Background: The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Methods: Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. Results: A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. Conclusion: This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Retinitis pigmentosa (RP) comprises several heritable diseases that involve photoreceptor, and ultimately retinal, degeneration. Currently, mutations in over 50 genes have known links to RP. Despite advances in clinical characterization, molecular characterization of RP remains challenging due to the heterogeneous nature of causal genes, mutations, and clinical phenotypes. In this study, we compiled large datasets of two important visual genes associated with RP: rhodopsin, which initiates the phototransduction cascade, and the retinoid isomerase RPE65, which regenerates the visual cycle. We used a comparative evolutionary approach to investigate the relationship between interspecific sequence variation and pathogenic mutations that lead to degenerative retinal disease. Using codon-based likelihood methods, we estimated evolutionary rates (d N/d S) across both genes in a phylogenetic context to investigate differences between pathogenic and nonpathogenic amino acid sites. In both genes, disease-associated sites showed significantly lower evolutionary rates compared to nondisease sites, and were more likely to occur in functionally critical areas of the proteins. The nature of the dataset (e.g., vertebrate or mammalian sequences), as well as selection of pathogenic sites, affected the differences observed between pathogenic and nonpathogenic sites. Our results illustrate that these methods can serve as an intermediate step in understanding protein structure and function in a clinical context, particularly in predicting the relative pathogenicity (i.e., functional impact) of point mutations and their downstream phenotypic effects. Extensions of this approach may also contribute to current methods for predicting the deleterious effects of candidate mutations and to the identification of protein regions under strong constraint where we expect pathogenic mutations to occur.

Ground squirrels are an increasingly important model for studying visual processing, retinal circuitry, and cone photoreceptor function. Here, we demonstrate that the photoreceptor mosaic can be longitudinally imaged noninvasively in the 13-lined ground squirrel (Ictidomys tridecemlineatus) using confocal and nonconfocal split-detection adaptive optics scanning ophthalmoscopy using 790 nm light. Photoreceptor density, spacing, and Voronoi analysis are consistent with that of the human cone mosaic. The high imaging success rate and consistent image quality in this study reinforce the ground squirrel as a practical model to aid drug discovery and testing through longitudinal imaging on the cellular scale.

Recent physiological studies coupled with intracellular staining have subdivided ON directionally selective (DS) ganglion cells of rabbit retina into two types. One exhibits more “transient” and more “brisk” responses (ON DS-t), and the other has more “sustained’ and more “sluggish” responses (ON DS-s), although both represent the same three preferred directions and show preference for low stimulus velocity, as reported in previous studies of ON DS ganglion cells in rabbit retina. ON DS-s cells have the morphology of ganglion cells previously shown to project to the medial terminal nucleus (MTN) of the accessory optic system, and the MTN-projecting, class IVus1 cells have been well-characterized previously in terms of their dendritic morphology, branching pattern, and stratification. ON DS-t ganglion cells have a distinctly different morphology and exhibit heterotypic coupling to amacrine cells, including axon-bearing amacrine cells, with accompanying synchronous firing, while ON DS-s cells are not coupled. The present study shows that ON DS-t cells are morphologically identical to the previously well-characterized, “orphan” class IIb1 ganglion cell, previously regarded as a member of the “brisk-concentric” category of ganglion cells. Its branching pattern, quantitatively analyzed, is similar to that of the morphological counterparts of X and Y cells, and very different from that of the ON DS-s ganglion cell. Close analysis of the dendritic stratification of class IIb1 ganglion cells together with fiducial cells indicates that they differ from that of the ON DS-s cells. In agreement with one of the three previous studies, class IIb1/ON DS-t cells, unlike class IVus1/ON DS-s ganglion cells, in the main do not co-stratify with starburst amacrine cells. As the present study shows, however, portions of their dendrites do deviate from the main substratum, coming within range of starburst boutons. Parsimony favors DS input from starburst amacrine cells both to ON DS-s and to ON DS-t ganglion cells, given the similarity of their DS responses, but further studies will be required to substantiate the origin of the DS responses of ON DS-t cells. Previously reported OFF DS responses in ON DS-t cells, unmasked by pharmacological agents, and mediated by gap junctions with amacrine cells, suggests an unusual trans-sublaminar organization of directional selectivity in the inner plexiform layer, connecting sublamina a and sublamina b.

