The roles of receptor noise and cone oil droplets in the photopic spectral sensitivity of the budgerigar, Melopsittacus undulatus.

Individual budgerigars (Melopsittacus undulatus) were taught to detect narrow bands of wavelengths under ambient illumination of known spectral composition. Because the cone pigments of this species of bird have been identified and data on carotenoid absorbance present in the cone oil droplets are available, predictions of the Vorobyev-Osorio equations can be calculated with reasonable confidence. Based on more than 27,600 individual choices made by several birds at 10 wavelengths, the photopic sensitivity (i.e., color thresholds) of these birds is found to be consistent with the hypothesis that threshold discrimination of colored targets is limited by receptor noise and that high sensitivity to near-ultraviolet wavelengths is in harmony with the relatively small number of ultraviolet cones present in the retina. The pronounced fine structure of the sensitivity spectrum is caused by the absorption of cone oil droplets. Under natural sunlight, containing more energy in the near-ultraviolet than is present in artificial indoor lighting, the birds' peak of sensitivity in the ultraviolet should be much less prominent than it is in laboratory experiments.

Spectral sensitivity of cones in the goldfish, Carassius auratus.

The spectral sensitivities of retinal cones isolated from goldfish (Carassius auratus) retinas were measured in the range 277-737 nm by recording membrane photocurrents with suction pipette electrodes (SPE). Cones were identified with lambda max (+/- S.D.) at 623 +/- 6.9 nm, 537 +/- 4.7 nm, 447 +/- 7.7 nm, and about 356 nm (three cells). Two cells (lambda max 572 and 576 nm) possibly represent genetic polymorphism. A single A2 template fits the alpha-band of P447(2), P537(2), and P623(2). HPLC analysis showed 4% retinal:96% 3-dehydroretinal. Sensitivity at 280 nm is nearly half that at the lambda max in the visible. The lambda max of the beta-band (in nm) is a linear function of the lambda max of the alpha-band and follows the same relation as found for A1-based cone pigments of a cyprinid fish.

Spectral and polarization sensitivity of photocurrents of amphibian rods in the visible and ultraviolet.

Photocurrents from isolated rods of adults and sub-adults of three species of amphibians, Rana pipiens, Ambystoma tigrinum, and Xenopus laevis, were measured with suction pipette electrodes. The intensity for a half-maximal response was 0.91+/-0.48 photons microm(-2) flash(-1) (mean +/- S.D., 10-ms flashes) for Rana, 0.92+/-0.44 for Ambystoma, and 6.14+/-1.33 for Xenopus. The mean number of photoisomerizations at half-saturation was 22+/-12 for Rana, 50+/-24 for Ambystoma, and 221+/-48 for Xenopus. The photocurrent per photoisomerization is several times smaller in Xenopus rods than in the other two species. Spectral sensitivity was measured from 277-737 nm with light polarized both parallel and perpendicular to the planes of the membrane disks. Dichroism fell in the near UV and was absent in the region of absorption by tryptophan and tyrosine. Maximum sensitivity of Rana was at 503.9+/-2.6 nm (n = 86), and of Ambystoma, 505.8+/-1.8 nm (n = 24). Animals from these same batches that were sampled by HPLC had no 3-dehydroretinal (retinal2). Xenopus containing about 94% retinal2 and 6% retinal1 had lambda(max) at 519.3+/-2.7 nm (n = 11). Spectral position of the beta-band, estimated by the method of Stavenga et al. (1993), appears to be at longer wavelengths in amphibian photoreceptors than in other vertebrates. Fits of log sensitivity to a normalized-frequency template that tracks the long-wavelength tail of the alpha-band (Lamb, 1995) show that the rod pigments of Rana and Ambystoma are slightly narrower than those found in the photoreceptors of fish and mammals.

Activity of long-wavelength cones under scotopic conditions in the cyprinid fish Danio aequipinnatus.

