS maximum change of an individual subject was moderate for single measures (intraclass correlation coefficient [ICC] = 0.730, 95% confidence interval [CI] = 0.376, 0.900, P<0.001) and good for average measures (ICC = 0.844, 95% CI = 0.546, 0.947, P<0.001). The mean area under the stimulus response curve with values of 14.8 ± 9.4 and 15.5 ± 7.5µm × minutes (P=0.782) showed excellent agreement between the stimulus response on D1 and D2. Intermittent dark adaptation of the retina led to an initial increase of the SRS by 6.1% (P=0.018) and thereafter showed a decrease toward baseline, despite continued dark adaptation. The data indicate the potential of commercial OCT in measuring slow IOS in the outer retina suggesting that the rod SRS could serve as a biomarker for photoreceptor function. The presented approach could provide an easily implementable clinical tool for the early detection of diseases affecting photoreceptor health.

Human retinal dark adaptation tracked in vivo with the electroretinogram: insights into processes underlying recovery of cone- and rod-mediated vision.

The substantial time taken for regaining visual sensitivity (dark adaptation) following bleaching exposures has been investigated for over a century. Psychophysical studies yielded the classic biphasic curve representing recovery of cone-driven and rod-driven vision. The electroretinogram (ERG) permits direct assessment of recovery at the level of the retina (photoreceptors, bipolar cells), with the first report over 70 years ago. Over the last two decades, ERG studies of dark adaptation have generated insights into underlying physiological processes. After large bleaches, rod photoreceptor circulating current, estimated from the rod-isolated bright-flash ERG a-wave, takes 30min to recover, indicating that products of bleaching, thought to be free opsin (unbound to 11-cis-retinal), continue to activate phototransduction, shutting off rod circulating current. In contrast, cone current, assessed with cone-driven bright-flash ERG a-waves, recovers within 100ms following similar exposures, suggesting that free opsin is less able to shut off cone current. The cone-driven dim-flash a-wave can be used to track recovery of cone photopigment, showing regeneration is 'rate-limited' rather than first order. Recoveries of the dim-flash ERG b-wave are consistent also with rate-limited rod photopigment regeneration (where free opsin, desensitising the visual system as an 'equivalent background', is removed by rate-limited delivery of 11-cis-retinal). These findings agree with psychophysical and retinal densitometry studies, although there are unexplained points of divergence. Post-bleach ERG recovery has been explored in age-related macular degeneration and in trials of visual cycle inhibitors for retinal diseases. ERG tracking of dark adaptation may prove useful in future clinical contexts.

Heinrich Müller (1820-1864) and the entoptic discovery of the site in the retina where vision is initiated

ABSTRACT Heinrich Müller was a nineteenth-century German retinal anatomist who, during his short career, was one of the discoverers of the rod photopigment rhodopsin and neuroglia in the retina, now known as Müller cells. He also described the ocular muscles and double foveae of some birds. An important, but largely neglected, insight by Müller was to combine careful psychophysical measurements and geometrical optics to find the location of the photosensitive layer of the retina in the living eye. Here, we provide translated passages from Müller’s (1855) publication and compare his entoptic observations with retinal imaging using optical coherence tomography. Müller correctly deduced from his careful experiments that vision is initiated in the photoreceptors located in the back of the retina.

Vision using multiple distinct rod opsins in deep-sea fishes.

Vertebrate vision is accomplished through light-sensitive photopigments consisting of an opsin protein bound to a chromophore. In dim light, vertebrates generally rely on a single rod opsin [rhodopsin 1 (RH1)] for obtaining visual information. By inspecting 101 fish genomes, we found that three deep-sea teleost lineages have independently expanded their RH1 gene repertoires. Among these, the silver spinyfin (Diretmus argenteus) stands out as having the highest number of visual opsins in vertebrates (two cone opsins and 38 rod opsins). Spinyfins express up to 14 RH1s (including the most blueshifted rod photopigments known), which cover the range of the residual daylight as well as the bioluminescence spectrum present in the deep sea. Our findings present molecular and functional evidence for the recurrent evolution of multiple rod opsin-based vision in vertebrates.

Behavioural guidance of Chinook salmon smolts: the variable effects of LED spectral wavelength and strobing frequency.

