Cone Photopigments Research Articles

Comment on "Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment"

Jacobs et al. (Reports, 23 March 2007, p. 1723) reported that plasticity in the mammalian visual system permitted the emergence of "a new dimension of sensory experience" in mice genetically engineered to express a human long-wavelength-sensitive cone photopigment. However, neither neural plasticity nor a new dimension of sensory experience is required to explain their results.

Response to Comment on "Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment"

Makous suggests that the novel color vision documented in knock-in mice neither requires visual system plasticity nor implies the emergence of a new dimension of sensory experience. We explain why we disagree.

Role of the Fas-Signaling Pathway in Photoreceptor Neuroprotection

To determine whether inhibiting the Fas proapoptosis pathway will result in increased photoreceptor survival after separation of the retina from the retinal pigment epithelium (RPE). Retina/RPE separation was induced in rat and mouse eyes by the subretinal injection of hyaluronic acid, 1%. Fas-pathway signaling was inhibited by the concomitant injection of a Fas receptor-neutralizing antibody, small inhibitory RNA against the Fas-receptor transcript (siFAS), or the use of the Fas-receptor defective mouse strain LPR. Indices of photoreceptor death included terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, cell counts, and retinal thickness measurements. Retinas were immunostained with antibodies against rhodopsin and cone opsin to evaluate rod and cone photopigment production, respectively. Inhibition of Fas signaling using Fas receptor-neutralizing antibody, siFas, or LPR mice resulted in a significant reduction in the number of TUNEL-positive photoreceptor cells as well as in a significant preservation of outer nuclear layer cell counts and thickness as compared with retina/RPE separation in eyes with intact Fas signaling. Fas-pathway inhibition resulted in preservation of both rhodopsin- and cone opsin-positive cells. Inhibition of the Fas proapoptosis pathway results in significant photoreceptor preservation after retinal separation from the RPE. Fas-pathway inhibition might serve as a novel mechanism for preserving photoreceptor cells during retinal disease.

Dissociation of rod and cone sensitivity by acute localized retinal pigment epithelium loss

To assess the impact of acute retinal pigment epithelium (RPE) loss on photopic and scotopic sensitivity. A 68-year-old woman who had been followed for drusenoid RPE detachment in age-related macular degeneration presented with an acute spontaneous retinal pigment epithelium tear. Three months later, she was seen for routine follow-up and was examined by manual photopic and scotopic threshold perimetry (a static 0.46-degree-diameter 660 nm stimulus under photopic conditions; then, following 25 min of dark adaptation, a static 0.46-degree-diameter 532 nm stimulus under scotopic conditions). The stimuli were applied over the RPE defect and at reference points of similar eccentricity in the opposite vertical haemifield of the same eye where the RPE remained present. Acute RPE loss was associated with only a marginal reduction of photopic sensitivity (-1.5 dB) but a pronounced loss of scotopic sensitivity (-19.5 dB). Our observations show that RPE is essential for scotopic but not for photopic retinal function, supporting the theory that cone photopigment regeneration occurs within the human neurosensory retina independently of the RPE.

Contributions of the mouse UV photopigment to the ERG and to vision

The mouse retina contains two classes of cone photopigment with respective peak sensitivities in the middle (M) wavelengths and in the ultraviolet (UV) portion of the spectrum. To examine the functional roles subserved by the UV pigment, the absorption of light by the mouse lens was measured and voltage versus intensity (V-log I) functions were derived from recordings of the flicker ERG made under test conditions designed to maximize the relative sensitivities of the two pigment types. These V-log I data accurately predict ERG-based spectral sensitivity functions, but they fail to provide a similarly accurate account of behaviorally based measurements of spectral sensitivity in that the ERG spectral sensitivity function has much higher sensitivity in the UV wavelengths than does the behavioral spectral sensitivity function. The disparity between these two is argued to be a consequence of the widespread receptor co-expression of the two types of cone pigment in the mouse and of the pattern of retinal wiring that is thought to be characteristic of all mammalian retinas.

Emergence of Novel Color Vision in Mice Engineered to Express a Human Cone Photopigment

Changes in the genes encoding sensory receptor proteins are an essential step in the evolution of new sensory capacities. In primates, trichromatic color vision evolved after changes in X chromosome-linked photopigment genes. To model this process, we studied knock-in mice that expressed a human long-wavelength-sensitive (L) cone photopigment in the form of an X-linked polymorphism. Behavioral tests demonstrated that heterozygous females, whose retinas contained both native mouse pigments and human L pigment, showed enhanced long-wavelength sensitivity and acquired a new capacity for chromatic discrimination. An inherent plasticity in the mammalian visual system thus permits the emergence of a new dimension of sensory experience based solely on gene-driven changes in receptor organization.