In carp (Cyprinus) and goldfish (Carassius), long-wavelength cones are reported to be active under scotopic conditions. Using the electroretinogram (ERG), we tested another cyprinid fish, Danio aequipinnatus, which contains A1-based visual pigments and for which we had previously measured the spectral sensitivities of individual cones. Dark adaptation curves show a rod/cone break at about 45 min. When thoroughly dark-adapted, the spectral sensitivity function is broader than can be accounted for by self-screening of rhodopsin, but it can be modeled by an additive combination of rods and the 560-nm cones. Dim, red background light causes adaptation of rods and a broadening of the spectral sensitivity function, which can be simulated by increasing the proportion of cones in the model. Brighter red backgrounds adapt the 560-nm cones. Because of the effect of red adapting lights, the ERG evidence for the participation of long-wavelength cones close to visual threshold appears to be different in Danio than in the goldfish Carassius.

On the mechanism of isomerization of ocular retinoids by the crayfish Procambarus clarkii.

The eyes of some crustaceans store substantial amounts of retinyl esters, with most of the retinol in the 11-cis configuration. Earlier work in this laboratory suggested that in lobster and crayfish the mechanism of isomerization of retinol to the 11-cis form involves the hydrolysis of all-trans retinyl esters. Although this is the same process as that occurring in the vertebrate eye, it is different from the retinal photoisomerase reaction known in other arthropods, specifically diurnal insects (Hymenoptera and probably Diptera). Using homogenates of crayfish, we have tested this proposed mechanism by inhibiting retinyl ester synthetase activity in the presence of exogenous all-trans retinol. Inhibition of lecithin:retinol acyl transferase with 5 mumol l-1 retinyl bromoacetate or 2 mmol l-1 phenylmethylsulfonyl fluoride blocks the formation of both all-trans and 11-cis retinyl esters as well as 11-cis retinol, as shown by direct assay and by the decrease in counts derived from tritiated all-trans retinol. The similarity of this isomerization to the mechanism in vertebrate pigment epithelium is thus an interesting example of convergent evolution in the biochemistry of visual pigments, in which the pigments themselves (the opsins) are largely conserved across phyla.

Sensitivity of cones from a cyprinid fish (Danio aequipinnatus) to ultraviolet and visible light.

Photocurrents of cones in the retinas of a small fish, Danio aequipinnatus (Cyprinidae) were recorded with suction pipette electrodes. Spectral sensitivity was measured between 277 and 697 nm. Four spectral classes of cone were found, with lambdamax at 560, 480, 408, and 358 nm. For the latter, we provide the first complete characterization of spectral sensitivity of a vertebrate ultraviolet (UV) photoreceptor. All cones responded with similar kinetics, except for a subset of the 560-nm cones, which were distinctly faster. The alpha-bands of the three cones absorbing maximally in the visible have the same bandwidth when log sensitivity is plotted versus normalized frequency, and in this respect they are indistinguishable from primate cones ("Mansfield's rule"). An eighth-degree polynomial in lambdamax/lambda based on this combined data set (fish, primate) is presented as a template that is likely to have predictive value in describing cone spectra from other vertebrates. The alpha-band of the UV cone, however, is somewhat narrower than predicted by this function, is similar to other UV visual pigments, and an eighth-degree polynomial that describes its shape is also presented. These measurements also provide information on the beta-band (i.e. cis peak region), difficult to obtain by microspectrophotometry. The beta-band of cone pigments is found at longer wavelengths as the alpha-band shifts toward the red. A secondary rise in cone sensitivity around 280 nm indicates that photons absorbed by aromatic amino acids in the opsin (gamma-band) excite the transduction cascade, but the quantum efficiency is not as high as when absorption occurs in the retinal-protein chromophore.

Formation and storage of 11-cis retinol in the eyes of lobster (Homarus) and crayfish (Procambarus).

Modes of storage and mechanisms of formation of 11-cis retinoids in the eyes of animals vary widely among the major phyla. We here describe evidence from two species of macruran decapod crustacea that point to different processes from those known in insects, the other group of arthropods for which there is extensive data. The eyes of the lobster (Homarus) contain about 300 pmol of retinal, somewhat less free retinol, and variable amounts (up to 1000+ pmol) of two retinyl esters, over 90% of which contain retinol in the 11-cis configuration. The major ester contains the long chain, polyunsaturated fatty acid docosahexaenoate (C22:6), but retinyl oleate (C18:1) is also present. Crayfish (Procambarus) contain the same retinyl esters, although in much smaller amounts. Homogenates of the eyes of both species are capable of isomerizing all-trans retinyl docosahexaenoate to the 11-cis configuration without using the energy of light. Crude fractionation of homogenates shows isomerase activity associated with membranes. The reaction mechanism has not been explored in detail, but on the basis of present evidence it may be similar to that found in vertebrate pigment epithelium. It is clearly different from the light-dependent processes known in insects (Hymenoptera and Diptera) and cephalopod mollusks, where isomerization takes place at the level of the aldehyde and 11-cis retinyl esters are not present as major storage reserves.

