[Color vision in animals : From color blind seals to tetrachromatic vision in birds]. / Farbensehen der Tiere : Von farbenblinden Seehunden und tetrachromatischen Vögeln.

BACKGROUND: The colors in which we see an object are not only dependent on the spectral composition of the reflected light but also represent an interpretation by our eyes and the trichromatic visual system. OBJECTIVE: How do animals of other species see the world? RESULTS: The majority of mammals do not have three but only two types of cones and therefore have dichromatic color vision. Marine mammals and some nocturnally active mammals even have only one type of cone and are completely color blind. In contrast, birds as well as many fish and reptiles see in the world in more color hues and with four types of cones. Many vertebrates, insects and crustaceans can see not only the spectrum perceived by us but also ultraviolet radiation as light. CONCLUSION: In order to understand how animals of other species see the world, their visual systems must be understood and the animals must be tested in behavioral investigations.

Protanopia (red color-blindness) in medaka: a simple system for producing color-blind fish and testing their spectral sensitivity.

BACKGROUND: Color perception is important for fish to survive and reproduce in nature. Visual pigments in the retinal photoreceptor cells are responsible for receiving light stimuli, but the function of the pigments in vivo has not been directly investigated in many animals due to the lack of color-blind lines and appropriate color-perception tests. METHODS: In this study, we established a system for producing color-blind fish and testing their spectral sensitivity. First, we disrupted long-wavelength-sensitive (LWS) opsins of medaka (Oryzias latipes) using the CRISPR/Cas9 system to make red-color-blind lines. Single guide RNAs were designed using the consensus sequences between the paralogous LWSa and LWSb genes to simultaneously introduce double-frameshift mutations. Next, we developed a non-invasive and no-prior-learning test for spectral sensitivity by applying an optomotor response (OMR) test under an Okazaki Large Spectrograph (OLS), termed the O-O test. We constructed an electrical-rotary cylinder with black/white stripes, into which a glass aquarium containing one or more fish was placed under various monochromatic light conditions. The medaka were irradiated by the OLS every 10 nm, from wavelengths of 700 nm to 900 nm, and OMR was evaluated under each condition. RESULTS: We confirmed that the lws - medaka were indeed insensitive to red light (protanopia). While the control fish responded to wavelengths of up to 830 nm (λ = 830 nm), the lws - mutants responded up to λ = 740 nm; however, this difference was not observed after adaptation to dark: both the control and lws - fish could respond up to λ = 820 ~ 830 nm. CONCLUSIONS: These results suggest that the lws - mutants lost photopic red-cone vision, but retained scotopic rod vision. Considering that the peak absorption spectra (λmax) of medaka LWSs are about 560 nm, but the light-adapted control medaka could respond behaviorally to light at λ = 830 nm, red-cone vision could cover an unexpectedly wide range of wavelengths, and behavioral tests could be an effective way to measure spectral sensitivity. Using the CRISPR/Cas9 and O-O systems, the establishment of various other color-blind lines and assessment of their spectra sensitivity could be expected to proceed in the future.

Novel retinopathy in related Gordon setters: a clinical, behavioral, electrophysiological, and genetic investigation.

PURPOSE: To conduct ophthalmic, behavioral, electrophysiological, and genetic testing on two related Gordon setters presented for day blindness and compare findings with those of nine related and unrelated Gordon setters. METHODS: All dogs underwent comprehensive ophthalmic examination. Maze testing was conducted under different light intensities. Rod and cone function was assessed electroretinographically. DNA samples were screened for five canine retinal disease gene mutations. RESULTS: Ophthalmic examination was unremarkable in all dogs. There was no notable difference between day blind dogs and the reference population in scotopic and mesopic maze tests. Day blind dogs performed worse in the photopic maze with slower course completion time and more obstacle collisions. Electroretinography revealed extinguished cone function in day blind dogs and depressed rod responses in all but two reference dogs. One reference population dog presented with day blindness 1 year after initial examination. Mutations that cause achromatopsia (in CNGB3) and cone-rod dystrophies (in ADAM9 and IQCB1) were not detected in any dog tested, although five reference dogs were carriers of the mutation in C2orf71 that causes rod-cone degeneration 4 (rcd4) in Gordon setters and in polski owczarek nizinny dogs. CONCLUSIONS: This report describes a novel retinopathy in related Gordon setters that has clinical signs and vision testing results consistent with achromatopsia but electroretinographic results suggestive of cone-rod dystrophy. The majority of Gordon setters in this study had low rod responses on electroretinography but it is unclear whether this was indicative of rod dysfunction or normal for the breed. Longer-term observation of affected individuals is warranted.

