Cone Oil Droplets Research Articles

Evolution, Development and Function of Vertebrate Cone Oil Droplets.

To distinguish colors, the nervous system must compare the activity of distinct subtypes of photoreceptors that are maximally sensitive to different portions of the light spectrum. In vertebrates, a variety of adaptations have arisen to refine the spectral sensitivity of cone photoreceptors and improve color vision. In this review article, we focus on one such adaptation, the oil droplet, a unique optical organelle found within the inner segment of cone photoreceptors of a diverse array of vertebrate species, from fish to mammals. These droplets, which consist of neutral lipids and carotenoid pigments, are interposed in the path of light through the photoreceptor and modify the intensity and spectrum of light reaching the photosensitive outer segment. In the course of evolution, the optical function of oil droplets has been fine-tuned through changes in carotenoid content. Species active in dim light reduce or eliminate carotenoids to enhance sensitivity, whereas species active in bright light precisely modulate carotenoid double bond conjugation and concentration among cone subtypes to optimize color discrimination and color constancy. Cone oil droplets have sparked the curiosity of vision scientists for more than a century. Accordingly, we begin by briefly reviewing the history of research on oil droplets. We then discuss what is known about the developmental origins of oil droplets. Next, we describe recent advances in understanding the function of oil droplets based on biochemical and optical analyses. Finally, we survey the occurrence and properties of oil droplets across the diversity of vertebrate species and discuss what these patterns indicate about the evolutionary history and function of this intriguing organelle.

Seeing red to being red: conserved genetic mechanism for red cone oil droplets and co-option for red coloration in birds and turtles.

Avian ketocarotenoid pigments occur in both the red retinal oil droplets that contribute to colour vision and bright red coloration used in signalling. Turtles are the only other tetrapods with red retinal oil droplets, and some also display red carotenoid-based coloration. Recently, the CYP2J19 gene was strongly implicated in ketocarotenoid synthesis in birds. Here, we investigate CYP2J19 evolution in relation to colour vision and red coloration in reptiles using genomic and expression data. We show that turtles, but not crocodiles or lepidosaurs, possess a CYP2J19 orthologue, which arose via gene duplication before turtles and archosaurs split, and which is strongly and specifically expressed in the ketocarotenoid-containing retina and red integument. We infer that CYP2J19 initially functioned in colour vision in archelosaurs and conclude that red ketocarotenoid-based coloration evolved independently in birds and turtles via gene regulatory changes of CYP2J19 Our results suggest that red oil droplets contributed to colour vision in dinosaurs and pterosaurs.

A complex carotenoid palette tunes avian colour vision

The brilliantly coloured cone oil droplets of the avian retina function as long-pass cut-off filters that tune the spectral sensitivity of the photoreceptors and are hypothesized to enhance colour discrimination and improve colour constancy. Although it has long been known that these droplets are pigmented with carotenoids, their precise composition has remained uncertain owing to the technical challenges of measuring these very small, dense and highly refractile optical organelles. In this study, we integrated results from high-performance liquid chromatography, hyperspectral microscopy and microspectrophotometry to obtain a comprehensive understanding of oil droplet carotenoid pigmentation in the chicken (Gallus gallus). We find that each of the four carotenoid-containing droplet types consists of a complex mixture of carotenoids, with a single predominant carotenoid determining the wavelength of the spectral filtering cut-off. Consistent with previous reports, we find that the predominant carotenoid type in the oil droplets of long-wavelength-sensitive, medium-wavelength-sensitive and short-wavelength-sensitive type 2 cones are astaxanthin, zeaxanthin and galloxanthin, respectively. In addition, the oil droplet of the principal member of the double cone contains a mixture of galloxanthin and two hydroxycarotenoids (lutein and zeaxanthin). Short-wavelength-absorbing apocarotenoids are present in all of the droplet types, providing filtering of light in a region of the spectrum where filtering by hydroxy- and ketocarotenoids may be incomplete. Thus, birds rely on a complex palette of carotenoid pigments within their cone oil droplets to achieve finely tuned spectral filtering.