Behavior of crayfish rhodopsin and metarhodopsin in digitonin: the 510 and 562 nm "visual pigments" are artifacts.

The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a long-lived intermediate (P'505) with absorption similar to metarhodopsin but with the retinal still in the cis configuration. Crayfish metarhodopsin (M515) is similarly unstable in digitonin, and a portion converts to M'508 while bleaching slowly in the dark. Both P'505 and M'508 are light sensitive and bleach through an intermediate absorbing at still shorter wavelengths, M'460. The photobleaching of M'508 is likely a two-photon process, possibly involving P'505 as an intermediate. The persistence of these altered forms of the pigment with lambda max near 510 nm has compromised earlier efforts to analyze extracts of crayfish rhodopsin by partial bleaching. First, because of the incomplete decay of M515 (a portion of which liners as M'508), the difference spectrum for a red light exposure followed by dark decay has lambda max at 562 nm, but this difference spectrum does not describe a pigment. Because of the photosensitivity of M'508, a second bleaching exposure reveals the presence of a pigment with lambda max near 510 nm, but it is not a visual pigment and it is not present in the extract initially.

Ultraviolet receptors and color vision: evolutionary implications and a dissonance of paradigms.

The discovery of visual sensitivity to UV dates from 1882 and was made in an insect, the ant, but in the last 15 years evidence for photoreceptors maximally sensitive in the UV has been found for many vertebrates. Studies of behavioral responses of insects that possess more than one spectral class of photoreceptor have generated the concept of wavelength-dependent behaviors. These phenomena are distinct from color vision, where chromatic information can be used in multiple associations. Recent work on vertebrates has shown a variety of behavioral responses that appear to be based on specific combinations of spectral classes of receptors. Among these are behavioral responses of birds that are maximally sensitive in the UV, surprising findings since the retinas of birds contain only relatively small numbers of cones with peak sensitivity in the UV. These and other examples, suggestive of both wavelength-dependent behaviors of arthropods and "releasers" of ethology, emphasize anew the need for explanatory concepts that reach beyond the paradigms of primate color vision and for greater attention to the ontogeny of visually-directed behavior in non-mammalian vertebrates. The rapidly accumulating data on the evolutionary relationships of opsins continue to suggest that within specific opsin lineages the absorption maxima of the retinal-based visual pigments lie within about 40 nm of each other. Some UV pigments may provide the first exception to this generalization.

Photocurrents in retinal rods of pigeons (Columba livia): kinetics and spectral sensitivity.

1. Membrane photocurrents were recorded from outer segments of isolated retinal rods of pigeons (Columba livia), the first such measurements on the photoreceptors of a bird. The amplitude of the response to 20 ms flashes of narrow wavelength bands of light increases linearly with intensity at low photon fluxes and saturates at higher intensities. The maximum (saturating) photocurrent observed in forty-nine rod cells was 50 pA. Larger responses with less variability in the intensity for half-maximal responses were observed when the physiological saline contained 20 mM bicarbonate (in addition to Hepes buffer). 2. The dependence of peak amplitude on intensity is well fitted by an exponential function; it is usually less well fitted by the Michaelis-Menten (Naka-Rushton) equation. 3. In the presence of bicarbonate, the average sensitivity of pigeon rods to dim flashes was 0.56 pA photon-1 microns -2. The effective collecting area per photon was 1.8 microns 2. About 83 +/- 26 (mean +/- S.D.) photoisomerizations were required for a half-saturating response. 4. The response kinetics of rods to dim flashes can be reasonably well described by a series of four to five either Poisson or independent filters. The time to peak, measured from the mid-point of a 20 ms flash, was 319 +/- 83 ms (mean +/- S.D.). The integration time of the response was 851 +/- 86 ms (mean +/- S.D.) with bicarbonate present and 572 +/- 126 ms in the absence of bicarbonate. The responses of pigeon rods appear to be slower than those of mammals at the same temperature. The fraction of current suppressed by a single photoisomerization is smaller in pigeon than in mammalian rods by a factor of at least two. 5. The spectral sensitivity function was measured between 680 and 330 nm. The maximum at about 505 nm (range 497-508 nm) corresponds to the alpha-band of a vertebrate rhodopsin and agrees with previous behavioural measurements of scotopic sensitivity of pigeons as well as the absorption spectrum of extracts of pigeon rhodopsin. There was no pronounced beta-band in the near-ultraviolet wavelengths.

