Ganglion cells in larval zebrafish retina integrate inputs from multiple cone types

We recently showed the presence of 7 physiological cone opsins - R1 (575nm), R2 (556nm), G1 (460nm), G3 (480nm), B1 (415nm), B2 (440nm), UV (358nm) - in ERG recordings of larval zebrafish (Danio rerio) retina. Larval ganglion cells (GCs) are generally thought to integrate only 4 cone opsin signals (red, green blue and UV). We address the question as to whether they may integrate 7 cone spectral signals. Here, we examined the 127 possible combinations of 7 cone signals to find the optimal representation, as based on impulse discharge datasets from GC axons in the larval optic nerve. We recorded four varieties of light-response waveform: sustained-ON, transient-ON, ON-OFF, and OFF, based on the time course of mean discharge rates to all stimulus wavelengths combined. Modeling of GC responses revealed each received 1-6 cone opsin signals, with a mean of 3.8 ± 1.3 cone signals/GC. Most onset or offset responses were opponent (ON, 80%; OFF, 100%). The most common cone signals were UV (93%), R2 (50%), G3 (55%), and G1 (60%). 73% of cone opsin signals were excitatory, 27% were inhibitory. UV signals favored excitation, while G3 and B2 signals favored inhibition. R1/R2, G1/G3 and B1/B2 opsin signals were selectively associated along a non-synergistic/opponent axis. Overall, these results suggest that larval zebrafish GC spectral responses are complex and use inputs from the 7 expressed opsins.

Behavioural red-light sensitivity in fish according to the optomotor response

Various procedures have been adopted to investigate spectral sensitivity of animals, e.g. absorption spectra of visual pigments, electroretinography, optokinetic response, optomotor response (OMR) and phototaxis. The use of these techniques has led to various conclusions about animal vision. However, visual sensitivity should be evaluated consistently for a reliable comparison. In this study, we retrieved behavioural data of several fish species using a single OMR procedure and compared their sensitivities to near-infrared light. Besides cavefish that lack eyes, some species were not appropriate for the OMR test because they either stayed still or changed swimming direction frequently. Eight of 13 fish species tested were OMR positive. Detailed analyses using medaka, goldfish, zebrafish, guppy, stickleback and cichlid revealed that all the fish were sensitive to light at a wavelength greater than or equal to 750 nm, where the threshold wavelengths varied from 750 to 880 nm. Fish opsin repertoire affected the perception of red light. By contrast, the copy number of long-wavelength-sensitive ( LWS ) genes did not necessarily improve red-light sensitivity. While the duplication of LWS and other cone opsin genes that has occurred extensively during fish evolution might not aid increasing spectral sensitivity, it may provide some other advantageous ophthalmic function, such as enhanced spectral discrimination.

Contribution of opsins and chromophores to cone pigment variation across populations of Lake Victoria cichlids

Adaptation to heterogeneous sensory environments has been implicated as a key parameter in speciation. Cichlid fish are a textbook example of divergent visual adaptation, mediated by variation in the sequences and expression levels of cone opsin genes (encoding the protein component of visual pigments). In some vertebrates including fish, visual sensitivity is also tuned by the ratio of Vitamin A1/A2-derived chromophores (i.e. the light-sensitive component of the visual pigment, bound to the opsin protein), where higher proportions of A2 cause a more red-shifted wavelength absorbance. Here, we explore variation in chromophore ratios across multiple cichlid populations in Lake Victoria, using as a proxy the enzyme CYP27C1 that catalyses the conversion of Vitamin A1- into A2. We focus on sympatric Pundamilia cichlids, where species with blue or red male coloration co-occur at multiple islands, but occupy different depths and consequently different visual habitats. In the red species, we found higher cyp27c1 expression in populations from turbid-water than from clear-water locations, but there was no such pattern in the blue species. Across populations, differences between the sympatric species in cyp27c1 expression had a consistent relationship with species differences in opsin expression patterns, but the red/blue identity reversed between clear- and turbid-water locations. To assess the contribution of heritable versus environmental causes of variation, we tested whether light manipulations induce a change in cyp27c1 expression in the laboratory. We found that cyp27c1 expression was not influenced by experimental light conditions, suggesting that the observed variation in the wild is due to genetic differences. Establishing the biological importance of this variation requires testing the link between cyp27c1 expression and A1/A2 ratios in the eye, as well as its consequences for visual performance.

Uniform trichromacy in Alouatta caraya and Alouatta seniculus: behavioural and genetic colour vision evaluation

Primate colour vision depends on a matrix of photoreceptors, a neuronal post receptoral structure and a combination of genes that culminate in different sensitivity through the visual spectrum. Along with a common cone opsin gene for short wavelengths (sws1), Neotropical primates (Platyrrhini) have only one cone opsin gene for medium-long wavelengths (mws/lws) per X chromosome while Paleotropical primates (Catarrhini), including humans, have two active genes. Therefore, while female platyrrhines may be trichromats, males are always dichromats. The genus Alouatta is inferred to be an exception to this rule, as electrophysiological, behavioural and molecular analyses indicated a potential for male trichromacy in this genus. However, it is very important to ascertain by a combination of genetic and behavioural analyses whether this potential translates in terms of colour discrimination capability. We evaluated two howler monkeys (Alouatta spp.), one male A. caraya and one female A. seniculus, using a combination of genetic analysis of the opsin gene sequences and a behavioral colour discrimination test not previously used in this genus. Both individuals completed the behavioural test with performances typical of trichromatic colour vision and the genetic analysis of the sws1, mws, and lws opsin genes revealed three different opsin sequences in both subjects. These results are consistent with uniform trichromacy in both male and female, with presumed spectral sensitivity peaks similar to Catarrhini, at ~ 430 nm, 532 nm, and 563 nm for S-, M- and L-cones, respectively.