Simultaneous measurement of current and calcium in the ultraviolet‐sensitive cones of zebrafish

In rods and visible cone photoreceptors, multiple measurements cannot be made of intracellular Ca2+ concentration from the same cell using fluorescent dyes, because a single exposure of the measuring light bleaches too large a fraction of the rod or cone photopigment. We have therefore identified and characterized UV-sensitive cones of the zebrafish, whose wavelength of maximum sensitivity is at 360 nm which is far enough from the wavelength of our measuring light (514.5 nm) so that it has been possible to make multiple determinations of photocurrent and Ca2+ concentration from the same cells. We show that for a limited number of measurements, for which the bleaching of the cone photopigment is too small to affect flash kinetics, the outer segment Ca2+ concentration closely follows the wave form of the flash response convolved with the dominant time constant for Ca2+ removal by Na+-Ca2+-K+ exchange. For a larger number of measurements, significant acceleration of the response kinetics by pigment bleaching inevitably occurs, but the Ca2+ concentration nevertheless rises and falls in approximate agreement with the flash wave form. During exposure to steady background light, the Ca2+ concentration falls in proportion to the steady-state current for dim backgrounds at all times and for bright backgrounds at steady state. At early times following the onset of bright backgrounds, however, the Ca2+ concentration is markedly higher than expected from the current of the cone. We show this to be the result of light-dependent Ca2+ release by bright background light, which can be abolished by pre-exposure of the cone to the membrane-permeant acetoxymethyl ester of the Ca2+ chelator BAPTA. Our results therefore demonstrate that the cone outer segment Ca2+ concentration is predominantly a function of the rate of influx and efflux of Ca2+ across the plasma membrane, but that a release of Ca2+ in bright light most probably from buffer sites within the cell can transiently elevate the Ca2+ concentration above the level expected from the open probability of the light-dependent channels.

Mutational changes in S‐cone opsin genes common to both nocturnal and cathemeralAotusmonkeys

Aotus is a platyrrhine primate that has been classically considered to be nocturnal. Earlier research revealed that this animal lacks a color vision capacity because, unlike all other platyrrhine monkeys, Aotus has a defect in the opsin gene that is required to produce short-wavelength sensitive (S) cone photopigment. Consequently, Aotus retains only a single type of cone photopigment. Other mammals have since been found to show similar losses and it has often been speculated that such change is in some fashion tied to nocturnality. Although most species of Aotus are indeed nocturnal, recent observations show that Aotus azarai, an owl monkey species native to portions of Argentina and Paraguay, displays a cathemeral activity pattern being active during daylight hours as frequently as during nighttime hours. We have sequenced portions of the S-cone opsin gene in A. azarai and Aotus nancymaae, the latter a typically nocturnal species. The S-cone opsin genes in both species contain the same fatal defects earlier detected for Aotus trivirgatus. On the basis of the phylogenetic relationships of these three species these results imply that Aotus must have lost a capacity for color vision early in its history and they also suggest that the absence of color vision is not compulsively linked to a nocturnal lifestyle.

Light‐Induced Melatonin Suppression in Humans with Polychromatic and Monochromatic Light

The relative contribution of rods, cones, and melanopsin to non‐image‐forming (NIF) responses under light conditions differing in irradiance, duration, and spectral composition remains to be determined in humans. NIF responses to a polychromatic light source may be very different to that predicted from the published human action spectra data, which have utilized narrow band monochromatic light and demonstrated short wavelength sensitivity. To test the hypothesis that only melanopsin is driving NIF responses in humans, monochromatic blue light (λmax 479 nm) was matched with polychromatic white light for total melanopsin‐stimulating photons at three light intensities. The ability of these light conditions to suppress nocturnal melatonin production was assessed. A within‐subject crossover design was used to investigate the suppressive effect of nocturnal light on melatonin production in a group of diurnally active young male subjects aged 18–35 yrs (24.9±3.8 yrs; mean±SD; n=11). A 30 min light pulse, individually timed to occur on the rising phase of the melatonin rhythm, was administered between 23:30 and 01:30 h. Regularly timed blood samples were taken for measurement of plasma melatonin. Repeated measures two‐way ANOVA, with irradiance and light condition as factors, was used for statistical analysis (n=9 analyzed). There was a significant effect of both light intensity (p<0.001) and light condition (p<0.01). Polychromatic light was more effective at suppressing nocturnal melatonin than monochromatic blue light matched for melanopsin stimulation, implying that the melatonin suppression response is not solely driven by melanopsin. The findings suggest a stimulatory effect of the additional wavelengths of light present in the polychromatic light, which could be mediated via the stimulation of cone photopigments and/or melanopsin regeneration. The results of this study may be relevant to designing the spectral composition of polychromatic lights for use in the home and workplace, as well as in the treatment of circadian rhythm disorders.