The retinoids of seven species of mantis shrimp.

Eyes of stomatopod crustaceans, or mantis shrimps, contain the greatest diversity of visual pigments yet described in any species, with as many as ten or more spectral classes present in a single retina. In this study, the eyes of seven species of mantis shrimp from three superfamilies of stomatopods were examined for their content of retinoids. Only retinal and retinol were found; neither hydroxyretinoids nor dehydroretinoids were detected. The principal isomers were 11-cis and all-trans. The eyes of most of these species contain stores of 11-cis retinol, principally as retinyl esters, and in amounts in excess of retinal. Squilla empusa is particularly noteworthy, with over 5000 pmoles of retinol per eye.

Packaging of rhodopsin and porphyropsin in the compound eye of the crayfish.

The distribution of 3-dehydroretinal (Ral2) in dorsal, middle, and ventral slices of eyes of the crayfish Procambarus clarkii was examined by HPLC. No pronounced differences were found. Similar results were obtained when the eyes were cut into anterior, intermediate, and posterior portions. Dichroic difference spectra were measured in single halves of microvillar layers of isolated rhabdoms and the proportions of rhodopsin (P1) and porphyropsin (P2) estimated by comparison with computer-generated mixtures of these pigments, whose spectra are known from previous work. The fraction of visual pigment that is porphyropsin appears to be uniform throughout individual retinular cells and among the retinular cells of individual rhabdoms, but various substantially among different rhabdoms from the same eye. The interommatidial variation in the amount of P2 greatly exceeds the gross regional variation in Ral2. This means there is an intermingling of ommatidia with different levels of P2. The variability in P2 among ommatidia is not likely to have important implications for the vision of the crayfish but suggests that in the metabolism of retinoids, individual ommatidia are quasi-independent metabolic units. The results are compatible with a single opsin for both crayfish rhodopsin and porphyropsin.

Retinoids in the lateral eye of Limulus: evidence for a retinal photoisomerase.

The lateral eyes of the horseshoe crab Limulus contain about 80 pmoles of retinal, 30 pmoles of retinol, and 4 pmoles of retinyl esters. More all-trans than 11-cis isomer was found in each category of retinoid. No consistent changes were observed in the amounts of retinal, retinol, or retinyl esters as a function of time of day. No 3,4-dehydro- nor hydroxyretinoids were found. Aqueous extracts of the eye support the stereospecific formation of 11-cis retinal from all-trans retinal when irradiated with light. The reaction requires a protein that is apparently recognized by polyclonal antibodies raised against the retinal photoisomerase extracted from honeybee eyes. The isomerase is able to use as substrate either endogenous all-trans retinal in the extract of retinal supplied in vesicles of phospholipid. The spectral efficiency of this isomerization has lambda max at 550 nm, but the spectrum appears too narrow compared with the absorbance spectrum of retinoid-binding proteins, probably because of inadequate correction for nonspecific isomerization at short wavelengths.

Localization of retinal photoisomerase in the compound eye of the honeybee.

The distribution of honeybee retinal photoisomerase, a soluble light-requiring enzyme that stereospecifically forms 11-cis retinal, was investigated by immunoelectron microscopy and by HPLC. Immunolocalization with polyclonal antibodies shows that the highest concentration of retinal photoisomerase is located in the proximal portion of the primary pigment cells in large aggregates (approximately 2 microns diameter). Photoisomerase is also located in the peripheral portion of the photoreceptor cells, laterally displaced from the rhabdom, but in much lower concentration. Because of the larger volume of the photoreceptor cells, about half of the total immunoreactivity is associated with the primary pigment cells. Dissection of the eye with the subsequent use of HPLC to assay for photoisomerase activity showed that most of the photoisomerase activity is associated with tissues near the cornea. The same tissue also supports the reduction of 11-cis retinal to 11-cis retinol. These biochemical findings are consistent with the immunolocalization of retinal photoisomerase to the high-concentration aggregates in the primary pigment cells that surround the crystalline cones. The major synthesis of 11-cis retinol therefore takes place in the primary pigment cells, and the retinoid must be moved into the photoreceptor cells to be available to newly synthesized opsin. The immunoreactivity of the photoreceptor cells appears to reflect the presence of some isomerase without an attached retinoid chromophore.

