The photoreceptors and visual pigments of the garter snake (Thamnophis sirtalis): a microspectrophotometric, scanning electron microscopic and immunocytochemical study

Scanning electron microscopy, immunocytochemistry, and single cell microspectrophotometry were employed to characterize the photoreceptors and visual pigments in the retina of the garter snake, Thamnophis sirtalis. The photoreceptor population was found to be comprised entirely of cones, of which four distinct types were identified. About 45.5% of the photoreceptors are double cones consisting of a large principal member joined near the outer segment with a much smaller accessory member. About 40% of the photoreceptors are large single cones, and about 14.5% are small single cones forming two subtypes. The outer segments of the large single cones and both the principal and accessory members of the doubles contain the same visual pigment, one with peak absorbance near 554 nm. The small single cones contain either a visual pigment with peak absorbance near 482 nm or one with peak absorbance near 360 nm. Two classes of small single cones could be distinguished also by immunocytochemistry and scanning electron microscopy. The small single cones with the 360-nm pigment provide the garter snake with selective sensitivity to light in the near ultraviolet region of the spectrum. This ultraviolet sensitivity might be important in localization of pheromone trails. Accepted: 10 March 1997     

The photoreceptors and visual pigments of two species of Acipenseriformes, the shovelnose sturgeon (Scaphirhynchus platorynchus ) and the paddlefish (Polyodon spathula )

Scanning electron microscopy, microspectrophotometry, and spectrophotometry of digitonin extracts were employed to characterize the photoreceptors and visual pigments of two freshwater Acipenseriformes. The retinas of the shovelnose sturgeon, Scaphirhynchus platorynchus (Acipenseridae), and the paddlefish, Polyodon spathula (Polyodontidae) are dominated by large rods with long, broad outer segments. A second rod, rare and much narrower than the dominant rod, is present in Scaphirhynchus but not seen in Polyodon. The absorbance maximum of the visual pigment in the rods of Polyodon is near 540 nm; that of Scaphirhynchus near 534 nm. The retinas of both species contain substantial numbers of large, single cones, about 33% of the photoreceptors in Scaphirhynchus; 37% in Polyodon. Scaphirhynchus cone pigments have absorbance maxima near 610 nm, 521 nm and 470 nm, respectively. Polyodon cone pigments absorb maximally near 607 nm and 535 nm, respectively. All visual pigments are based on vitamin A₂. The data are compared to those from other Acipenseriformes and are discussed in terms of lifestyle and behavior. Accepted: 7 October 1998     

Retinal photoreceptors and visual pigments in Boa constrictor imperator

The photoreceptors of Boa constrictor, a boid snake of the subfamily Boinae, were examined with scanning electron microscopy and microspectrophotometry. The retina of B. constrictor is duplex but highly dominated by rods, cones comprising 11% of the photoreceptor population. The rather tightly packed rods have relatively long outer segments with proximal ends that are somewhat tapered. There are two morphologically distinct, single cones. The most common cone by far has a large inner segment and a relatively stout outer segment. The second cone, seen only infrequently, has a substantially smaller inner segment and a finer outer segment. The visual pigments of B. constrictor are virtually identical to those of the pythonine boid, Python regius. Three different visual pigments are present, all based on vitamin A(1.) The visual pigment of the rods has a wavelength of peak absorbance (lambda(max)) at 495 +/- 2 nm. The visual pigment of the more common, large cone has a lambda(max) at 549 +/- 1 nm. The small, rare cone contains a visual pigment with lambda(max) at 357 +/- 2 nm, providing the snake with sensitivity in the ultraviolet. We suggest that B. constrictor might employ UV sensitivity to locate conspecifics and/or to improve hunting efficiency. The data indicate that wavelength discrimination above 430 nm would not be possible without some input from the rods.     