Distribution of Retinal Cone Photoreceptor Oil Droplets, and Identification of Associated Carotenoids in Crow (Corvus macrorhynchos)

The topography of cone oil droplets and their carotenoids were investigated in the retina of jungle crow (Corvus macrorhynchos). Fresh retina was sampled for the study of retinal cone oil droplets, and extracted retinal carotenoids were saponified using methods adapted from a recent study, then identified with reverse-phase high-performance liquid chromatography (HPLC). To assess the effects of saponification conditions on carotenoid recovery from crow retina, we varied base concentration and total time of saponification across a wide range of conditions, and again used HPLC to compare carotenoid concentrations. Based on colors, at least four types of oil droplets were recognized, i.e., red, orange, green, and translucent, across the retina. With an average of 91,202 /mm(2), density gradually declines in an eccentric manner from optic disc. In retina, the density and size of droplets are inversely related. In the peripheral zone, oil droplets were significantly larger than those of the central area. The proportion of orange oil droplets (33%) was higher in the central area, whereas green was predominant in other areas. Three types of carotenoid (astaxanthin, galloxanthin and lutein), together with one unknown carotenoid, were recovered from the crow retina; astaxanthin was the dominant carotenoid among them. The recovery of carotenoids was affected by saponification conditions. Astaxanthin was well recovered in weak alkali (0.06 M KOH), in contrast, xanthophyllic carotenoids were best recovered in strong alkali (0.6 M KOH) after 12 h of saponification at freeze temperature.

Cone photoreceptor oil droplet pigmentation is affected by ambient light intensity

The cone photoreceptors of many vertebrates contain spherical organelles called oil droplets. In birds, turtles, lizards and some lungfish the oil droplets are heavily pigmented and function to filter the spectrum of light incident upon the visual pigment within the outer segment. Pigmented oil droplets are beneficial for colour discrimination in bright light, but at lower light levels the reduction in sensitivity caused by the pigmentation increasingly outweighs the benefits generated by spectral tuning. Consequently, it is expected that species with pigmented oil droplets should modulate the density of pigment in response to ambient light intensity and thereby regulate the amount of light transmitted to the outer segment. In this study, microspectrophotometry was used to measure the absorption spectra of cone oil droplets in chickens (Gallus gallus domesticus) reared under bright (unfiltered) or dim (filtered) sunlight. Oil droplet pigmentation was found to be dependent on the intensity of the ambient light and the duration of exposure to the different lighting treatments. In adult chickens reared in bright light, the oil droplets of all cone types (except the violet-sensitive single cones, whose oil droplet is always non-pigmented) were more densely pigmented than those in chickens reared in dim light. Calculations show that the reduced levels of oil droplet pigmentation in chickens reared in dim light would increase the sensitivity and spectral bandwidth of the outer segment significantly. The density of pigmentation in the oil droplets presumably represents a trade-off between the need for good colour discrimination and absolute sensitivity. This might also explain why nocturnal animals, or those that underwent a nocturnal phase during their evolution, have evolved oil droplets with low pigment densities or no pigmentation or have lost their oil droplets altogether.

Color vision of the budgerigar (Melopsittacus undulatus): hue matches, tetrachromacy, and intensity discrimination

Budgerigars, Melopsittacus undulatus, were trained to discriminate monochromatic lights from mixtures of two comparison lights. The addition of small amounts of UV (365 nm) to blue or yellow lights dramatically changed the color for the birds. Hue matches showed the birds to be dichromatic both at long wavelengths (only P565 and P508 active) and at short wavelengths (only P370 and P445 active because of screening of P508 and P565 by cone oil droplets). In mid-spectrum (only P445 and P508 active), a hue match was achieved, but the results were more complicated because two opponent neural processes were activated. All observed hue matches were in quantitative agreement with calculations of relative quantum catch in the pairs of participating single cones and point to the presence of a minimum of three opponent neural processes. For the hue matches at mid- and short wavelengths, the calculations also predict peak values of absorbance of the cone oil droplets associated with P508 and P445. Relative intensity of the training light affected difficult matches at long but not short wavelengths, likely due to achromatic signals from the double cones. With suitable training, birds could make intensity discriminations at short wavelengths, where the double cones have diminished sensitivity.