Variations in photoreceptor throughput to mouse visual cortex and the unique effects on tuning

Visual input to primary visual cortex (V1) depends on highly adaptive filtering in the retina. In turn, isolation of V1 computations requires experimental control of retinal adaptation to infer its spatio-temporal-chromatic output. Here, we measure the balance of input to mouse V1, in the anesthetized setup, from the three main photoreceptor opsins—M-opsin, S-opsin, and rhodopsin—as a function of two stimulus dimensions. The first dimension is the level of light adaptation within the mesopic range, which governs the balance of rod and cone inputs to cortex. The second stimulus dimension is retinotopic position, which governs the balance of S- and M-cone opsin input due to the opsin expression gradient in the retina. The fitted model predicts opsin input under arbitrary lighting environments, which provides a much-needed handle on in-vivo studies of the mouse visual system. We use it here to reveal that V1 is rod-mediated in common laboratory settings yet cone-mediated in natural daylight. Next, we compare functional properties of V1 under rod and cone-mediated inputs. The results show that cone-mediated V1 responds to 2.5-fold higher temporal frequencies than rod-mediated V1. Furthermore, cone-mediated V1 has smaller receptive fields, yet similar spatial frequency tuning. V1 responses in rod-deficient (Gnat1−/−) mice confirm that the effects are due to differences in photoreceptor opsin contribution.

Multiple ancestral and a plethora of recent gene duplications during the evolution of the green sensitive opsin genes (RH2) in teleost fishes

Vertebrates have four visual cone opsin classes that, together with a light-sensitive chromophore, provide sensitivity from the ultraviolet to the red wavelengths of light. The rhodopsin-like 2 (RH2) opsin is sensitive to the centre blue-green part of the spectrum, which is the most prevalent light underwater. While various vertebrate groups such as mammals and sharks have lost the RH2 gene, in teleost fishes this opsin has continued to proliferate. By investigating the genomes of 115 teleost species, we find that RH2 shows an extremely dynamic evolutionary history with repeated gene duplications, gene losses and gene conversion affecting entire orders, families and species. At least four ancestral duplications provided the substrate for todays RH2 diversity with duplications occurring in the common ancestors of Clupeocephala, Neoteleostei, and Acanthopterygii. Following these events, RH2 has continued to duplicate both in tandem and during lineage specific genome duplications. However, it has also been lost many times over so that in the genomes of extant teleosts, we find between zero to eight RH2 copies. Using retinal transcriptomes in a phylogenetic representative dataset of 30 species, we show that RH2 is expressed as the dominant green-sensitive opsin in almost all fish lineages. The exceptions are the Osteoglossomorpha (bony tongues and mooneyes) and several characin species that have lost RH2, and tarpons, other characins and gobies which do not or only lowly express the gene. These fishes instead express a green-shifted long-wavelength-sensitive LWS opsin. Our study highlights the strength of using modern genomic tools within a comparative framework to elucidate the detailed evolutionary history of gene families.

Multiple ancestral duplications of the red-sensitive opsin gene (LWS) in teleost fishes and convergent spectral shifts to green vision in gobies

Photopigments, consisting of an opsin protein bound to a light-sensitive chromophore, are at the centre of vertebrate vision. The vertebrate ancestor already possessed four cone opsin classes involved in colour perception during bright-light conditions, which are sensitive from the ultraviolet to the red-wavelengths of light. Teleosts experienced an extra round of whole genome duplication (3R) at their origin, and while most teleosts maintained only one long-wavelength-sensitive opsin gene (LWS1), the second ancestral copy (LWS2) persisted in characins and osteoglossomorphs. Following 3R, teleost opsins have continued to expand and diversify, which is thought to be a consequence of the different light environment fishes inhabit, from clear streams to the relative darkness of the deep-sea. Although many recent and a few ancestral opsin duplicates can be found, none predating the 3R were thought to exist. In this study we report on a second, previously unnoticed ancestral duplication of the red-sensitive opsin (LWS3), which predates the teleost-specific genome duplication and only persists in gobiid fishes. This is surprising, since it implies that LWS3 has been lost at least 19-20 times independently along the teleost phylogeny. Mining 109 teleost genomes we also uncover a third lineage, the elopomorphs, that maintained the LWS2 copy. We identify convergent amino acid changes that green-shift ancestral and recent LWS copies, leading to adaptive differentiation and the functional replacement of the original green-sensitive RH2 opsin. Retinal transcriptomes and in-situ hybridisation show that LWS3 is expressed to various extents in gobies and in the case of the whitebarred goby, Amblygobius phalaena, it occurs in a separate photoreceptor to LWS1. Our study highlights the importance of comparative studies to comprehend evolution of gene function.