Photoreceptors and visual pigments in the retina of the fully anadromous green sturgeon (<Emphasis Type="Italic">Acipenser medirostrus</Emphasis>) and the potamodromous pallid sturgeon (<Emphasis Type="Italic">Scaphirhynchus albus</Emphasis>)

Green sturgeon and pallid sturgeon photoreceptors were studied with scanning electron microscopy (SEM), microspectrophotometry and, in the case of the green sturgeon, retinal whole-mounts. The retinas of both species contain both rods and cones: cones comprise between 23% (whole-mount) and 36% (SEM) of the photoreceptors. The cone population of both species is dominated by large single cones, but a rare small single cone is also present. In both species, most rods have long outer segments of large diameter. A rod with a relatively thin outer segment is present in the pallid sturgeon retina. Mean cone packing density for the entire green sturgeon retina is 4,690±891 cones/mm², with the dorsal retina 14% more dense than the ventral. There is evidence for a horizontal visual streak just above and including the optic disc. Mean rod packing density is 16,006±1,668 rods/mm² for the entire retina, and fairly uniform throughout. Both species have rods with peak absorbance near 540 nm, as well as short-wavelength-sensitive cones (green: 464.5±0.7 nm; pallid: 439.7±3.5 nm); middle-wavelength-sensitive cones (green: 538.0±1.4 nm; pallid: 537.0±1.7 nm); and long-wavelength-sensitive cones (green: 613.9±3.0 nm; pallid: 617.8±7.6 nm).     

The cone photoreceptors and visual pigments of chameleons

Visual pigments, oil droplets and photoreceptor types in the retinas of four species of true chameleons have been examined by microspectrophotometry. The species occupy different photic environments: two species of Chamaeleo are from Madagascar and two species of Furcifer are from Africa and the Arabian Peninsula. In addition to double cones, four spectrally distinct classes of single cone were identified. No rod photoreceptors were observed. The visual pigments appear to be mixtures of rhodopsins and porphyropsins. Double cones contained a pale oil droplet in the principle member and both outer segments contained a long-wave-sensitive visual pigment with a spectral maximum between about 555 nm and 610 nm, depending on the rhodopsin/porphyropsin mixture. Long-wave-sensitive single cones contained a visual pigment spectrally identical to the double cones, but combined with a yellow oil droplet. The other three classes of single cone contained visual pigments with maxima at about 480–505, 440–450 and 375–385 nm, combined with yellow, clear and transparent oil droplets respectively. The latter two classes were sparsely distributed. The transmission of the lens and cornea of C. dilepis was measured and found to be transparent throughout the visible and near ultraviolet, with a cut off at about 350 nm.     

Visual cycle: Dependence of retinol production and removal on photoproduct decay and cell morphology

The visual cycle is a chain of biochemical reactions that regenerate visual pigment following exposure to light. Initial steps, the liberation of all-trans retinal and its reduction to all-trans retinol by retinol dehydrogenase (RDH), take place in photoreceptors. We performed comparative microspectrophotometric and microfluorometric measurements on a variety of rod and cone photoreceptors isolated from salamander retinae to correlate the rates of photoproduct decay and retinol production. Metapigment decay rate was spatially uniform within outer segments and 50-70 times faster in the cells that contained cone-type pigment (SWS2 and M/LWS) compared to cells with rod-type pigment (RH1). Retinol production rate was strongly position dependent, fastest at the base of outer segments. Retinol production rate was 10-40 times faster in cones with cone pigments (SWS2 and M/LWS) than in the basal OS of rods containing rod pigment (RH1). Production rate was approximately five times faster in rods containing cone pigment (SWS2) than the rate in basal OS of rods containing the rod pigment (RH1). We show that retinol production is defined either by metapigment decay rate or RDH reaction rate, depending on cell type or outer segment region, whereas retinol removal is defined by the surface-to-volume ratio of the outer segment and the availability of retinoid binding protein (IRBP). The more rapid rates of retinol production in cones compared to rods are consistent with the more rapid operation of the visual cycle in these cells.     

Visual pigments, oil droplets, ocular media and cone photoreceptor distribution in two species of passerine bird: the blue tit (Parus caeruleus L.) and the blackbird (Turdus merula L.)