Exploiting species-specific behavioural responses of fish to light is an increasingly promising technique to reduce the entrainment or impingement of fish that results from the diversion of water for human activities, such as hydropower or irrigation. Whilst there is some evidence that white light can be an effective deterrent for Chinook salmon smolts, the results have been mixed. There is a need to test the response of fish to different spectra and strobing frequencies to improve deterrent performance. We tested the movement and spatial response of groups of four fish to combinations of light-emitting diode (LED) spectra (red, green, blue and white light) during the day and night, and strobing frequencies (constant and 2Hz) during the day, using innovative LED technology intended as a behavioural guidance device for use in the field. Whilst strobing did not alter fish behaviour when compared to constant light, the red light had a repulsive effect during the day, with fish under this treatment spending significantly less time in the half of the arena closest to the behavioural guidance device compared to both the control and blue light. Importantly, this effect disappeared at night, where there were no differences in movement and space use found between spectra. There was some evidence of a potential attractive response of fish to the blue and green light during the day. Under these light treatments, fish spent the highest amount of time closest to the behavioural guidance device. Further tests manipulating the light intensity in the different spectra are needed to verify the mechanistic determinants of the observed behaviours. Results are discussed in reference to the known spectral sensitivities of the cone and rod photopigments in these fish, and further experiments are suggested to better relate the work to mitigating the effects on fish of infrastructure used for hydropower and irrigation.

Spectral sensitivity of the electroretinogram b-wave in dark-adapted Prussian carp (Carassius gibelio Bloch, 1782).

One of the purposes of this study was to examine whether b-wave measurements can be used in the evaluation of scotopic spectral sensitivity in Prussian carp measurements when the eyes were surgically deprived of cornea, lens, and most of the vitreous. Another goal was testing the new fitting procedure for A2-based photopigments. Using fitted amplitude-log intensity functions for threshold calculation, and two models for computer-assisted fitting of spectral sensitivity curves, no significant differences in λ(max) were found between rod photopigments and b-wave-based spectral sensitivity.

Middle- and long-wavelength discrimination declines with rod photopigment regeneration

Hue-discrimination functions were derived from hue-naming data (480-620 nm, 20 nm steps) obtained in 4 min intervals from 4 min to 28 min postbleach at 10° temporal retinal eccentricity. Hue-naming data were also obtained in the fovea. Hue-discrimination functions derived at the 4, 8, and 12 min intervals were very similar to those derived in the fovea. As time postbleach exceeded 12 min and rod sensitivity increased, the shape of the hue-discrimination functions changed. Most notably, the minimum between 560-580 nm disappeared and the just noticeable differences (JNDs) for the longer wavelength stimuli increased. The long-wavelength suppression in hue discrimination may be due to rod input in the magnocellular pathway interacting and affecting the long-wavelength sensitivity of the parvocellular pathway.

Diversity of Color Vision: Not All Australian Marsupials Are Trichromatic

Color vision in marsupials has recently emerged as a particularly interesting case among mammals. It appears that there are both dichromats and trichromats among closely related species. In contrast to primates, marsupials seem to have evolved a different type of trichromacy that is not linked to the X-chromosome. Based on microspectrophotometry and retinal whole-mount immunohistochemistry, four trichromatic marsupial species have been described: quokka, quenda, honey possum, and fat-tailed dunnart. It has, however, been impossible to identify the photopigment of the third cone type, and genetically, all evidence so far suggests that all marsupials are dichromatic. The tammar wallaby is the only Australian marsupial to date for which there is no evidence of a third cone type. To clarify whether the wallaby is indeed a dichromat or trichromatic like other Australian marsupials, we analyzed the number of cone types in the “dichromatic” wallaby and the “trichromatic” dunnart. Employing identical immunohistochemical protocols, we confirmed that the wallaby has only two cone types, whereas 20–25% of cones remained unlabeled by S- and LM-opsin antibodies in the dunnart retina. In addition, we found no evidence to support the hypothesis that the rod photopigment (rod opsin) is expressed in cones which would have explained the absence of a third cone opsin gene. Our study is the first comprehensive and quantitative account of color vision in Australian marsupials where we now know that an unexpected diversity of different color vision systems appears to have evolved.

