A visual pigment expressed in both rod and cone photoreceptors.

Rods and cones contain closely related but distinct G protein-coupled receptors, opsins, which have diverged to meet the differing requirements of night and day vision. Here, we provide evidence for an exception to that rule. Results from immunohistochemistry, spectrophotometry, and single-cell RT-PCR demonstrate that, in the tiger salamander, the green rods and blue-sensitive cones contain the same opsin. In contrast, the two cells express distinct G protein transducin alpha subunits: rod alpha transducin in green rods and cone alpha transducin in blue-sensitive cones. The different transducins do not appear to markedly affect photon sensitivity or response kinetics in the green rod and blue-sensitive cone. This suggests that neither the cell topology or the transducin is sufficient to differentiate the rod and the cone response.

Photoreceptors, visual pigments, and ellipsosomes in the killifish, Fundulus heteroclitus: a microspectrophotometric and histological study.

The photoreceptor layer of F. heteroclitus was examined by light and electron microscopy. We identified four cone visual pigments with maximum absorbance (lambda(max)) in the UV (363 nm), short (400 nm), middle (463 nm), and long (563 nm) wavelength regions of the spectrum and a rod visual pigment that peaked in the middle wavelengths (503 nm). Electron-dense bodies, ellipsosomes and pseudoellipsosomes, were present in the distal ellipsoids of long/middle (L/M) and long/long (L/L) wavelength double cones and in single short wavelength (S) cones, respectively. The light absorption of ellipsosomes indicated the presence of reduced cytochrome-c with the highest optical densities found in the M members of L/M double cones. By contrast, S cones contained pseudo-ellipsosomes which had very low optical density. UV cones were present everywhere as part of square or row mosaics in the retina of F. heteroclitus. Cone packing was on average higher for locations in the upper half of the retina while the highest cone density was found in the centro-ventral retina. An analysis of potential quantum catches for each cone type as a function of retinal sector and underwater irradiance characteristics revealed higher overall quantum catches for cones in the upper retina when the light field was assumed homogeneous, and higher quantum catches for cones in the lower retina when downwelling, horizontal, and upwelling irradiances were considered separately. At dusk, quantum catch was highest for M cones and the contribution to the overall retinal quantum catch by UV and S cones was much greater than during daylight hours. We propose that UV and S cones may be used to detect targets of interest against the background irradiance sensed by double cones.

Visual pigments of African cichlid fishes: evidence for ultraviolet vision from microspectrophotometry and DNA sequences.

We have found evidence for ultraviolet visual capabilities in a Lake Malawi cichlid fish, Metriaclima zebra. Microspectrophotometry of single cones revealed a visual pigment with peak sensitivity at 368+/-4 nm. M. zebra also expresses a putative ultraviolet opsin gene whose sequence is closely related to the SWS-1 opsin for other fishes. Several other African cichlids have a functional copy of this UV gene in their genomic DNA, but do not appear to express this gene as adults. These results suggest that ultraviolet vision is important for some cichlid fishes. UV wavelengths should therefore be included in future studies of cichlid vision, behavior and color patterns.

Sickling of anoxic red blood cells in fish.

The occurrence of the mutant hemoglobin Hb S in human red blood cells results in sickle cell anemia. This disease, including its genetic and molecular bases, has been extensively investigated and is well understood (1,2). The presence of deoxy-induced sickling of animal erythrocytes is largely unknown, however. We examined red blood cells (RBCs) from several fish species in vitro under aerated and anoxic conditions. Our polarized light microscopic techniques were aimed at establishing correlations between erythrocyte morphology, state of oxygenation, spectral absorbance, linear dichroism, and linear birefringence. We found no fish with intracellular HbO2 polymerization; but there were intraerythrocytic aggregations of deoxy Hb with a high degree of either molecular order or disorder. The ordered aggregates in the RBCs of Atlantic cod, haddock, and toadfish were remarkably similar in dichroic ratio magnitudes and birefrigence to those in human RBCs that contain HbS. Therefore, fish hemoglobins appear to be good models of sickling disorders and polymerization-related phenomena. The consequences of sickling on animal health and fish aquaculture remain to be studied.

Double-cone internal reflection as a basis for polarization detection in fish.