The spectral absorption characteristics of the retinal photoreceptors of the blue tit (Parus caeruleus) and blackbird (Turdus merula) were investigated using microspectrophotometry. The retinae of both species contained rods, double cones and four spectrally distinct types of single cone. Whilst the visual pigments and cone oil droplets in the other receptor types are very similar in both species, the wavelength of maximum sensitivity (λ_max) of long-wavelength-sensitive single and double cone visual pigment occurs at a shorter wavelength (557 nm) in the blackbird than in the blue tit (563 nm). Oil droplets located in the long-wavelength-sensitivesingle cones of both species cut off wavelengths below 570–573 nm, theoretically shifting cone peak spectral sensitivity some 40 nm towards the long-wavelength end of the spectrum. This raises the possibility that the precise λ_max of the long-wavelength-sensitive visual pigment is optimised for the visual function of the double cones. The distribution of cone photoreceptors across the retina, determined using conventional light and fluorescence microscopy, also varies between the two species and may reflect differences in their visual ecology. Accepted: 8 January 2000     

Developmental Changes in the Visual Pigments of the Yellowfin Tuna, Thunnus Albacares

Previous studies have suggested that adult tunas have only two visual pigments in their retinas - a rod pigment with a wavelength at maximum absorbance ( λmax ) around 485 nm and one with similar λmax in both twin and single cones inferred from extraction data. Using microspectrophotometry we confirm the presence of a λmax 483 nm visual pigment in the rods of adult yellowfin tuna and a λmax 485 nm pigment in both members of the twin cones. However, all single cones contain a previously undetected violet visual pigment with λmax 426 nm making the adult yellowfin tuna a photopic dichromat. The situation for larvae and early juveniles is different from that of the adults. The all single-cone retina of preflexion larvae shows a wide distribution in individual cone absorbances suggesting not only mixtures of the two adult cone pigments, but the presence of at least a third visual pigment with λmax greater than 560 nm. With growth, the variation in cone absorbances decreases with convergence to the adult condition coincident with cone twinning. The significance of λmax variability, multiple visual pigment expression and age-related differences are discussed in terms of the visual ecology of larval, juvenile and adult tunas.     

The ecology of the visual pigments of snappers (Lutjanidae) on the Great Barrier Reef

The visual pigments in the retinal photoreceptors of 12 species of snappers of the genus Lutjanus (Teleostei; Perciformes; Lutjanidae) were measured by microspectrophotometry. All the species were caught on the Great Barrier Reef (Australia) but differ in the colour of the water in which they live. Some live in the clear blue water of the outer reef, some in the greener water of the middle and inshore reefs and some in the more heavily stained mangrove and estuarine water. All the species had double cones, each member of the pair containing a different visual pigment. Using Baker's and Smith's (1982) model to predict the spectral distribution of ambient light from chlorophyll and dissolved organic matter it was found that the absorption spectra of the visual pigments in the double cones were close to those that confer the maximum sensitivity in the different water types. Single cones contained a blue or violet-sensitive visual pigment. The visual pigments in the rods showed little variation, their wavelength of maximum absorption always being in the region 489–502 nm.Abbreviations DOC dissolved organic carbon - DOM dissolved organic material - MSP microspectrophotometry deceased     

Developmental Changes in the Visual Pigments of the Yellowfin Tuna,Thunnus Albacares

Previous studies have suggested that adult tunas have only two visual pigments in their retinas - a rod pigment with a wavelength at maximum absorbance (u max) around 485 nm and one with similar u max in both twin and single cones inferred from extraction data. Using microspectrophotometry we confirm the presence of a u max 483 nm visual pigment in the rods of adult yellowfin tuna and a u max 485 nm pigment in both members of the twin cones. However, all single cones contain a previously undetected violet visual pigment with u max 426 nm making the adult yellowfin tuna a photopic dichromat. The situation for larvae and early juveniles is different from that of the adults. The all single-cone retina of preflexion larvae shows a wide distribution in individual cone absorbances suggesting not only mixtures of the two adult cone pigments, but the presence of at least a third visual pigment with u max greater than 560 nm. With growth, the variation in cone absorbances decreases with convergence to the adult condition coincident with cone twinning. The significance of u max variability, multiple visual pigment expression and age-related differences are discussed in terms of the visual ecology of larval, juvenile and adult tunas.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