The ability to noninvasively image the cone photoreceptor mosaic holds significant potential as a diagnostic for retinal disease. Central to the realization of this potential is the development of sensitive metrics for characterizing the organization of the mosaic. Here we evaluated previously-described and newly-developed (Fourier- and Radon-based) methods of measuring cone orientation in simulated and real images of the parafoveal cone mosaic. The proposed algorithms correlated well across both simulated and real mosaics, suggesting that each algorithm provides an accurate description of photoreceptor orientation. Despite high agreement between algorithms, each performed differently in response to image intensity variation and cone coordinate jitter. The integration property of the Fourier transform allowed the Fourier-based method to be resistant to cone coordinate jitter and perform the most robustly of all three algorithms. Conversely, when there is good image quality but unreliable cone identification, the Radon algorithm performed best. Finally, in cases where the cone coordinate reliability was excellent, the method previously described by Pum and colleagues performed best. These descriptors are complementary to conventional descriptive metrics of the cone mosaic, such as cell density and spacing, and have the potential to aid in the detection of photoreceptor pathology.

The endogenous cannabinoid system plays important roles in the retina of mice and monkeys via their classic CB1 and CB2 receptors. We have previously reported that the G protein-coupled receptor 55 (GPR55), a putative cannabinoid receptor, is exclusively expressed in rod photoreceptors in the monkey retina, suggesting its possible role in scotopic vision. To test this hypothesis, we recorded full-field electroretinograms (ERGs) after the intravitreal injection of the GPR55 agonist lysophosphatidylglucoside (LPG) or the selective GPR55 antagonist CID16020046 (CID), under light- and dark-adapted conditions. Thirteen vervet monkeys (Chlorocebus sabaeus) were used in this study: four controls (injected with the vehicle dimethyl sulfoxide, DMSO), four injected with LPG and five with CID. We analyzed amplitudes and latencies of the a-wave (photoreceptor responses) and the b-wave (rod and cone system responses) of the ERG. Our results showed that after injection of LPG, the amplitude of the scotopic b-wave was significantly higher, whereas after the injection of CID, it was significantly decreased, compared to the vehicle (DMSO). On the other hand, the a-wave amplitude, and the a-wave and b-wave latencies, of the scotopic ERG responses were not significantly affected by the injection of either compound. Furthermore, the photopic ERG waveforms were not affected by either drug. These results support the hypothesis that GPR55 plays an instrumental role in mediating scotopic vision.

Detection of illusory contours (ICs) such as Kanizsa figures is known to depend primarily upon the lateral occipital complex. Yet there is no universal agreement on the role of the primary visual cortex in this process; some existing evidence hints that an early stage of the visual response in V1 may involve relative suppression to Kanizsa figures compared with controls. Iso-oriented luminance borders, which are responsible for Kanizsa illusion, may evoke surround suppression in V1 and adjacent areas leading to the reduction in the initial response to Kanizsa figures. We attempted to test the existence, as well as to find localization and timing of the early suppression effect produced by Kanizsa figures in adult nonclinical human participants. We used two sizes of visual stimuli (4.5 and 9.0°) in order to probe the effect at two different levels of eccentricity; the stimuli were presented centrally in passive viewing conditions. We recorded magnetoencephalogram, which is more sensitive than electroencephalogram to activity originating from V1 and V2 areas. We restricted our analysis to the medial occipital area and the occipital pole, and to a 40–120 ms time window after the stimulus onset. By applying threshold-free cluster enhancement technique in combination with permutation statistics, we were able to detect the inverted IC effect—a relative suppression of the response to the Kanizsa figures compared with the control stimuli. The current finding is highly compatible with the explanation involving surround suppression evoked by iso-oriented collinear borders. The effect may be related to the principle of sparse coding, according to which V1 suppresses representations of inner parts of collinear assemblies as being informationally redundant. Such a mechanism is likely to be an important preliminary step preceding object contour detection.