Spatial and temporal patterns of growth and differentiation of cone oil droplets in the chick retina

Avian cone photoreceptors have an oil droplet in the outer portion of their inner segment that acts as a long-pass cut-off filter between incident light and visual pigment. Chick cone droplets are mainly red, orange, yellow, green, and colorless, and the colors are due to three carotenoid pigments with characteristic absorption spectra. Little is known of the differentiation of this organelle, the natural marker of cones, and the little that is known is largely controversial. We used flat whole-mounts of fresh retinas to study the time and place of the appearance of droplets, their growth rates, the sequence of droplet color differentiation, and the spatial distribution of these colors. We show that droplet differentiation starts on embryonic Day 10 (E10) in a relatively small area above the optic nerve head. The differentiation spreads to the rest of the retina in a manner similar to that of photoreceptor neurogenesis, with three decreasing gradients of droplet size and color between E13-E20: from central to peripheral, dorsal to ventral, and temporal to nasal. The rate of growth of the droplets was not constant, but showed a maximum between E17 and postnatal Day 1 (P1) in most of the retinal zones. Color differentiation started at E16-E17, 5-6 days after their appearance, when the droplets were already of considerable size. Initially, all droplets were colorless, and then turned pale green or yellow to acquire progressively the mature colors. Differentiation ended in the whole retina by P15, with ventral droplets of larger diameter than dorsal ones, the peripheral ones generally larger than the central ones, and with the color distribution varying with the retinal area. Our results show that growth and color differentiation of the droplets is regulated temporally and spatially, and the cones complete differentiation at P15 rather than at prenatal stages, as is thought generally.

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.

Vision in the peafowl (Aves: Pavo cristatus).

The visual sense of the Indian blue-shouldered peafowl Pavo cristatus was investigated with respect to the spectral absorption characteristics of the retinal photoreceptors, the spectral transmittance of the ocular media and the topographic distribution of cells in the retinal ganglion cell layer. Microspectrophotometry revealed a single class of rod, four spectrally distinct types of single cone and a single class of double cone. In the case of the single cone types, which contained visual pigments with wavelengths of maximum absorbance (lambda(max)) at 424, 458, 505 and 567 nm, spectral filtering by the ocular media and the different cone oil droplets with which each visual pigment is associated gives predicted peak spectral sensitivities of 432, 477, 537 and 605 nm, respectively. Topographic analysis of retinal ganglion cell distribution revealed a large central area of increased cell density (at peak, 35,609 cells mm(-2)) with a poorly defined visual streak extending nasally. The peafowl has a calculated maximum spatial resolution (visual acuity) in the lateral visual field of 20.6 cycles degrees(-1). These properties of the peafowl eye are discussed with respect to its visual ecology and are compared with those of other closely related species.

Novel identification of the different types of cones in the retina of the chicken.

1. Although single cones and double cones in the chicken retina had been documented for more than 30 years, the exact morphology of these cells had never been studied by the scanning electronmicroscopy. In this brief report, we present the evidence for the first time the existence of two types of single cones and three types of double cones (termed as types A, B. and C), each with distinct morphology. 2. The proportion of type A:type B:type C double cones, as estimated from the midperipheral and central regions of our scanning specimens, was about 30%:50%:20%. 3. Based on the literature data that red oil droplets reside in single cones and yellow oil droplets in chief cones of the double cones, the proportion of single to double cones was deciphered and the relative proportion was estimated to be 1:0.91 in the central region, 1:0.92 in the midperiphery, and 1:0.84 in the periphery.

The Visual Ecology of Avian Photoreceptors

The spectral sensitivities of avian retinal photoreceptors are examined with respect to microspectrophotometric measurements of single cells, spectrophotometric measurements of extracted or in vitro regenerated visual pigments, and molecular genetic analyses of visual pigment opsin protein sequences. Bird species from diverse orders are compared in relation to their evolution, their habitats and the multiplicity of visual tasks they must perform. Birds have five different types of visual pigment and seven different types of photoreceptor-rods, double (uneven twin) cones and four types of single cone. The spectral locations of the wavelengths of maximum absorbance (lambda(max)) of the different visual pigments, and the spectral transmittance characteristics of the intraocular spectral filters (cone oil droplets) that also determine photoreceptor spectral sensitivity, vary according to both habitat and phylogenetic relatedness. The primary influence on avian retinal design appears to be the range of wavelengths available for vision, regardless of whether that range is determined by the spectral distribution of the natural illumination or the spectral transmittance of the ocular media (cornea, aqueous humour, lens, vitreous humour). Nevertheless, other variations in spectral sensitivity exist that reflect the variability and complexity of avian visual ecology.