The Photoreceptor Outer Segments of the River Lamprey (Lampreta fluviatilis). An Electron-, Fluorescence- and Light Microscopic Study

The outer segment of long and short photoreceptors in the retina of the river lamprey, Lampetra fluviatilis, were studied by light- and fluorescence microscopy together with some different electron microscopic methods. The outer segments show characteristics of both rods and cones and are suggested to represent intermediate kinds of photoreceptors.     

Photoreceptors and eyes of pikeperch Sander lucioperca,pike Esox lucius,perch Perca fluviatilis and roach Rutilus rutilus from a clear and a brown lake

The photoreceptors and eyes of four fish species commonly cohabiting Fennoscandian lakes with different light transmission properties were compared: pikeperch Sander lucioperca, pike Esox lucius, perch Perca fluviatilis and roach Rutilus rutilus. Each species was represented by individuals from a clear (greenish) and a humic (dark brown) lake in southern Finland: Lake Vesijärvi (LV; peak transmission around 570 nm) and Lake Tuusulanjärvi (LT; peak transmission around 630 nm). In the autumn, all species had almost purely A2-based visual pigments. Rod absorption spectra peaked at c.526 nm (S. lucioperca), c. 533 nm (E. lucius) and c. 540 nm (P. fluviatilis and R. rutilus), with no differences between the lakes. Esox lucius rods had remarkably long outer segments, 1.5–2.8-fold longer than those of the other species. All species possessed middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) cone pigments in single, twin or double cones. Rutilus rutilus also had two types of short-wavelength sensitive (SWS) cones: UV-sensitive [SWS1] and blue-sensitive (SWS2) cones, although in the samples from LT no UV cones were found. No other within-species differences in photoreceptor cell complements, absorption spectra or morphologies were found between the lakes. However, E. lucius eyes had a significantly lower focal ratio in LT compared with LV, enhancing sensitivity at the expense of acuity in the dark-brown lake. Comparing species, S. lucioperca was estimated to have the highest visual sensitivity, at least two times higher than similar-sized E. lucius, thanks to the large relative size of the eye (pupil) and the presence of a reflecting tapetum behind the retina. High absolute sensitivity will give a competitive edge also in terms of short reaction times and long visual range.     

Visual cells and visual pigments of the lamprey,Lampetra fluviatilis

Summary The long and short photoreceptors in the lamprey retina possess similar cone-like outer segments where many disks are infoldings of the outer plasmic membrane. Following the treatment by the Hartwig's (1967) method, outer segments of the long receptors are stained red, and those of the short receptor are stained blue, like the cones and rods in higher vertebrates, resp. (Fig. 1). Microspectrophotometry has shown that the short cells contain P517₁ whereas the long receptors possess P555₁ (Fig. 3). Spectral sensitivity of the dark-adapted retina measured by electroretinographic b-wave and aspartate-isolated receptor potential, corresponds to P517 (Figs. 5, 8). Judging from the receptor potential, the short receptors do not saturate at high illuminances and contribute to the retinal function in photopic conditions as well (Fig. 7). Photopic ERG is of a typical cone-dominant shape (Fig. 4).It is concluded that the long photoreceptors of the lamprey retina are cones whereas the short cells should be regarded as a peculiar kind of rods which possess cone ultrastructure and can operate in scotopic as well as in photopic conditions.Abbreviation LRP late receptor potential     

The retinal pigment epithelium and photoreceptor cells light-and electron microscopic studies on monkey eyes