Some species of fish are able to discriminate, in addition to intensity and wavelength (color), the direction of polarization of visible light. Optical experiments on axially oriented retinal cones from trout and sunfish with use of two types of polarization microscope indicate anisotropic light transmission through paired cones. The measured linear birefringence of paired cone ellipsoids is consistent with the presence of membranous partitions. It is proposed that the partition between the two members of a paired cone, which often appears extensive and flat, functions as a dielectric mirror and that polarization-dependent reflection and refraction at this partition constitutes the underlying mechanism in the transduction of polarization into intensity variation at the photoreceptor's outer segments. We support this hypothesis with linear birefringence and linear dichroism measurements, histological evidence, large-scale optical model measurements, and theoretical calculations based on Fresnel's formulas.

Visual pigment types and quantum-catch ratios: implications from three marine teleosts.

Experimental data on photoreceptor cells and visual pigments are the basis for model calculations performed to assess photoreceptor quantum catches under disparate irradiance conditions. Three unrelated species of fish--black sea bass (Centropristis striata), sea raven (Hemitripterus americanus), and adult winter flounder (Pseudopleuronectes americanus)--are considered. In each case, receptor type quantum catches are compared for various water types and depths. By associating the habit and habitat of an organism with the physical properties of its photoreceptors, quantum-catch ratios are found as possible criteria in the selection of pigment peaks (lambda max). In addition to integrated ("total") quantum catches by the receptor types, rates of quantum catches are determined as a function of wavelength. The latter functions are replotted as pairwise difference spectra. These, in turn, are used to assess the ability of receptor types to participate in wavelength discrimination.

An analysis of two spectral properties of vertebrate visual pigments.

Spectral data are scrutinized on two properties: (1) the chromophore-induced wavelength shift, i.e. the variation in absorbance maximum (lambda max) upon exchanging the vitamin A1-based chromophore with one based on vitamin A2 in the same opsin; (2) the half-band width (HBW) variation as a function of the reciprocal of the peak absorbance (lambda max)-1. It is shown that in an extended spectral range that includes the UV and when data are plotted in wavenumbers, the chromophore-induced shifts can be approximated with a parabola, whose minimum occurs near 23,000 cm-1 (approximately 430 nm). Similarly, HBW variations can also be fitted with parabolas, however, these show maxima near 430 nm. The theoretical implications of the two phenomena concerning lambda max tuning in vertebrate visual pigments are discussed.

Spectral characteristics of visual pigments in rainbow trout (Oncorhynchus mykiss).

We investigated retina preparations of young rainbow trout (Oncorhynchus mykiss) with body wt 5-40 g. Rods, single and double cones were measured in side-on orientation by microspectrophotometry, identifying five spectrally distinct visual pigments (or photoreceptors containing mixtures of visual pigments). The mean wavelength of peak absorbance (lambda max) of the alpha-bands were 365 and 434 nm in single cones, 531 and 576 nm in double cones, and 521 nm in the rods. The half-band width (HBW) of the main absorption bands were broader than expected of retinal- (vitamin A1-) based visual pigments, and thus, they were indicative of a mixed chromophore pool derived from both the vitamin A1 and A2 forms. One consequence of the utilization of mixed chromophores is the broadening of the alpha-band absorption in each pigment type. And yet, we obtained exceptionally narrow HBW for the UV-type pigment, when compared with HBW values expected on the basis of the linear trend seen in visual pigments absorbing in the visible spectrum. We conclude that the UV pigment in rainbow trout has an unusually narrow HBW. Nevertheless, this species is not exceptional in this regard, for the UV-absorbing visual pigments in other vertebrate species also have narrow HBW.

Photoreceptor spectral absorbance in larval and adult winter flounder (Pseudopleuronectes americanus).

The habitat occupied by larval winter flounder (Pseudopleuronectes americanus) differs considerably in light regime from that of the adult. To understand how the visual system has adapted to such changes, photoreceptor spectral absorbance was measured microspectrophotometrically in premetamorphic and postmetamorphic specimens of winter flounder. Before metamorphosis, larval flounder retinas contain only one kind of photoreceptor which is morphologically cone-like with peak absorbance at 519 nm. After metamorphosis, the adult retina has three types of photoreceptors: single cones, double cones, and rods. The visual pigment in single cones has a peak absorbance at lambda max = 457 nm, the double cones at lambda max = 531 and 547 nm, and the rod photoreceptors at lambda max = 506 nm. Double cones were morphologically identical, but the two members contained either different (531/547 nm) or identical pigments (531/531 nm). The latter type were found only in the dorsal retina. The measured spectral half-bandwidths (HBW) were typical of visual pigments with chromophores derived from vitamin A1 with the possible exception of the long-wavelength absorbing pigment in double cones which appeared slightly broader. Because the premetamorphic pigment absorbance has a different lambda max than those of the postmetamorphic pigments, different opsin genes must be expressed before and after metamorphosis.