Summary of the findings of the RLBP1 mutations affecting the visual cycle known so far with extremely prolonged dark adaptation in the RP of Bothnia type

Abstract Purpose The retinal diseases in the RP group associated with affected recycling of retinoids in the visual cycle is now being recognized in populations worldwide. The RLBP1 mutations are here summarized and the phenotype known so far is presented. Methods A presentation of literature in the RLBP1 mutations and the phenotype of Bothnia Dystrophy (BD) with ophthalmologic findings and full‐field electroretinography (ERGs) findings after 24 hours of dark adaptation are presented. Results The phenotype (BD) in this group is described, an early progressive maculopathy, retinitis punctata albescens (RPA) as well as peripheral retinal degenerative changes is found. An extremely prolonged dark adaptation (24h) in the BD disease is presented and the full‐field ERG (24h) show the rod b‐wave and the mixed rod‐cone a‐wave responses reached normal but delayed amplitudes in the ERGs, the increase of the oscillatory response up to normal level was found and no recovery of the cone response was found. Conclusion The phenotypes of RLBP 1 mutations worldwide and in Sweden show similarities in the expression and may be represented by the phenotype of BD. The unique findings of extremely prolonged DA and a significant and additional capacity of recovery of rod function and activity in the inner retinal layer and a continuous but slow regeneration of rod photopigment seems to occur at least up to 24h. The visual process in the RPE is retarded but also the Müller cells of the retina seem to be involved. The findings support an extremely slow synthesis of photopigments and an irreversibly disturbed cone function early in BD.

Macular pigment, photopigments, and melanin: Distributions in young subjects determined by four-wavelength reflectometry

We have developed an objective procedure, using a modified retinal camera, to determine macular pigment (MP) optical density distributions in the human retina. Using two multi-band filters, reflectance maps of the retinas of young subjects (<25 years old) were obtained at 460, 528, 610 and 670nm, without pupil dilation. The log-transformed maps were combined linearly to yield optical density maps of MP, cone and rod photopigments, and melanin. MP optical density and heterochromatic flicker photometry results for 22 subjects were in reasonable agreement. Cone photopigments, like MP, showed similar, well-defined peaks at the fovea, whereas rod photopigment showed a minimum. Melanin was more broadly distributed.

Effects of prolonged dark adaptation in patients with retinitis pigmentosa of Bothnia type: an electrophysiological study

Bothnia dystrophy (BD) is a variant of recessive retinitis punctata albescens (RPA), caused by the missense mutation R233W in cellular retinaldehyde-binding protein (CRALBP), which is localized in the retinal pigment epithelium (RPE) and Müller cells of the retina. The purpose of this study was, by examining the electrophysiological responses of the retina, to evaluate the capacity of recovery of the whole retinal area and different cell types induced by extremely prolonged dark adaptation (DA) in BD disease and to gain further understanding of the pathogenesis of BD. Six young patients underwent bilateral full-field ERGs after 24 h of DA in one eye and standard DA in the fellow eye. The results were also compared with the effect of prolonged DA (10 h), previously studied in the same patients. After extremely prolonged DA (24 h) the rod b-wave and the mixed rod-cone a-wave responses reached normal though delayed amplitudes. An increase, up to normal level, in the oscillatory response was found. There was no obvious recovery of the cone response. We conclude that in young BD patients during extremely prolonged DA there is a significant additional capacity of recovery of rod function and also significant gain of activity in the inner retinal layer. A continuous but slow regeneration of rod photopigment seems to occur at least up to 24 h. The visual process in the RPE is retarded and CRALBP acts in this process; also, the Müller cells of the retina seem to be involved. The findings also support an extremely slow synthesis of photopigments and irreversibly disturbed cone function early in BD.

A proposed role for all- trans retinal in regulation of rhodopsin regeneration in human rods

In order to account for the multi-phasic dynamics of photopigment regeneration in human rods, we developed a new model of the retinoid cycle. We first examined the relative roles of the classical and channeling mechanisms of metarhodopsin decay in establishing these dynamics. We showed that neither of these mechanisms alone, nor a linear combination of the two, can adequately account for the dynamics of rhodopsin regeneration at all bleach levels. Our new model adds novel inhibitory interactions in the cycle of regeneration of rhodopsin that are consistent with the 3D structure of rhodopsin. Our analyses show that the dynamics of human rod photopigment regeneration can be accounted for by end-product regulation of the channeling mechanism where all- trans retinal ( tral) inhibits the binding of 11- cis retinal to the opsin. tral complex.