A low-power, LED-based, high-brightness anomaloscope

Color matches made with a Nagel anomaloscope are used in the differentiation of color vision deficiencies. When these color matches are made over a wide range of retinal illuminances, the changes in the color match provide information about the regeneration kinetics and the absorption spectra of the middle- and long-wavelength cone photopigments. These steady-state color matches vary with a variety of conditions, and may have value in screening for eye disease. Recently, high-brightness LEDs have become available that allowed us to construct a LED-based, high-brightness anomaloscope. We used inexpensive, low-energy components to replicate an earlier instrument, getting a maximum retinal illuminance over 5.6 log Trolands.

Convergence of experimental multiple Rayleigh matches to peak L- and M-photopigment sensitivity estimates

Shift in the wavelength of peak sensitivity of the cone photo pigments is a major cause of inter-individual variations in the Rayleigh match. Normal color observers performed multiple Rayleigh matches (i.e., a series of seven Rayleigh-type color matches using various sets of test and primary lights) in order to derive estimates of the M- and L-photo pigment wavelengths of maximum absorbance. We predicted the ratio of incident radiances Pr/Pg for a range of plausible values in terms of wavelength of the peak sensitivity of the M-cone and L-cone. An algorithm allowed us to adjust the values of the peak wavelength of the cone photo pigments in order to minimize the sum of squared differences between predicted and real results of matches. To create candidate M-cone and L-cone photo pigments that best predict the set of equation values, we used the low density spectral absorbance curves of the M-cone and L-cone photo pigments respectively, as tabulated by Stockman and Sharpe (2000), and shifted each template along a logarithmic wavelength scale. For all observers, the system of seven Rayleigh-like equations converges to a unique solution. M- and L-cone photo pigments are estimated separately. The distribution of the wavelength of maximum sensitivity of the photo pigments includes a cluster. Only a few women's results lie outside the cluster. The choice of the template has a considerable influence on the convergence of the algorithm.

L and M cone proportions in polymorphic New World monkeys

Platyrrhine monkeys typically have only a single X-chromosome opsin gene. Alleles of this gene code for multiple versions of middle- to long-wavelength cone photopigments. X-chromosome inactivation provides heterozygous females with a retinal mosaic of cones containing either of two types of M and L pigment, thus establishing the photopigment basis for trichromatic color vision. This study examined the proportions of L and M cones created by this process. For that purpose, electroretinogram flicker photometry was used to obtain complete spectral sensitivity functions from 60 heterozygous female monkeys drawn from seven genera of platyrrhine monkeys. To obtain estimates of cone proportions, these functions were subsequently fit with linear combinations of L and M cone fundamentals that were derived from similar recordings made on conspecific animals having only one type of M/L pigment. Consistent with a random X-chromosome inactivation process, the average L:M cone weighting across the sample was close to unity. At the same time, there were significant individual variations in L:M cone proportions. The genesis of this variation and its implications for seeing are discussed.

A novel mutation in the short-wavelength-sensitive cone pigment gene associated with a tritan color vision defect

Inherited tritan color vision deficiency is caused by defects in the function of the short-wavelength-sensitive (S) cones. This heterozygous group of disorders has an autosomal dominant pattern of inheritance. Amino acid variations of the S cone opsin are rare and all that have been identified thus far are associated with inherited tritan color vision defects. Here we report the identification of a 30-year-old male who made errors on standard color vision tests consistent with the presence of a mild tritan color vision deficiency. We tested the hypothesis that his color vision impairment was due to a mutation in the S cone photopigment gene. He was found to be heterozygous for a mutation that caused the amino acid proline to be substituted in place of a highly conserved leucine at amino acid position 56 in the S cone opsin. This mutation was absent in 564 S cone photopigment genes from 282 subjects who did not make tritan errors. Thus, we conclude that this mutation disrupts the normal function of S cones.

Cone photopigment absorption assessed from retinal afterimage

Cone photopigment absorption assessed from retinal afterimage

Cone excitation ratios correlate with color discrimination performance in the horse (Equus caballus).

Six horses (Equus caballus) were trained to discriminate color from grays in a counterbalanced sequence in which lightness cues were irrelevant. Subsequently, the pretrained colors were presented in a different sequence. Two sets of novel colors paired with novel grays were also tested. Performance was just as good in these transfer tests. Once the horse had learned to select the chromatic from the achromatic stimulus, regardless of the specific color, they were immediately able to apply this rule to novel stimuli. In terms of the underlying visual mechanisms, the present study showed for the first time that the spectral sensitivity of horse cone photopigments, measured as cone excitation ratios, was correlated with color discrimination performance, measured as accuracy, repeated errors, and latency of approach.

