Search

The distribution of cone photoreceptor subtypes (important for color vision and vision quality) varies widely in different carnivore species, but there have been limited studies on bear (ursid) cone distribution. A previous behavioral study suggests that American black bears (Ursus americanus) are dichromatic, indicating that they possess two cone subtypes, although the retinal distribution of cones is unknown. The purpose of this study was to examine the subtype and topography of cones in American black bear retinas to further predict the nature of their color vision and image resolution. We studied 10 eyes from seven individual legally hunted black bears in northeastern North Carolina. Cryosections and retinal wholemounts were labeled using antibodies targeting two cone opsin subtypes: long/medium (L/M) wavelength sensitive and short (S) wavelength sensitive. Cones in fluorescent microscopy images were counted and density maps were created for retinal wholemounts. The black bear retina contains both cone subtypes and L/M cones outnumber S cones by at least 3:1, a finding confirmed in retinal frozen sections. There are higher concentrations of S cones present than typically seen in other carnivores with some evidence for co-expression of L/M and S cones. A cone-dense area centralis is present dorsotemporal to the optic nerve, similar to other carnivores. These results confirm that American black bears are predicted to have a dichromatic vision with high acuity indicated by the presence of a dorsotemporally located area centralis., (© 2020 American Association for Anatomy.)

The canine species has dichromatic color vision comprising short-wavelength (S-) and long/medium (L/M-) wavelength-sensitive cones with peak spectral sensitivity of 429-435 nm and 555 nm respectively. Although differentiation of rod- and cone-mediated responses by electroretinogram (ERG) in dogs is commonly performed, and standards have been developed based on standards for human observers, methods to differentiate S- and L/M-cone responses in dogs have not been described. We developed flicker protocols derived from previously published rod and cone spectral sensitivities. We used a double silent substitution paradigm to isolate responses from each of the 3 photoreceptor subclasses. ERG responses were measured to sine-wave modulation of photoreceptor excitation at different temporal frequencies (between 4 and 56 Hz) and mean luminance (between 3.25 and 130 cd/m 2 ) on 6 different normal dogs (3 adult female, and 3 adult male beagles) and one female beagle dog with suspected hereditary congenital stationary night blindness (CSNB). Peak rod driven response amplitudes were achieved with low frequency (4 Hz, maximal range 4-12 Hz) and low mean luminance (3.25 cd/m 2 ). In contrast, peak L/M-cone driven response amplitudes were achieved with high frequency (32 Hz, maximal range 28-44 Hz) and high mean luminance (32.5-130 cd/m 2 ). Maximal S-cone driven responses were obtained with low frequency stimuli (4 Hz, maximal range 4-12 Hz) and 32.5-130 cd/m 2 mean luminance. The dog with CSNB had reduced rod- and S-cone-driven responses, but normal/supernormal L/M cone-driven responses. We have developed methods to differentiate rod, S- and L/M-cone function in dogs using silent substitution methods. The influence of temporal frequency and mean luminance on the ERGs originating in each photoreceptor type can now be studied independently. Dogs and humans have similar L/M cone responses, whereas mice have significantly different L/M responses. This work will facilitate a greater understanding of canine retinal electrophysiology and will complement the study of canine models of human hereditary photoreceptor disorders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Retinal epithelium-specific protein 65 kDa (RPE65)-deficient dogs are a valuable large animal model species that have been used to refine gene augmentation therapy for Leber congenital amaurosis type-2 (LCA2). Previous studies have suggested that retinal degeneration in the dog model is slower than that observed in humans. However, the area centralis of the dog retina is a cone and rod photoreceptor rich region comparable to the human macula, and the effect of RPE65 deficiency specifically on this retinal region, important for high acuity vision, has not previously been reported., Methods: Spectral-domain optical coherence tomography, fundus photography, and immunohistochemistry of retinal wholemounts and sagittal frozen sections were used to define the time-course and cell-types affected in degeneration of the area centralis in affected dogs., Results: Area centralis photoreceptor degeneration was evident from 6 weeks of age, and progressed to involve the inner retina. Immunohistochemistry showed that RPE65-deficient dogs developed early loss of S-cone outer segments, with slower loss of L/M-cone outer segments and rods., Conclusions: Early-onset severe photoreceptor degeneration in the area centralis of dogs with RPE65-deficiency offers a model of the early foveal/perifoveal degeneration in some patients with LCA2. This model could be used to refine interventions aiming to improve function and halt the progression of foveal/perifoveal photoreceptor degeneration.

The cat is emerging as a promising large animal model for preclinical testing of retinal dystrophy therapies, for example, by gene therapy. However, there is a paucity of studies investigating viral vector gene transfer to the feline retina. We therefore sought to study the tropism of recombinant adeno-associated viral (rAAV) vectors for the feline outer retina. We delivered four rAAV serotypes: rAAV2/2, rAAV2/5, rAAV2/8 and rAAV2/9, each expressing green fluorescent protein (GFP) under the control of a cytomegalovirus promoter, to the subretinal space in cats and, for comparison, mice. Cats were monitored for gene expression by in vivo imaging and cellular tropism was determined using immunohistochemistry. In cats, rAAV2/2, rAAV2/8 and rAAV2/9 vectors induced faster and stronger GFP expression than rAAV2/5 and all vectors transduced the retinal pigment epithelium (RPE) and photoreceptors. Unlike in mice, cone photoreceptors in the cat retina were more efficiently transduced than rod photoreceptors. In mice, rAAV2/2 only transduced the RPE whereas the other vectors also transduced rods and cones. These results highlight species differences in cellular tropism of rAAV vectors in the outer retina. We conclude that rAAV serotypes are suitable for use for retinal gene therapy in feline models, particularly when cone photoreceptors are the target cell.

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Purpose: The canine is an important large animal model of human retinal genetic disorders. Studies of ganglion cell distribution in the canine retina have identified a visual streak of high density superior to the optic disc with a temporal area of peak density known as the area centralis. The topography of cone photoreceptors in the canine retina has not been characterized in detail, and in contrast to the macula in humans, the position of the area centralis in dogs is not apparent on clinical funduscopic examination. The purpose of this study was to define the location of the area centralis in the dog and to characterize in detail the topography of rod and cone photoreceptors within the area centralis. This will facilitate the investigation and treatment of retinal disease in the canine., Methods: We used peanut agglutinin, which labels cone matrix sheaths and antibodies against long/medium wavelength (L/M)- and short wavelength (S)-cone opsins, to stain retinal cryosections and flatmounts from beagle dogs. Retinas were imaged using differential interference contrast imaging, fluorescence, and confocal microscopy. Within the area centralis, rod and cone size and density were quantified, and the proportion of cones expressing each cone opsin subtype was calculated. Using a grid pattern of sampling in 9 retinal flatmounts, we investigated the distribution of cones throughout the retina to predict the location of the area centralis., Results: We identified the area centralis as the site of maximal density of rod and cone photoreceptor cells, which have a smaller inner segment cross-sectional area in this region. L/M opsin was expressed by the majority of cones in the retina, both within the area centralis and in the peripheral retina. Using the mean of cone density distribution from 9 retinas, we calculated that the area centralis is likely to be centered at a point 1.5 mm temporal and 0.6 mm superior to the optic disc. For clinical funduscopic examination, this represents 1.2 disc diameters temporal and 0.4 disc diameters superior to the optic disc., Conclusions: We have described the distribution of rods and cone subtypes within the canine retina and calculated a predictable location for the area centralis. These findings will facilitate the characterization and treatment of cone photoreceptor dystrophies in the dog.