Retinoid cycles in the cone-dominated chicken retina

In past decades, the role of retinoids in support of rod photopigment regeneration has been extensively characterized. In the rhodopsin cycle, retinal chromophore from bleached rod pigments is reduced to retinol and transferred to the retinal pigment epithelium (RPE) to store as all-trans retinyl ester. This ester pool is subsequently utilized for visual pigment regeneration. However, there is a lack of information on the putative cone visual cycle. In the present study, we provide experimental evidence in support of a novel retinoid cycle for cone photopigment regeneration. In the cone-rich chicken, light exposure resulted in the accumulation of 11-cis retinyl esters to the retina and all-trans retinyl esters to the RPE. Both the rate of increase and the amount of 11-cis retinyl esters in the retina far exceeded those of the all-trans retinyl esters in the RPE. In response to dark adaptation, this 11-cis retinyl ester pool in the retina depletes at a rate several times faster than the all-trans retinyl ester pool in the RPE. In vitro, isolated, dark-adapted retinas devoid of RPE show both an accumulation of 11-cis retinyl ester and a concomitant reduction of 11-cis retinal chromophore in response to light exposure. Finally, we provide experimental results to elucidate a cone visual cycle in chicken by relating the change in retinoids (retinal and retinyl ester) with time during light and dark adaptation. Our results support a new paradigm for cone photopigment regeneration in which the 11-cis retinyl ester pool in the retina serves as the primary source of visual chromophore for cone pigment regeneration.

Are there advantages in implanting a yellow IOL to reduce the risk of AMD?

Are there advantages in implanting a yellow IOL to reduce the risk of AMD?

Recovery of Visual Functions in a Mouse Model of Leber Congenital Amaurosis

The visual process is initiated by the photoisomerization of 11-cis-retinal to all-trans-retinal. For sustained vision the 11-cis-chromophore must be regenerated from all-trans-retinal. This requires RPE65, a dominant retinal pigment epithelium protein. Disruption of the RPE65 gene results in massive accumulation of all-trans-retinyl esters in the retinal pigment epithelium, lack of 11-cis-retinal and therefore rhodopsin, and ultimately blindness. We reported previously (Van Hooser, J. P., Aleman, T. S., He, Y. G., Cideciyan, A. V., Kuksa, V., Pittler, S. J., Stone, E. M., Jacobson, S. G., and Palczewski, K. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8623-8628) that in Rpe65-/- mice, oral administration of 9-cis-retinal generated isorhodopsin, a rod photopigment, and restored light sensitivity to the electroretinogram. Here, we provide evidence that early intervention by 9-cis-retinal administration significantly attenuated retinal ester accumulation and supported rod retinal function for more than 6 months post-treatment. In single cell recordings rod light sensitivity was shown to be a function of the amount of regenerated isorhodopsin; high doses restored rod responses with normal sensitivity and kinetics. Highly attenuated residual rod function was observed in untreated Rpe65-/- mice. This rod function is likely a consequence of low efficiency production of 11-cis-retinal by photo-conversion of all-trans-retinal in the retina as demonstrated by retinoid analysis. These studies show that pharmacological intervention produces long lasting preservation of visual function in dark-reared Rpe65-/- mice and may be a useful therapeutic strategy in recovering vision in humans diagnosed with Leber congenital amaurosis caused by mutations in the RPE65 gene, an inherited group of early onset blinding and retinal degenerations.

Rapid restoration of visual pigment and function with oral retinoid in a mouse model of childhood blindness

Mutations in the retinal pigment epithelium gene encoding RPE65 are a cause of the incurable early-onset recessive human retinal degenerations known as Leber congenital amaurosis. Rpe65-deficient mice, a model of Leber congenital amaurosis, have no rod photopigment and severely impaired rod physiology. We analyzed retinoid flow in this model and then intervened by using oral 9-cis-retinal, attempting to bypass the biochemical block caused by the genetic abnormality. Within 48 h, there was formation of rod photopigment and dramatic improvement in rod physiology, thus demonstrating that mechanism-based pharmacological intervention has the potential to restore vision in otherwise incurable genetic retinal degenerations.

Origin and functional impact of dark noise in retinal cones.

Spontaneous fluctuations in the electrical signals of the retina's photoreceptors impose a fundamental limit on visual sensitivity. While noise in the rods has been studied extensively, relatively little is known about the noise of cones. We show that the origin of the dark noise in salamander cones varies with cone type. Most of the noise in long wavelength-sensitive (L) cones arose from spontaneous activation of the photopigment, which is a million-fold less stable than the rod photopigment rhodopsin. Most of the noise in short wavelength-sensitive (S) cones arose in a later stage of the transduction cascade, as the photopigment was relatively stable. Spontaneous pigment activation effectively light adapted L cones in darkness, causing them to have a smaller and briefer dim flash response than S cones.

Mutations in the gene encoding 11-cis retinol dehydrogenase cause delayed dark adaptation and fundus albipunctatus.