Visual pigments in the sea lamprey, Petromyzon marinus.

We present microspectrophotometric evidence for the existence of two distinct visual pigments residing in two different morphological types of photoreceptor of the sea lamprey. In the upstream migrant Petromyzon marinus, the pigment found in short receptors has a wavelength of peak absorbance (lambda max) of 525 nm, whereas the pigment located in long receptors has a lambda max of 600 nm. Although the former appears to be pure porphyropsin, the latter is akin to visual pigments found in the red-absorbing cones of amphibian and teleost retinae. The kinship is more than superficial pertaining to lambda max, however, because the long receptor pigment, like the others, shows the typical sensitivity to the anionic milieu. Lampreys belong to the class Cyclostomata, which now becomes the sixth phylogenetic class of vertebrates with anion-sensitive as well as anion-insensitive visual pigments. This finding strengthens the hypothesis that sensitivity to anions is an integral property of all long-wavelength-absorbing vertebrate pigments and that these pigments form a distinct group in which an external Cl- ion is utilized in tuning the lambda max of the alpha-band absorbance to its native maximum value. The presence of an anion-sensitive and an anion-insensitive pigment in a retina implies the expression of two distinct opsin genes. We infer this from several examples of correlation between anion sensitivity and opsin sequence groupings. Moreover, the presence of two distinct opsin genes expressed throughout six vertebrate classes implies their existence in a common ancestor to all.

Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization.

In many vertebrates, UV-sensitive photoreceptors have been identified by microspectrophotometry and UV-visual sensitivity has been identified by behavioral studies, but as yet no vertebrate UV-sensitive pigment gene has been isolated. We have sequenced a cDNA clone that hybridizes to short single cone cells in the zebrafish (Brachydanio rerio). These cells, which make up 25% of the cone population in zebrafish retinae, are UV-sensitive (lambda max approximately 360 nm). The visual pigment encoded by this gene is unusual in that its amino acid sequence is more homologous to the rod pigment rhodopsin (up to 89%) than to other cone pigments (35-83%). Like all other vertebrate visual pigments, it contains a lysine residue at position 296, the presumptive retinal binding site, and a glutamate residue at position 113. However, it is unique in possessing a lysine residue at position 126, which may account for the UV-sensitivity of the pigment.

Anion sensitivity and spectral tuning of cone visual pigments in situ.

We tested the effect of anions on the absorbance spectrum of native visual pigments as measured by microspectrophotometry in individual cone outer segments of four species of fish and one species of amphibian. In all species tested, the long-wavelength-absorbing cone pigments were anion sensitive, and their lambda max could be tuned over a range of 55 nm depending on the identity of the anion present. Cl- and Br- were the only anions that produced native pigment spectra by red shifting lambda max from its value under anion-free conditions. Lyotropic anions such as NO3-, SCN-, BF4-, and ClO4- caused substantial and graded blue shifts of lambda max. The apparent Kd of binding sites on the pigment for Cl- and for ClO4- was approximately 2 mM. Taken together with previous findings on three visual pigments from the reptilian, avian, and amphibian classes, our results support the hypothesis that all long-wavelength-absorbing vertebrate visual pigments are spectrally tuned in part through the binding of a chloride ion. We propose that the site of anion tuning is near the protonated Schiff base of the chromophore, whose counterion may be complex and include Cl- as an exchangeable anion. This counterion configuration may resemble the one present in the light-driven Cl- pump halorhodopsin.

Visual Cells and Pigments in a Demersal Fish, the Black Sea Bass (Centropristis striata).