The selection of coloured stimuli by the horse (Equus caballus)

Colour is an important feature that increases the visibility of objects and may aid recognition of con-specifics. The adaptive significance of the ability to utilize colour cues has been demonstrated in a number of species, in particular in relation to food selection. The evolution of trichromatic colour vision in primates has been associated with the advantages that it provided in the selection of yellow and orange fruits (Mollon, 1989) or tender young red foliage (Dominy and Lucas, 2001) from a background of green. The horse is a generalist herbivore that must both select nutritious plants and avoid toxic ones. Familiarity is an important factor in this selection process (Augneret al., 1998), although it is not clear exactly what features are used to recognise previously ingested and “safe” herbage.The horse has been shown to possess dichromatic colour vision. There are two types of cone photopigment in the equine retina, with spectral peaks at 429 and 545 nanometres (Macuda, 2000).

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.

Organization of the Human Trichromatic Cone Mosaic

Using high-resolution adaptive-optics imaging combined with retinal densitometry, we characterized the arrangement of short- (S), middle- (M), and long- (L) wavelength-sensitive cones in eight human foveal mosaics. As suggested by previous studies, we found males with normal color vision that varied in the ratio of L to M cones (from 1.1:1 to 16.5:1). We also found a protan carrier with an even more extreme L:M ratio (0.37:1). All subjects had nearly identical S-cone densities, indicating independence of the developmental mechanism that governs the relative numerosity of L/M and S cones. L:M cone ratio estimates were correlated highly with those obtained in the same eyes using the flicker photometric electroretinogram (ERG), although the comparison indicates that the signal from each M cone makes a larger contribution to the ERG than each L cone. Although all subjects had highly disordered arrangements of L and M cones, three subjects showed evidence for departures from a strictly random rule for assigning the L and M cone photopigments. In two retinas, these departures corresponded to local clumping of cones of like type. In a third retina, the L:M cone ratio differed significantly at two retinal locations on opposite sides of the fovea. These results suggest that the assignment of L and M pigment, although highly irregular, is not a completely random process. Surprisingly, in the protan carrier, in which X-chromosome inactivation would favor L- or M-cone clumping, there was no evidence of clumping, perhaps as a result of cone migration during foveal development.

Polymorphic New World monkeys with more than three M/L cone types

Most New World (platyrrhine) monkeys have M/L cone photopigment polymorphisms that map directly into individual variations in visual sensitivity and color vision. We used electroretinogram flicker photometry to examine M/L cone photopigments in the New World monkey Callicebus moloch (the dusky Titi). Like other New World monkeys, this species has an M/L cone photopigment polymorphism that reflects the presence of X-chromosome opsin gene alleles. However, unlike other platyrrhines in which three M/L photopigments are typical, Callicebus has a total of five M/L cone photopigments. The peak sensitivity values for these pigments extend across the range from 530 to 562 nm. The result is an enhanced array of potential color vision phenotypes in this species.

Autofluorescence Characteristics of Normal Foveas and Reconstruction of Foveal Autofluorescence from Limited Data Subsets

To develop mathematical and geometric models of the nonuniform autofluorescence (AF) patterns of foveas of normal subjects and to reconstruct these models from limited subsets of data. Confocal scanning laser ophthalmoscope (cSLO) AF fundus images of normal maculae were obtained from both eyes of 10 middle-aged subjects. They were filtered and contrast enhanced, to obtain elliptical isobars of equal gray levels (GLs) and determine the isobars' resolutions, eccentricities, and angles of orientation. The original image data were fit with a mathematical model of elliptic quadratic polynomials in two equal zones: the center and the remaining annulus. The AF images segmented into nested concentric GL isobars with GLs that increased radially from the least-fluorescent center. The mean isobar resolution was 31 +/- 7 mum. The geometric eccentricity of the ellipses increased from 0.42 +/- 0.12 centrally to 0.52 +/- 0.14 peripherally (P = 0.0005), with mean axes of orientation peripherally 97.12 +/- 15.46 degrees . The model fits to the complete image data had mean absolute normalized errors ranging from 3.6% +/- 3.7% to 7.3% +/- 7.1%. The model fits to small subsets (1% to 2% of total image data) had mean absolute errors ranging from 3.7% +/- 3.8% to 7.3% +/- 7.2%. Normal AF fundus images show finely resolved, concentric, elliptical foveal patterns consistent with the anatomic distribution of fluorescent lipofuscin, light-attenuating macular pigment (MP), cone photopigment, and retinal pigment epithelial (RPE) pigment in the fovea. A two-zone, elliptic, quadratic polynomial model can accurately model foveal data. This model may be useful for image analysis and for automated segmentation of pathology.