Achromatopsia in three sibling Labrador Retrievers in the UK.

Achromatopsia was identified in three Labrador Retriever littermates. The dogs demonstrated day blindness, negotiating obstacles under low-light conditions, but apparently blind when outdoors. One of the dogs presented with immature bilateral diffuse posterior cortical cataracts and clinical signs of day blindness became apparent following cataract extraction surgery. Electroretinography demonstrated an absence of a cone photoreceptor response to a bright stimulus and a flicker response of 30 Hz in all three dogs. No fundic lesions have been apparent ophthalmoscopically in any of the dogs as the initial presentation of each case. No abnormalities were detected with DNA screening for known mutations of the CNGB3 gene in any of the dogs.

Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.

Flicker cone function in normal and day blind sheep: a large animal model for human achromatopsia caused by CNGA3 mutation.

PURPOSE: Recently we reported on day blindness in sheep caused by a mutation in the CNGA3 gene, thus making affected sheep a naturally occurring large animal model for therapeutic intervention in CNGA3 achromatopsia patients. The purpose of this study was to characterize flicker cone function in normal and day blind sheep, with the aim of generating a normative data base for ongoing gene therapy studies. METHODS: Electoretinographic (ERG) cone responses were evoked with full-field conditions in 10 normal, 6 heterozygous carriers and 36 day blind sheep. Following light adaptation (10 min, 30 cd/m(2)), responses were recorded at four increasing light intensities (1, 2.5, 5 and 10 cd s/m(2)). At each of these intensities, a single photopic flash response followed by 8 cone flicker responses (10-80 Hz) was recorded. Results were used to generate a normative data base for the three groups. Differences between day blind and normal control animals were tested in two age-matched groups (n = 10 per group). RESULTS: The normal sheep cone ERG wave is bipartite in nature, with critical flicker fusion frequency (CFF) >80 Hz. In all four flash intensities, the single photopic flash a-wave and b-wave amplitudes were significantly lower (p < 0.005), and implicit times significantly delayed (p < 0.0001), in day blind animals. In all four flash intensities, CFF values were significantly lower (p < 0.0001) in day blind sheep. CONCLUSIONS: Cone function is severely depressed in day blind sheep. Our results will provide a normative data base for ongoing gene therapy studies.

Genomic deletion of CNGB3 is identical by descent in multiple canine breeds and causes achromatopsia.

BACKGROUND: Achromatopsia is an autosomal recessive disease characterized by the loss of cone photoreceptor function that results in day-blindness, total colorblindness, and decreased central visual acuity. The most common causes for the disease are mutations in the CNGB3 gene, coding for the beta subunit of the cyclic nucleotide-gated channels in cones. CNGB3-achromatopsia, or cone degeneration (cd), is also known to occur in two canine breeds, the Alaskan malamute (AM) and the German shorthaired pointer. RESULTS: Here we report an in-depth characterization of the achromatopsia phenotype in a new canine breed, the miniature Australian shepherd (MAS). Genotyping revealed that the dog was homozygous for a complete genomic deletion of the CNGB3 gene, as has been previously observed in the AM. Identical breakpoints on chromosome 29 were identified in both the affected AM and MAS with a resulting deletion of 404,820 bp. Pooled DNA samples of unrelated purebred Australian shepherd, MAS, Siberian husky, Samoyed and Alaskan sled dogs were screened for the presence of the affected allele; one Siberian husky and three Alaskan sled dogs were identified as carriers. The affected chromosomes from the AM, MAS, and Siberian husky were genotyped for 147 SNPs in a 3.93 Mb interval within the cd locus. An identical shared affected haplotype, 0.5 Mb long, was observed in all three breeds and defined the minimal linkage disequilibrium (LD) across breeds. This supports the idea that the mutated allele was identical by descent (IBD). CONCLUSION: We report the occurrence of CNGB3-achromatopsia in a new canine breed, the MAS. The CNGB3-deletion allele previously described in the AM was also observed in a homozygous state in the affected MAS, as well as in a heterozygous carrier state in a Siberian husky and Alaskan sled dogs. All affected alleles were shown to be IBD, strongly suggesting an affected founder effect. Since the MAS is not known to be genetically related to the AM, other breeds may potentially carry the same cd-allele and be affected by achromatopsia.