Using a single-beam, wavelength-scanning, dichroic microspectrophotometer, we measured absolute absorbance, bleaching difference absorbance, and linear dichroism spectra from isolated retinal receptors of the black sea bass, Centropristis striata. We determined, among other properties, the wavelength of peak α-band absorbance (λmax) of the pigment of the receptor cells. Out of well over 100 recordings, we found only 3 spectral types of visual pigment. The shortest-wavelength-absorbing type (λmax = 463 +/- 2 nm) was present only in single cones. Both members of the double cones contained the longest-wavelength-absorbing pigment of the three, with λmax = 527 +/- 5 nm. Rods were found to bear a typical rhodopsin, with λmax = 498 +/- 2 nm. Thus, the retina of this predatory demersal fish appears to use a set of three closely spaced visual pigments, with λmax clustering about 500 +/- 30 nm. This remarkable feature is discussed in relation to photic conditions in the habitat.

Solubility of retinoids in water.

Spectrophotometric and radioactive techniques were used to measure the water solubility of retinaldehyde, retinol (vitamin A), and retinoic acid under physiological conditions. Hydration decreases the molar extinction coefficient of these substances and shifts their absorption peak bathochromically (10 nm for retinal and approximately 1 nm for the rest). We find their solubility to be about 0.1 microM at room temperature, pH 7.3 (with experimental values being 0.11 microM for retinaldehyde, 0.06 microM for retinol, and 0.21 microM for retinoic acid). To prevent oxidative degradation of retinol, which is the most labile retinoid, our argon-saturated buffer solutions contained physiological levels of ascorbate or alpha-tocopherol. To the best of our knowledge, water solubility of these compounds has not yet been previously reported. Although the measured solubilities are relatively low, they are significant and may account for the movement of retinoids through the aqueous phase as observed by others during exchange with binding proteins and during intervesicular transfer in the absence of binding proteins. Diffusion of uncomplexed retinoids through the aqueous phase can be a major pathway for transport over subcellular distances.

Ultraviolet photoreception in carp: microspectrophotometry and behaviorally determined action spectra.

This study demonstrates correlations between u.v. sensitivity and microspectrophotometric absorption spectra determined sequentially for the same group of individuals. We used the heart-rate conditioning technique to measure spectral sensitivity of carp, a species known to have u.v.-sensitive photoreceptors. Mean spectral sensitivity (n = 3) determined with a spectrally-broad background (450 nm long pass filter) revealed a small but consistent u.v. peak (lambda max of 380 nm) in addition to the other long wavelength peaks. An intense blue-green background (490 nm) produced a more prominent u.v. peak (lambda max of 400 nm) when a 450 nm longpass filter was added to the background. Microspectrophotometric measurements of u.v.-sensitive photoreceptors from one individual, which belonged to the group used in the spectral sensitivity experiments, revealed an average lambda max of 377.5 nm (SD +/- 4.5 nm, n = 5 cells). Bleaching and dichroic measurements of these receptors ensured that we were examining typical vertebrate visual pigments and not stable photoproducts. The mean spectral sensitivity points were compared with the u.v. and blue-sensitive visual pigment absorption spectra. A linear subtractive model and ocular media absorption were used in this comparison for the various photic conditions used in the heart-rate conditioning experiments. The model successfully described the sensitivity of the test fish in two cases but in a third case there was some discrepancy. The model generated curve was broader than the spectral sensitivity of the u.v.-sensitive cone mechanism on the shortwave side even though the ocular media corrections had been accounted for.

Visual pigments of the tree shrew (Tupaia belangeri) and greater galago (Galago crassicaudatus): a microspectrophotometric investigation.

Optical density, linear dichroism and bleaching difference spectra were measured in photoreceptors from the cone-dominated retina of the tree shrew (Tupaia belangeri) and from the rod-dominated retina of the greater galago (Galago crassicaudatus) using a single-beam, wavelength-scanning, dichroic microspectrophotometer. In Tupaia, we obtained spectral records from 272 cone receptors (from 10 eyes), of which 264 were long-wave sensitive (lambda max = 555 +/- 6 nm) and 8 were short-wave sensitive (lambda max = 428 +/- 15 nm). Also, one anatomically-recognizable rod receptor was encountered and showed a peak absorption at approx. 496 nm. No mid-wave sensitive cone pigment was found, as would be expected in deutan-type dichromats like the tree shrew. Pre-retinal absorption by the cornea and lens was maximal at 370 nm and negligible beyond 430 nm. In Galago, all outer segments measured were rod-like in appearance (lambda max near 501 nm). Measurements of pre-retinal absorption yielded a single-peaked function with a maximum at 363 nm.