The visual pigment rhodopsin (rh1) constitutes the first step in the sensory transduction cascade in the rod photoreceptors of the vertebrate eye, forming the basis of vision at low light levels. In most vertebrates, rhodopsin is a single-copy gene whose function in rod photoreceptors is highly conserved. We found evidence for a second rhodopsin-like gene (rh1-2) in the zebrafish genome. This novel gene was not the product of a zebrafish-specific gene duplication event and contains a number of unique amino acid substitutions. Despite these differences, expression of rh1-2in vitro yielded a protein that not only bound chromophore, producing an absorption spectrum in the visible range (λmax ≈ 500 nm), but also activated in response to light. Unlike rh1, rh1-2 is not expressed during the first 4 days of embryonic development; it is expressed in the retina of adult fish but not the brain or muscle. Similar rh1-2 sequences were found in two other Danio species, as well as a more distantly related cyprinid, Epalzeorhynchos bicolor. While sequences were only identified in cyprinid fish, phylogenetic analyses suggest an older origin for this gene family. Our study suggests that rh1-2 is a functional opsin gene that is expressed in the retina later in development. The discovery of a new previously uncharacterized opsin gene in zebrafish retina is surprising given its status as a model system for studies of vertebrate vision and visual development.

Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander (Ambystoma tigrinum). The delay period (Te) that preceded recovery of the dark current by a criterion amount (3 pA) was analyzed in relation to the flash intensity (If), and to the corresponding fractional bleach (R*0/Rtot) of the visual pigment; R*0/Rtot was compared with R*s/Rtot the fractional bleach at which the peak level of activated transducin approaches saturation. Over an approximately 8 In unit range of If that included the predicted value of R*s/Rtot, Te increased linearly with In If. Within the linear range, the slope of the function yielded an apparent exponential time constant (TC) of 1.7 ± 0.2 s (mean ± S.D.). Background light reduced the value of Tc measured at a given flash intensity but preserved a range over which Tc increased linearly with In If; the linear-range slope was similar to that measured in the absence of background light. The intensity dependence of Tc resembles that of a delay (Td) seen in light-scattering experiments on bovine retinas, which describes the period of essentially complete activation of transducin following a bright flash; the slope of the function relating Td and In flash intensity is thought to reflect the lifetime of photoactivated visual pigment (R*) (Pepperberg et al., 1988; Kahlert et al., 1990). The present data suggest that the electrophysiological delay has a similar basis in the deactivation kinetics of R*, and that Tc represents TR* the lifetime of R* in the phototransduction process. The results furthermore suggest a preservation of the “dark-adapted” value of TR* within the investigated range of background intensity.

R1–6 dominated electroretinographic (ERG) spectral sensitivities were determined as a function of days posteclosion from carotenoid deprived and replaced white-eyed Drosophila. The sensitivity of flies deprived from egg to adult waxed (about 1.5 log units by day 3), and then waned gradually from 3–11 days (over 2 log units by day 11). Carotenoid replacement (feeding nothing but carrot juice) effected recovery to near the replete control' level in about 1 day throughout (tested at 0, 4, and 11 days). The normal yellow cornmeal-agar-molasses-brewers yeast fly food (in our laboratory, supplemented with β-carotene) renders a slower recovery (requiring 7–9 days) since it is a medium designed largely for larval growth. Placing replete adults on deprivational medium did not create a deprivational syndrome in over 11 days. At 3–7 days, deprived flies reared and maintained in constant darkness had substantially enhanced sensitivity, beyond the 1.5 log unit increment already described for cyclic light rearing conditions. All spectral analyses are consistent with the ultraviolet (UV) sensitization of the blue (480 nm) rhodopsin by a replacement-dependent retinoid including two unexpected findings: (1) sensitivity recovery with carrot juice was so fast that the UV peak was already high at 6 h; and (2) the waxing of the deprived fly's sensitivity in dark rearing was so great that the UV peak was present at 4–7 days.

Following bright flashes, rod photoreceptors exhibit a period of photocurrent saturation that increases linearly with the logarithm of flash intensity. In a recent report, Pepperberg et al. (1992) presented evidence that the slope of the function relating the saturation period (T) to the natural logarithm of flash intensity (In If) represents the exponential lifetime (τ) of photoactivated visual pigment: τ = ΔT/Δ[ln If]. In salamander rods, 11 -cis 9-desmethylretinal combines with opsin to form 9-desmethyl rhodopsin. Dim flash responses mediated by this analogue visual pigment exhibited slow recovery kinetics relative to those of native pigment (Corson et al., 1991). This observation raises the hypothesis that the physiological lifetime of photoactivated 9-desmethyl rhodopsin is substantially longer than that of native visual pigment. To test this hypothesis, we have examined the relation between the period of photocurrent saturation and flash intensity in salamander rods containing a mixture of the two pigments. Brief stimuli at two widely separated wavelengths (440 and 640 nm) elicited saturating photocurrent responses that were preferentially mediated by 9-desmethyl rhodopsin or residual native pigment, respectively. Plots of T vs. In If revealed a linear increase in the period of response saturation over a large range of saturating intensities at both wavelengths. However, the slope of the relation between T and In If with 440-nm flashes was more than twice as large (4.1 ± 0.5 s, n = 5) as that measured with 640-nm flashes (1.7 ± 0.4 s). For rods subjected only to bleaching of the native pigment, or to bleaching and resensitization with 11-cis retinal, the slope of the relation between T and In If remained independent of wavelength and indistinguishable from that of native pigment in unbleached cells. The data provide support for the hypothesis that the slope parameter τ represents the lifetime of photoactivated pigment, and specifically suggest that the lifetime of photoactivated 9-desmethyl rhodopsin is abnormally long.