Retinal photoreceptors of paleognathous birds: the ostrich ( Struthio camelus) and rhea ( Rhea americana)

Microspectrophotometry was used to determine the absorbance spectra of both rod and cone visual pigments and oil droplets from the retinae of the ostrich ( Struthio camelus) and rhea ( Rhea americana). Light and fluorescence microscopy of whole fresh tissue mounts were used to determine the relative numbers and distribution of oil droplets in the retinae. Both species possessed rods, double cones and four classes of single cone identified by their oil droplets. The rods had λ max at about 505 nm, whereas three cone pigments were recorded with λ max at 570, 505 and 445 nm. The P570 pigment was located in both members of the double cones and in a class of single cone containing an R-type oil droplet ( λ cut at 555 nm). The P505 and P445 cone pigments were found in populations of single cones containing Y-type and C-type oil droplets ( λ cut of 500 and 420 nm, respectively). The fourth class of single cone contained a T-type droplet and in the ostrich contained a visual pigment with λ max at about 405 nm. Double cones possessed a P-type droplet in the principal member and an A-type droplet in the accessory member. The complement of visual pigments and oil droplets, and the ratio of cone types in the ostrich and rhea, are remarkably similar to those found in many groups of neognathous birds.

Visual pigments, cone oil droplets, ocular media and predicted spectral sensitivity in the domestic turkey ( Meleagris gallopavo)

A microspectrophotometric survey conducted on the retinal photoreceptors of the domestic turkey ( Meleagris gallopavo) revealed the presence of five different types of vitamin A 1-based visual pigment (rhodopsin) in seven different types of photoreceptor. A single class of rod contained a medium wavelength-sensitive visual pigment (wavelength of maximum absorbance, λ max, 504 nm). Four different types of single cone contained visual pigment maximally sensitive to wavelengths in either the long (LWS, λ max 564 nm), medium (MWS, λ max 505 nm), short (SWS, λ max 460 nm) or violet (VS, λ max 420 nm) spectral ranges. The LWS, MWS and SWS single cones contained pigmented oil droplets with cut-off wavelengths ( λ cut) at 514, 490 and 437 nm, respectively. The VS single cone contained a transparent oil droplet which displayed no significant absorbance above 330 nm. A single class of double cone was also identified, both the principal and accessory members of which contained the LWS cone visual pigment. The principal member contained an oil droplet with a λ cut at 436 nm. No oil droplet was observed in the accessory member. The use of a glycerol-based cell mountant, which reduced wavelength dependent measurement artefacts in the microspectrophotometric measurements, is described. Predictions of cone effective spectral sensitivity, incorporating measurements of the spectral transmission of the ocular media, suggest that turkeys have considerable sensitivity to wavelengths in the ultraviolet-A (UV-A, 315–400 nm) spectral range. This has implications for both the visual ecology of wild birds and the welfare of intensively farmed individuals.

Ultraviolet cues affect the foraging behaviour of blue tits

The function of avian ultraviolet (UV) vision is only just beginning to be understood. One plausible hypothesis is that UV vision enhances the foraging ability of birds. To test this, we carried out behavioural experiments using wild–caught blue tits foraging for cabbage moth and winter moth caterpillars on natural and artificial backgrounds. The light environment in our experiments was manipulated using either UV–blocking or UV–transmitting filters. We found that the blue tits tended to find the first prey item (out of four) more quickly when UV cues were present. This suggests that UV vision offers benefits to birds when searching for cryptic prey, despite the prey and backgrounds reflecting relatively little UV. Although there was no direct effect of UV on the time taken to find all four prey items in a trial, search performance in the absence of UV wavelengths tended to increase over the course of an experiment. This may reflect changes in the search tactics of the birds. To our knowledge, these are the first data to suggest that birds use UV cues to detect cryptic insect prey, and have implications for our understanding of protective coloration.