Evaluation of a behavioral method for objective vision testing and identification of achromatopsia in dogs.

OBJECTIVE: To develop a quantifiable behavioral test for identification of achromatopsic dogs based on visual performance. ANIMALS: 14 dogs. PROCEDURES: A 3.6-m-long obstacle-avoidance course with 6 obstacle panels was developed from a preliminary 2.4-m-long course. Achromatopsic and visually normal control dogs were run through the course at 4 ambient light intensities (from dim to bright: 0.2, 25, 65, and 646 lux). Completion of 4 runs ranging from dimmest to brightest light intensity constituted 1 complete trial. Each dog underwent 3 trials. Transit times were measured and compared between dog groups and between light intensities by use of a generalized linear model and ANOVA. RESULTS: At the 3 highest light intensities, the achromatopsic dogs needed significantly more time to pass through the obstacle course than the control animals. Compared with the mean transit time at the lowest light intensity, mean transit times were 2.6 times as long at 25 lux, 3.2 times as long at 65 lux, and 5.7 times as long at 646 lux. The achromatopsic dogs had signs of increasing difficulty navigating around the obstacle panels with increasing light intensities; this was not the situation for the control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: A 3.6-m-long obstacle-avoidance course with 6 movable obstacle panels allowed identification of achromatopsic dogs at ambient light intensities >or= 25 lux based on transit times. This test could be helpful in the evaluation of new cone photoreceptor specific treatments.

Detection of cone dysfunction induced by digoxin in dogs by multicolor electroretinography.

It is difficult to detect discrete cone function with the present conventional electroretinography (ERG) examination. In this study, we developed contact electrodes with a built-in color (red (644 nm), green (525 nm), or blue (470 nm)) light source (color LED-electrode), and evaluated an experimental model of digoxin in the dog. First, 17 normal Beagle dogs were used to determine which electrode works well for color ERG measurement on dogs. Then, color ERG was performed on seven normal Beagle dogs at various points during a 14-day period of digoxin administration. A single daily dose of 0.0125 mg/kg/day, which is within the recommended oral maintenance dosage range for dogs, was administered orally for 2 weeks. Ophthalmic examination, measurement of plasma concentration of digoxin, and color ERG examination were performed. On first examination, amplitudes of all responses were significantly (P < 0.01) lower with the red, than with the blue and green electrodes during ERG recording. In ERG using the red electrode, the standard deviation was large. According to these preliminary results, the red electrode was not used in the experimental dog model with digoxin. In the digoxin administrated animals, no significant change was observed in the ophthalmic examination findings. The digoxin level increased steadily throughout the dosing period but was always within the therapeutic range for dogs. In rod ERG, no abnormalities were detected with any electrode. In standard combined ERG, decreased amplitude of the a-wave was detected with every electrode. In single flash cone ERG, prolongation of implicit time was detected by color ERG with the blue and green electrodes. In 30-Hz flicker ERG, decreased amplitude was detected only by color ERG with the blue electrode. The decreased amplitude and prolonged implicit time recovered after termination of digoxin administration. Cone dysfunction induced by digoxin in the dog was revealed by multicolor ERG using blue and green LED-electrodes. Multi-color ERG was useful for detecting cone type-specific dysfunction in the dog.