We examined a white-eyed strain of the norpA mutant (norpA;cn bw) and white (w)norpA+ controls using microspectrophotometry (MSP), electron microscopy (EM), and electroretinography (ERG). These studies revealed that light mediates receptor demise in norpA even though norpA lacks phototransduction. Rhodopsin and the rhabdomere which houses it decrease with increasing age in norpA but not in w with rearing on a 12 h light/12-h dark cycle or in constant light. At higher temperature in norpA;cn bw and w reared in constant light, visual pigment decreases, rhabdomeres diminish, and cells die. Importantly, dark rearing blocked visual pigment loss in norpA;cn bw; the M-potential, an ERG reflection of visual pigment level, corroborated this finding. MSP showed that norpA's visual pigment loss was not due to acute loss of metarhodopsin, rhodopsin's photoproduct. NorpA blocks certain processes expected to be light elicited. The alteration of visual pigment as a function of time of day, present in w controls, is absent in white-eyed norpA, suggesting that light-induced depolarization may be necessary to entrain the rhythm. Microspectrofluorometry using the fluorescent dye, Lucifer yellow, suggested that norpA lacks a light-induced uptake mechanism; using control flies, we determined the stimulus parameters required for uptake in vivo. An attempt to “cure” norpA;cn bw by replacement “therapy” using phospholipase C, missing in norpA's phototransduction cascade, was largely unsuccessful.

A photon-counting retinal densitometer is described that has been designed optically and electronically for improved sensitivity and reliability. The device allows measurement of visual pigments through the undilated natural pupils of subjects at relatively low levels of measuring lights, and serves also as an adaptometer for direct comparisons between pigment bleaching or regeneration and light or dark adaptation. Instrumental control and data collection are by computer to permit rapid and simple data analysis and comparisons between subjects. The methods by which the sensitivity and reliability have been enhanced are described in detail, and some examples of experimental results are presented.

Stomatopod crustaceans possess apposition compound eyes that contain more photoreceptor types than any other animal described. While the anatomy and physiology of this complexity have been studied for more than two decades, few studies have investigated the molecular aspects underlying the stomatopod visual complexity. Based on previous studies of the structure and function of the different types of photoreceptors, stomatopod retinas are hypothesized to contain up to 16 different visual pigments, with 6 of these having sensitivity to middle or long wavelengths of light. We investigated stomatopod middle- and long-wavelength-sensitive opsin genes from five species with the hypothesis that each species investigated would express up to six different opsin genes. In order to understand the evolution of this class of stomatopod opsins, we examined the complement of expressed transcripts in the retinas of species representing a broad taxonomic range (four families and three superfamilies). A total of 54 unique retinal opsins were isolated, resulting in 6–15 different expressed transcripts in each species. Phylogenetically, these transcripts form six distinct clades, grouping with other crustacean opsins and sister to insect long-wavelength visual pigments. Within these stomatopod opsin groups, intra- and interspecific clusters of highly similar transcripts suggest that there has been rampant recent gene duplication. Some of the observed molecular diversity is also due to ancient gene duplication events within the stem crustacean lineage. Using evolutionary trace analysis, 10 amino acid sites were identified as functionally divergent among the six stomatopod opsin clades. These sites form tight clusters in two regions of the opsin protein known to be functionally important: six in the chromophore-binding pocket and four at the cytoplasmic surface in loops II and III. These two clusters of sites indicate that stomatopod opsins have diverged with respect to both spectral tuning and signal transduction.