Filtering effect of cone oil droplets detected in the P-111 response spectra of japanese quail

While absorption spectra of bird cone visual pigments have been well studied, physiological study of bird cone cells has been less advanced owing to their small sizes. We measured the P-III components of electroretinograms (ERG) from isolated retinas of Japanese quail. We recorded responses to monochromatic flashes of equal photon numbers, and found that the shape of the response spectrum is dependent on the incident direction of the flashes. The spectrum obtained with the flashes from the cornea side had a steeper peak around 500 nm than that with flashes from the receptor side. This is clear electrophysiological evidence of the filtering effect of the oil droplets in the cone cells, which has long been suspected. We analyzed these spectra with respect to the absorption spectra of cone pigments and transmittance spectra of oil droplets.

The visual ecology of avian cone oil droplets

Microspectrophotometric (msp) measurements were made of retinal oil droplets of 15 species of birds from 5 orders. The droplets were assigned to six categories on the basis of their cut-off wavelengths. Counts of oil droplets from the retinae of different species revealed large variations in the proportions of oil droplets of different categories. Cluster analysis was used to demonstrate relationships between 12 species of birds on the basis of their oil droplet complements. This analysis linked species in ways which were best explained by ecological factors and which only sometimes reflected phylogeny.

Relation of spectral types to oil droplets in cones of turtle retina.

The spectral sensitivities and color of oil droplets of cone photoreceptors in the retina of the red-eared turtle (Pseudemys scripta elegans) were investigated by intracellular recording and injections of Lucifer yellow dye. Six morphological types of cones could be distinguished by the color of the oil droplets located in the outermost inner segments. Single cones containing either red or pale green oil droplets were sensitive to red light, cones with yellow oil droplets to green, and cones with clear oil droplets to blue. Contrary to previous reports, both principal and accessory members of double cones were sensitive to red, and no diffusion of dye was detected between the two apposed members. Thus, the oil droplets provide a reliable morphological basis for further investigation of the neuroanatomical networks underlying the processing of color information in the vertebrate retina.

A new method for determining peak absorbance of dense pigment samples and its application to the cone oil droplets ofEmydoidea blandingii

A new method measures the spectral absorption of small volume samples having peak optical densities (Dp) too high (up to 50 and higher) to be measured by standard direct or indirect methods. This method specifically corrects for the effect on the microspectrophotometer (MSP) of wavelength-independent light which bypasses the sample. The method involves; (1) measuring by MSP the long wavelength cutoff of the dense samples and the absorption spectrum of thinned samples of the same pigment, (2) calculating Dp from these. The method's application is illustrated for the colored oil droplets in the retinal cones of the turtle, Emydoidea blandingii.

Pigment types, densities and concentrations in cone oil droplets of Emydoidea blandingii.

The peak optical densities (Dp) of colored oil droplets in the retinal cones of the turtle, Emydoidea blandingii, were found to range from 1.2 to 8.8 for yellow droplets, 3.2 to 11.4 for orange, and 8.8 to 58 for red. The yellow droplets' spectra matched that of zeaxanthin, the red and orange matched astaxanthin. One type of clear droplet absorbed negligibly in the visible spectrum. A second type had a Dp approximately 0.9 somewhere between 370 and 400 nm. The accessory cone had no oil droplet, but its ellipsoid contained a pigment with the absorption spectrum of zeaxanthin and Dp approximately 1.1.

The cone oil droplets of avian retinas

The cone oil droplets of 19 species of birds from 11 families were examined by microspectrophotometry. Individual droplets were expanded with mineral oil, suspended in aqueous glycerol, and absorbance spectra measured between 700 and 320 nm. A classification of oil droplets is proposed, in which objective measurements of their carotenoids are related to the size, position and visual appearance of the droplets under the microscope. Some droplets contain no carotenoid and are transparent at wavelengths longer than 320 nm. Other droplets appear colorless but contain carotenoids absorbing at 385 or 402 nm. The pale droplets that have traditionally been described as greenish contain a mixture of two carotenoids. All of these types are distinct from yellow and red droplets. Red droplets contain astaxanthin esters, and yellow droplets contain a carotenoid with a spectrum similar to zeaxanthin. The 402 nm chromophore is galloxanthin, a C 27 apo-carotenoid with 8 double bonds. The in vivo optical densities are 1–4 in the paler droplets, range up to about 8 in the yellow droplets, and can exceed 20 in the red droplets. All droplets that contain carotenoid can exhibit substantial absorption in the near u.v. The frequencies of the several droplet types in the retinas of different species suggests that these organelles respond readily to natural selection and may be involved in more than one function.