Sequence analysis and ontogenetic expression patterns of cone opsin genes in the bluefin killifish (Lucania goodei)

Sensory systems allow for the transfer of environmental stimuli into internal cues that can alter physiology and behaviour. Many studies of visual systems focus on opsins to compare spectral sensitivity among individuals, populations, and species living in different lighting environments. This requires an understanding of the cone opsins, which can be numerous. The bluefin killifish is a good model for studying the interaction between environments and visual systems as they are found in both clear springs and tannin-stained swamps. We conducted a genome-wide screening and demonstrated that the bluefin killifish has nine cone opsins: one SWS1 (354 nm), two SWS2 (SWS2B: 359 nm, SWS2A: 448 nm), two RH2 (RH2-2: 476 nm, RH2-1: 537 nm), and four LWS (LWS-1: 569 nm, LWS-2: 524 nm, LWS-3: 569 nm, LWS-R: 560 or 569 nm). These nine cone opsins were located on four scaffolds. One scaffold contained the two SWS2 and three of the four LWS opsins in the same syntenic order as found in other cyprinodontoid fishes. We also compared opsin expression in larval and adult killifish under clear water conditions, which mimic springs. Two of the newly discovered opsins (LWS-2 and LWS-3) were expressed at low levels (< 0.2 %). Whether these opsins make meaningful contributions to visual perception in other contexts (i.e., swamp conditions) is unclear. In contrast, there was an ontogenetic change from using LWS-R to LWS-1 opsin. Bluefin killifish adults may be slightly more sensitive to longer wavelengths, which might be related to sexual selection and/or foraging preferences.

Not just shades of grey: life is full of colour for the ocellate freshwater river stingray (Potamotrygon motoro)

Previous studies have shown that marine stingrays have the anatomical and physiological basis for colour vision, with cone spectral sensitivities in the blue to green range of the visible spectrum. Behavioural studies on Glaucostegus typus also showed that blue and grey can be perceived and discriminated. The present study is the first to assess visual opsin genetics in the ocellate river stingray (Potamotrygon motoro) and test if individuals perceive colour in two alternative forced choice experiments. Retinal transcriptome profiling using RNA-Seq and quantification demonstrated the presence of lws and rh2 cone opsin genes and a highly expressed single rod (rh1) opsin gene. Spectral tuning analysis predicted these vitamin-A1 based visual photopigments to exhibit spectral absorbance maxima at 461 nm (rh2), 496 nm (rh1), and 555 nm (lws); suggesting the presence of dichromacy in this species. Indeed, P. motoro demonstrates the potential to be equally sensitive to wavelengths from 380 nm to 600 nm of the visible spectrum. Behavioural results showed that red and green plates, as well as blue and yellow plates, were readily discriminated based on colour; however, brightness differences also played a part in the discrimination of blue and yellow. Red hues of different brightness were distinguished significantly above chance level from one another. In conclusion, the genetic and behavioural results support prior data on marine stingrays. However, this study suggests that freshwater stingrays of the family Potamotrygonidae may have a visual colour system that has ecologically adapted to a riverine habitat.

Functional identification of an opsin kinase underlying inactivation of the pineal bistable opsin parapinopsin in zebrafish

In the pineal organ of zebrafish larvae, the bistable opsin parapinopsin alone generates color opponency between UV and visible light. Our previous study suggested that dark inactivation of the parapinopsin photoproduct, which activates G-proteins, is important for the regulation of the amount of the photoproduct. In turn, the photoproduct is responsible for visible light sensitivity in color opponency. Here, we found that an opsin kinase or a G-protein-coupled receptor kinase (GRK) is involved in inactivation of the active photoproduct of parapinopsin in the pineal photoreceptor cells of zebrafish larvae. We investigated inactivation of the photoproduct in the parapinopsin cells of various knockdown larvae by measuring the light responses of the cells using calcium imaging. We found that GRK7a knockdown slowed recovery of the response of parapinopsin photoreceptor cells, whereas GRK1b knockdown or GRK7b knockdown did not have a remarkable effect, suggesting that GRK7a, a cone-type GRK, is mainly responsible for inactivation of the parapinopsin photoproduct in zebrafish larvae. We also observed a similar knockdown effect on the response of the parapinopsin photoreceptor cells of mutant larvae expressing the opsin SWS1, a UV-sensitive cone opsin, instead of parapinopsin, suggesting that the parapinopsin photoproduct was inactivated in a way similar to that described for cone opsins. We confirmed the immunohistochemical distribution of GRK7a in parapinopsin photoreceptor cells by comparing the immunoreactivity to GRK7 in GRK7a-knockdown and control larvae. These findings suggest that in pineal photoreceptor cells, the cone opsin kinase GRK7a contributes greatly to the inactivation of parapinopsin, which underlies pineal color opponency.