Identification of a protanomalous chimpanzee by molecular genetic and electroretinogram analyses.

We determined the structures of long (L)-wavelength-sensitive and middle (M)-wavelength-sensitive opsin gene array of 58 male chimpanzees and we investigated relative sensitivity to red and green lights by electroretinogram flicker photometry. One subject had protanomalous color vision, while others had normal color vision. Unlike in humans, a polymorphic difference in the copy number of the genes and a polymorphic base substitution at amino acid position 180 were not frequently observed in chimpanzees.

CNGB3 mutations account for 50% of all cases with autosomal recessive achromatopsia.

Achromatopsia is a congenital, autosomal recessively inherited disorder characterized by a lack of color discrimination, low visual acuity (<0.2), photophobia, and nystagmus. Mutations in the genes for CNGA3, CNGB3, and GNAT2 have been associated with this disorder. Here, we analyzed the spectrum and prevalence of CNGB3 gene mutations in a cohort of 341 independent patients with achromatopsia. In 163 patients, CNGB3 mutations could be identified. A total of 105 achromats carried apparent homozygous mutations, 44 were compound (double) heterozygotes, and 14 patients had only a single mutant allele. The derived CNGB3 mutation spectrum comprises 28 different mutations including 12 nonsense mutations, eight insertions and/or deletions, five putative splice site mutations, and three missense mutations. Thus, the majority of mutations in the CNGB3 gene result in significantly altered and/or truncated polypeptides. Several mutations were found recurrently, in particular a 1 bp deletion, c.1148delC, which accounts for over 70% of all CNGB3 mutant alleles. In conclusion, mutations in the CNGB3 gene are responsible for approximately 50% of all patients with achromatopsia. This indicates that the CNGB3/ACHM3 locus on chromosome 8q21 is the major locus for achromatopsia in patients of European origin or descent.

Day-blindness in three dogs: clinical and electroretinographic findings.

A 6-month-old Rhodesian ridgeback-cross, a 6-year-old Chihuahua and a 12-month-old Australian cattle dog were presented to the authors with a history of colliding with obstacles in daylight. Ophthalmic examination was normal and all three dogs successfully negotiated obstacle courses in dim light. In daylight the dogs became suddenly blind and repeatedly collided with obstacles. Elecroretinography (ERG) revealed no retinal activity to high frequency (30 Hz), bright intensity blue light retinal stimulation by any dog, confirming cone dysfunction. Achromatopsia has previously been recorded in Alaskan malamutes and miniature poodles. This clinical case series illustrates the characteristic behavioral presentation and the electroretinographic findings of severe day-blindness and demonstrates that this condition may exist in other breeds of dogs.

Variations in long- and middle-wavelength-sensitive opsin gene loci in crab-eating monkeys.

We analyzed variations in long (L)- and middle (M)-wavelength-sensitive opsin gene loci in crab-eating monkeys. Unlike humans, most monkeys have a single L and a single M gene. Two variant genotypes, one with only one opsin gene (dichromatic) and one with tandemly arrayed multiple genes, were also found in the monkeys. However, the frequency of the former was 0.47%, and that of the latter was 5% in the monkeys, while 2% and 66%, respectively, in Caucasian males. The two variants were found only in Java Island, Indonesia, and South Thailand, respectively. The data suggest that the frequency of each genotype is different among Old World primates.

Visual sensitivity: significant within-species variations in a nonhuman primate.

Among squirrel monkeys (Saimiri sciureus) there are significant sex-related differences in visual sensitivity. As measured behaviorally in an increment-thershold task, a sample of males was found to be substantially less sensitive to long-wavelength (640-nanometer) light than a group of females tested in the same way, although the two groups showed no significant differences in sensitivity to a middle-wavelength (540-nanometer) light. The two group also differed on a test designed to measure the effects of chromatic adaptation.