During their complex life history, anguilliform eels go through a major metamorphosis when developing from a fresh water yellow eel into a deep-sea silver eel. In addition to major changes in body morphology, the visual system also adapts from a fresh water teleost duplex retina with rods and cones, to a specialized deep-sea retina containing only rods. The history of the rods is well documented with an initial switch from a porphyropsin to a rhodopsin (P5232 to P5011) and then a total change in gene expression with the down regulation of a “freshwater” opsin and its concomitant replacement by the expression of a typical “deep-sea” opsin (P5011 to P4821). Yellow eels possess only two spectral classes of single cones, one sensitive in the green presumably expressing an RH2 opsin gene and the second sensitive in the blue expressing an SWS2 opsin gene. In immature glass eels, entering into rivers from the sea, the cones contain mixtures of rhodopsins and porphyropsins, whereas the fully freshwater yellow eels have cone pigments that are almost pure porphyropsins with peak sensitivities at about 540–545 nm and 435–440 nm, respectively. However, during the early stages of metamorphosis, the pigments switch to rhodopsins with the maximum sensitivity of the “green”-sensitive cone shifting to about 525 nm, somewhat paralleling, but preceding the change in rods. During metamorphosis, the cones are almost completely lost.

The retina of salmonid fishes has two types of cone photoreceptors: single and double cones. At the nuclear level, these cones are distributed in a square mosaic such that the double cones form the sides of the square and the single cones occupy positions at the centre and at the corners of the square. Double cones consist of two members, one having visual pigment protein maximally sensitive to green light (RH2 opsin), the other maximally sensitive to red light (LWS opsin). Single cones can have opsins maximally sensitive to ultraviolet (UV) or blue light (SWS1 and SWS2 opsins, respectively). In Pacific salmonids, all single cones express UV opsin at hatching. Around the time of yolk sac absorption, single cones start switching opsin expression from UV to blue, in an event that proceeds from the ventral to the dorsal retina. This transformation is accompanied by a loss of single corner cones such that the large juvenile shows corner cones and UV opsin expression in the dorsal retina only. Previous research has shown that adult Pacific salmon have corner cones over large areas of retina suggesting that these cones may be regenerated and that they may express UV opsin. Here we used in-situ hybridization with cRNA probes and RT-PCR to show that: (1) all single cones in non-growth zone areas of the retina express blue opsin and (2) double cone opsin expression alternates around the square mosaic unit. Our results indicate that single cone driven UV sensitivity in adult salmon must emanate from stimulation of growth zone areas.

The retinas of anchovies have two unique photoreceptor types: “bifid” and “long” cones (Fineran & Nicol, 1976). The outer segments of these cells contain multiple layers of membranes (lamellae) oriented longitudinally (axially). This orientation is distinct from that in all other vertebrate rods and cones, where the lamellae are stacked transversely with their planes perpendicular to the incident light path. Although the common arrangement provides optimal absorption for normally incident light rays, it is also insensitive to the rays' direction of vibration (i.e. their polarization). In contrast, the two mutually perpendicular sets of axially oriented lamellae segregated into bifid and long cones could function as the principal analyzers for linearly polarized light, as previously hypothesized (Fineran & Nicol, 1976, 1978). Here, we report on a microspectrophotometric study that shows (1) the presence of two spectrally distinct visual pigments in the three photoreceptor types of the bay anchovy retina; these are typical vertebrate pigments in that they bleach, when exposed to light, and have absorption spectra like all other vitamin A1-based visual pigments; (2) that the rods and cones exhibit dichroic absorption of light in accordance with their lamellar orientation, and (3) that the two cone types of the retina contain a spectrally indistinguishable pigment with peak absorbance (λmax) around 540 nm, while the rods contain a rhodopsin-like pigment with λmax near 500 nm. Compared to other vertebrates, anchovies are remarkable for using a monochromatic cone system with unusual specializations supportive of a polarization detection system.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (λmax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a λmax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of λmax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

The retinae of insectivores have been rarely studied, and their photoreceptor arrangements and expression patterns of visual pigments are largely unknown. We have determined the presence and distribution of cones in three species of shrews (common shrew Sorex araneus, greater white-toothed shrew Crocidura russula, dark forest shrew Crocidura poensis; Soricidae) and in the lesser hedgehog tenrec Echinops telfairi (Tenrecidae). Spectral cone types were identified and quantified in flattened whole retinae by antisera/antibodies recognizing the middle-to-long-wavelength-sensitive (M/L-)cone opsin and the short-wavelength-sensitive (S-)cone opsin, respectively. A combination of immunocytochemistry with conventional histology was used to assess rod densities and cone/rod ratios. In all four species the rods dominate at densities of about 230,000–260,000/mm2. M/L- and S-cones are present, comprising between 2% of the photoreceptors in the nocturnal Echinops telfairi and 13% in Sorex araneus that has equal diurnal and nocturnal activity phases. This suggests dichromatic color vision like in many other mammals. A striking feature in all four species are dramatically higher S-cone proportions in ventral than in dorsal retina (0.5% vs. 2.5–12% in Sorex, 5–15% vs. 30–45% in Crocidura poensis, 3–12% vs. 20–50% in Crocidura russula, 10–30% vs. 40–70% in Echinops). The functional and comparative aspects of these structural findings are discussed.