Summary In the perifoveal retina of the monkey, Cercopithecus aethiops, the melanin granules are accumulated in apical cytoplasmatic protrusions of the pigment epithelial cells, facing the end of the cones. The rods are inserted deeper into the pigment epithelium than the cones; they reach the bottom of the infoldings of the apical surface membrane of the pigment epithelial cells. No melanin granules or other inclusions are situated at the end of the rods. The outer extremity of the rods is considerably inclined and in sections often appears as groups of rod discs which are incompletely or completely separated from the main part of the outer segments. This separation is regarded as an artifact caused by the inclination of the rods, and it is therefore not considered to represent phagocytosis of the outer segments by the pigment epithelium.The inclusions of the pigment epithelial cells are classified in five categories which seem to be related to each other owing to their shared structural characteristics. It is suggested that melanin granules are produced, modified and destroyed by the pigment epithelial cells of the adult.Because of the relations between the photoreceptors and the melanin granules it is suggested that light scattered by the melanin granules may pass backwards through the outer segments of the cones, but not of the rods.This investigation was supported in part by the Danish Foundation for the Advancement of Science and by the Danish Medical Research Council.     

THE VISUAL CELLS AND VISUAL PIGMENT OF THE MUDPUPPY, NECTURUS

Electron microscopy of the visual cells of the mudpuppy Necturus have revealed several new or hitherto neglected features of organization: (a) A system of deeply staining micelles in virtually crystalline array, probably located in the lamellae of the rod outer segments. These particles may contain the visual pigment, porphyropsin. Counts of the micelles, and microspectrophotometric measurements of porphyropsin in the retina and single rods yield the estimate that each lamellar micelle may contain about 50 molecules of porphyropsin. (b) Systems of about 30 cytoplasmic filaments (here called dendrites), continuous with the cytoplasm of the inner segment, and standing like a palisade about the outer segments of the rods and cones. In the rods, one such filament stands in the mouth of each of the approximately 30 deep fissures that carve the outer segment into a radial array of lobules. (c) A system of deeply staining particles in the membranes of the dendrites, and another in the membranes of the pigment epithelial processes. It is suggested that these may have a part in interchanges of material with the outer segments. The ciliary process is found to penetrate more deeply than is commonly supposed into the outer segments of the rods and cones. The edge of each double-membrane disc in rods forms a differentiated rim structure, both around the disc circumference and bordering the fissures. These anatomical arrangements are summarized in Figs. 13 and 14, and the relevant measurements in Table I. The dilution of visual pigment in Necturus rods and cones and a general consideration of their microstructures make it seem unlikely that such typically solid state processes as exciton migration or photoconduction can transport the effects of light far from the site of absorption. Excitation must, therefore, be conveyed to the receptor as a whole by some axial structure. Among axial structures, the plasma membrane is most likely to be the site of nervous excitation. The ciliary process probably plays its main role in the embryogenesis and regeneration of outer segments; and the dendrites and pigment epithelial processes in exchanges of material with the outer segments and perhaps with one another.     

Characterization of photoreceptor cell types in the little brown bat Myotis lucifugus (Vespertilionidae)

We report the expression of three visual opsins in the retina of the little brown bat (Myotis lucifugus, Vespertilionidae). Gene sequences for a rod-specific opsin and two cone-specific opsins were cloned from cDNA derived from bat eyes. Comparative sequence analyses indicate that the two cone opsins correspond to an ultraviolet short-wavelength opsin (SWS1) and a long-wavelength opsin (LWS). Immunocytochemistry using antisera to visual opsins revealed that the little brown bat retina contains two types of cone photoreceptors within a rod-dominated background. However, unlike other mammalian photoreceptors, M. lucifugus cones and rods are morphologically indistinguishable by light microscopy. Both photoreceptor types have a thin, elongated outer segment. Using microspectrophotometry we classified the absorption spectrum for the ubiquitous rods. Similar to other mammals, bat rhodopsin has an absorption peak near 500 nm. Although we were unable to confirm a spectral range, cellular and molecular analyses indicate that M. lucifugus expresses two types of cone visual pigments located within the photoreceptor layer. This study provides important insights into the visual capacity of a nocturnal microchiropteran species.     