The metabolic pathways that produce 11-cis retinal are important for vision because this retinoid is the chromophore residing in rhodopsin and the cone opsins. The all-trans retinal that is generated after cone and rod photopigments absorb photons of light is recycled back to 11-cis retinal by the retinal pigment epithelium and Müller cells of the retina. Several of the enzymes involved have recently been purified and molecularly cloned; here we focus on 11-cis retinol dehydrogenase (encoded by the gene RDH5; chromosome 12q13-14; ref. 4), the first cloned enzyme in this pathway. This microsomal enzyme is abundant in the retinal pigment epithelium, where it has been proposed to catalyse the conversion of 11-cis retinol to 11-cis retinal. We evaluated patients with hereditary retinal diseases featuring subretinal spots (retinitis punctata albescens and fundus albipunctatus) and patients with typical dominant or recessive retinitis pigmentosa for mutations in RDH5. Mutations were found only in two unrelated patients, both with fundus albipunctatus; they segregated with disease in the respective families. Recombinant mutant 11-cis retinol dehydrogenases had reduced activity compared with recombinant enzyme with wild-type sequence. Our results suggest that mutant alleles in RDH5 are a cause of fundus albipunctatus, a rare form of stationary night blindness characterized by a delay in the regeneration of cone and rod photopigments.

Opsin localization and rhodopsin photochemistry in a transgenic mouse model of retinitis pigmentosa

The VPP mouse is a transgenic strain carrying three mutations (P23H, V20G, P27L) near the N-terminus of opsin, the apoprotein of rhodopsin, the rod photopigment. These animals exhibit a slowly progressive degeneration of the rod photoreceptors, and concomitant changes in retinal function that mimic those seen in humans with autosomal dominant retinitis pigmentosa resulting from a point mutation (P23H) in opsin. In the present study we attempted to determine whether the disease process prevents the translocation of mutant opsin to the rod outer segments of transgenic mice, and whether it affects the photochemical properties of the rhodopsin present within their rod outer segments. Immunocytochemistry with a monoclonal antibody against a region of the C-terminus that recognizes epitopes common to both normal and mutant opsin (monoclonal antibody-1D4), and a polyclonal antibody that reacts preferentially with the mutant opsin (anti-VPP), were used to identify the opsin present in the rods of three-week-old VPP mice and normal littermates. Absorbance spectra, photosensitivity, and regeneration kinetics of rhodopsin in rod outer segment disc membranes were analysed by spectrophotometry. Western blot analysis with anti-VPP antibody indicated the specific binding of this antibody to the mutant opsin. Immunolocalization with monoclonal antibody-1D4 and anti-VPP antibodies suggested a normal translocation of the mutant protein to the outer segments. Aside from a small disparity in the absorbance spectra of rhodopsin obtained from normal and VPP retinas, there were no significant differences in either the ability of opsin to bind 11- cis retinal chromophore, or in the photic sensitivity of rhodopsin. The results indicate that mutant opsin is translated and incorporated into the rod outer segment disc membranes of VPP mice, and that the photochemical properties of rhodopsin in the rods of VPP retinas are similar to those of rhodopsin in normal retinas.

Variation in Sensitivity, Absorption and Density of the Central Rod Distribution with Eccentricity

Purpose: To assess the human rod photopigment distribution and sensitivity with high spatial resolution within the central ±15° and to compare the results of pigment absorption, sensitivity and rod density distribution (number of rods per square degree). Method: Rod photopigment density distribution was measured with imaging densitometry using a modified Rodenstock scanning laser ophthalmoscope. Dark-adapted sensitivity profiles were measured with green stimuli (17′ arc diameter, 1° spacing) using a Tübingen manual perimeter. Sensitivity profiles were plotted on a linear scale and rod photopigment optical density distribution profiles were converted to absorption profiles of the rod photopigment layer. Results: Both the absorption profile of the rod photopigment and the linear sensitivity profile for green stimuli show a minimum at the foveal center and increase steeply with eccentricity. The variation with eccentricity corresponds to the rod density distribution. Conclusion: Rod photopigment absorption profiles, retinal sensitivity profiles, and the rod density distribution are linearly related within the central ±15°. This is in agreement with theoretical considerations. Both methods, imaging retinal densitometry using a scanning laser ophthalmoscope and dark-adapted perimetry with small green stimuli, are useful for assessing the central rod distribution and sensitivity. However, at present, both methods have limitations. Suggestions for improving the reliability of both methods are given.