The role of retinal photoisomerase in the visual cycle of the honeybee.

The compound eye of the honeybee has previously been shown to contain a soluble retinal photoisomerase which, in vitro, is able to catalyze stereospecifically the photoconversion of all-trans retinal to 11-cis retinal. In this study we combine in vivo and in vitro techniques to demonstrate how the retinal photoisomerase is involved in the visual cycle, creating 11-cis retinal for the generation of visual pigment. Honeybees have approximately 2.5 pmol/eye of retinal associated with visual pigments, but larger amounts (4-12 pmol/eye) of both retinal and retinol bound to soluble proteins. When bees are dark adapted for 24 h or longer, greater than 80% of the endogenous retinal, mostly in the all-trans configuration, is associated with the retinal photoisomerase. On exposure to blue light the retinal is isomerized to 11-cis, which makes it available to an alcohol dehydrogenase. Most of it is then reduced to 11-cis retinol. The retinol is not esterified and remains associated with a soluble protein, serving as a reservoir of 11-cis retinoid available for renewal of visual pigment. Alternatively, 11-cis retinal can be transferred directly to opsin to regenerate rhodopsin, as shown by synthesis of rhodopsin in bleached frog rod outer segments. This retinaldehyde cycle from the honeybee is the third to be described. It appears very similar to the system in another group of arthropods, flies, and differs from the isomerization processes in vertebrates and cephalopod mollusks.

Optimization, constraint, and history in the evolution of eyes.

Several features of the evolution of eyes and photoreceptors are examined in an effort to explore the relative roles of adaptation and historical and developmental constraints. Optical design shows clear evidence of adaptation, which in some respects approaches optima predictable from physics. The primate fovea, on the other hand, illustrates how adaptation can be channeled by developmental heritage. The primary structures of opsins reveal multiple evolutionary lineages within both Drosophila and humans. The pigments of vertebrae rods comprise a subset of opsins whose evolutionary relationships map onto the phylogeny of the parent species. The evolutionary reasons for why most rod pigments absorb maximally at 500 +/- 10 nm are obscure, as there is no convincing explanation based on adaptation alone. Rods are appropriately distinguished from cones on the basis of which opsin gene is expressed. This criterion is likely to be in conflict with other definitions in phyletic lines (e.g., geckos, snakes) that have long diurnal or nocturnal histories accompanied by loss of one or more opsin genes, followed by a secondary adaptation to life in a different photic environment. Color vision--a generalizable perception associated with the spectral composition of light--is usefully distinguished from wavelength-specific behaviors. The latter are also based on multiple visual pigments and more than one spectral class of receptors but cannot be altered by learning. The distinction is particularly forceful in bees, which exhibit both kinds of behavior. The evolution of primate color vision has been shaped by historical factors involving an extensive period of early mammalian nocturnality. Birds, by contrast, have more elaborate cones and a richer set of visual pigments. Avian color space can be represented in a tetrahedron.

On polar auxin transport in plant cells.

We present here explicit mathematical formulas for calculating the concentration, mass, and velocity of movement of the center of mass of the plant growth regulator auxin during its polar movement through a linear file of cells. The results of numerical computations for two cases, (a) the conservative, in which the mass in the system remains constant and (b) the non-conservative, in which the system acquires mass at one end and loses it at the other, are graphically presented. Our approach differs from that of Mitchison's (Mitchison 1980) in considering both initial effects of loading and end effects of substance leaving the file of cells. We find the velocity varies greatly as mass is entering or leaving the file of cells but remains constant as long as most of the mass is within the cells. This is also the time for which Mitchison's formula for the velocity, which neglects end effects, reflects the true velocity of auxin movement. Finally, the predictions of the model are compared with two sets of experimental data. Movement of a pulse of auxin through corn coleoptiles is well described by the theory. Movement of auxin through zucchini shoots, however, shows the need to take into account immobilization of auxin by this tissue during the course of transport.