NEW EVIDENCE SUPPORTING THE LINKAGE TO EXTRACELLULAR SPACE OF OUTER SEGMENT SACCULES OF FROG CONES BUT NOT RODS

Previous electron microscopic examinations of outer segments of photoreceptors suggest that many flattened saccules of cones are continuous with the cell membrane and that their lumina connect with the extracellular compartment but that most saccules in rods appear to lack these connections. The saccules probably contain photolabile pigment, and certain potentials appear to result from dipole formation during pigment bleaching. The detection of dipoles from rod saccules may require that the lumina of rod saccules connect with extracellular space, and questions have been raised whether the interpretation of micrographs is correct or the isolation of rod saccules is the result of artifact. Accordingly, lanthanum and barium precipitates were produced near fixed and unfixed frog photoreceptors. Lanthanum precipitates appeared to infiltrate the saccules of fixed cones and the few surviving cones exposed prior to fixation, but no rod saccules were infiltrated except occasional, most basal saccules or saccules within narrow zones of probable damage. Barium precipitates did not infiltrate saccules of either variety of unfixed photoreceptor, but they did occasionally infiltrate around the saccules at points of damage in rod outer segments. The results thus support the view of the patency of saccules of frog cones and are consistent with, but do not prove, the isolation of saccules of frog rods.     

Photoreceptors and visual pigments in three species of newts

Photoreceptor composition and retinal visual pigments in three newt (Caudata, Salamandridae, Pleurodelinae) species (Pleurodeles waltl, Lissotriton (Triturus) vulgaris, and Cynops orientalis) were studied by light microscopy and single-cell microspectrophotometry. Retinas of all three species contain “red” (rhodopsin/porphyropsin) rods, large and small single cones, and double cones. Large single cones and both components of double cones contain red-sensitive (presumably LWS) visual pigment whose absorption spectrum peaks between 593 and 611 nm. Small single cones are either blue- (SWS2, maximum absorption between 470 and 489 nm) or UV-sensitive (SWS1, maximum absorption between 340 and 359 nm). Chromophore composition of visual pigments (A₁ vs. A₂) was assessed both from template fitting of absorption spectra and by the method of selective bleaching. All pigments contained a mixture of A₁ (11-cis retinal) and A₂ (11-cis-3,4-dehydroretinal) chromophore in the proportion depending on the species and cell type. In all cases, A₂ was dominant. However, in C. orientalis rods the fraction of A₁ could reach 45%, while in P. waltl and L. vulgaris cones it did not exceed 5%. Remarkably, the absorption of the newt blue-sensitive visual pigment was shifted by up to 45 nm toward the longer wavelength, as compared with all other amphibian SWS2-pigments. We found no “green” rods typical of retinas of Anura and some Caudata (ambystomas) in the three newt species studied.     

Transduction noise induced by 4-hydroxy retinals in rod photoreceptors.

New visual pigments were formed with 4-hydroxy retinals in isolated vertebrate rod photoreceptors by exposing bleached rods from the tiger salamander, Ambystoma tigrinum, to lipid vesicles containing the analogues. Formation of physiologically active pigment was demonstrated by the restoration of sensitivity and by a shift of approximately 50 nm in the peak of both the visual pigment absorptance spectrum and rod spectral sensitivity spectrum from approximately 520 to approximately 470 nm for 11-cis 4-hydroxy retinal. Membrane current recordings from the inner segments of isolated rods revealed excess fluctuations in membrane current after formation of the new pigment in bleached cells or after exposure of unbleached cells to flashes in the presence of the analogue. The excess current fluctuations are similar to the fluctuations elicited by steady light producing a few discrete responses per second, a rate approximately 100 times greater than the normal rate of spontaneous events in darkness. These results suggest that analogues of retinal can produce alterations in the frequency of production of discrete responses in darkness in rod photoreceptors.