The medial part of the nucleus of Edinger–Westphal (EWM) in birds mediates light-regulated adaptive increases in choroidal blood flow (ChBF). We sought to characterize the effect of loss of EWM-mediated ChBF regulation on photoreceptor health in pigeons housed in either moderate intensity diurnal or constant light (CL). Photoreceptor abundance following complete EWM destruction was compared to that following a lesion in the pupil control circuit (as a control for spread of EWM lesions to the nearby pupil-controlling lateral EW) or following no EW damage. Birds were housed post-lesion in a 12 h 400 lux light/12 h dark light cycle for up to 16.5 months, or in constant 400 lux light for up to 3 weeks. Paraformaldehyde–glutaraldehyde fixed eyes were embedded in plastic, sectioned, slide-mounted, and stained with toluidine blue/azure II. Blinded analysis of photoreceptor outer segment abundance was performed, with outer segment types distinguished by oil droplet tint and laminar position. Brains were examined histologically to assess lesion accuracy. Disruption of pupil control had no adverse effect on photoreceptor outer segment abundance in either diurnal light or CL, but EWM destruction led to 50–60% loss of blue/violet cone outer segments in both light conditions, and a 42% loss of principal cone outer segments in CL. The findings indicate that adaptive regulation of ChBF by the EWM circuit plays a role in maintaining photoreceptor health and mitigates the harmful effect of light on photoreceptors, especially short wavelength-sensitive cone photoreceptors.

Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 −/− mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.

Studies into the mechanisms underlying the active emmetropization process by which neonatal refractive errors are corrected, have described rapid, compensatory changes in the thickness of the choroidal layer in response to imposed optical defocus. While high frequency A-scan ultrasonography, as traditionally used to characterize such changes, offers good resolution of central (on-axis) changes, evidence of local retinal control mechanisms make it imperative that more peripheral, off-axis changes also be tracked. In this study, we used in vivo high resolution spectral domain-optical coherence tomography (SD-OCT) imaging in combination with the Iowa Reference Algorithms for 3-dimensional segmentation, to more fully characterize these changes, both spatially and temporally, in young, 7-day old chicks (n = 15), which were fitted with monocular +15 D defocusing lenses to induce choroidal thickening. With these tools, we were also able to localize the retinal area centralis, which was used as a landmark along with the ocular pectin in standardizing the location of scans and aligning them for subsequent analyses of choroidal thickness (CT) changes across time and between eyes. Values were derived for each of four quadrants, centered on the area centralis, and global CT values were also derived for all eyes. Data were compared with on-axis changes measured using ultrasonography. There were significant on-axis choroidal thickening that was detected after just one day of lens wear (∼190 µm), and regional (quadrant-related) differences in choroidal responses were also found, as well as global thickness changes 1 day after treatment. The ratio of global to on-axis choroidal thicknesses, used as an index of regional variability in responses, was also found to change significantly, reflecting the significant central changes. In summary, we demonstrated in vivo high resolution SD-OCT imaging, used in combination with segmentation algorithms, to be a viable and informative approach for characterizing regional (spatial), time-sensitive changes in CT in small animals such as the chick.

Zebrafish (Danio rerio) provide many advantages as a model organism for studying ocular disease and development, and there is great interest in the ability to non-invasively assess their photoreceptor mosaic. Despite recent applications of scanning light ophthalmoscopy, fundus photography, and gonioscopy to in vivo imaging of the adult zebrafish eye, current techniques either lack accurate scaling information (limiting quantitative analyses) or require euthanizing the fish (precluding longitudinal analyses). Here we describe improved methods for imaging the adult zebrafish retina using spectral domain optical coherence tomography (OCT). Transgenic fli1:eGFP zebrafish were imaged using the Bioptigen Envisu R2200 broadband source OCT with a 12-mm telecentric probe to measure axial length and a mouse retina probe to acquire retinal volume scans subtending 1.2 × 1.2 mm nominally. En face summed volume projections were generated from the volume scans using custom software that allows the user to create contours tailored to specific retinal layer(s) of interest. Following imaging, the eyes were dissected for ex vivo fluorescence microscopy, and measurements of blood vessel branch points were compared to those made from the en face OCT images to determine the OCT lateral scale as a function of axial length. Using this scaling model, we imaged the photoreceptor layer of five wild-type zebrafish and quantified the density and packing geometry of the UV cone submosaic. Our in vivo cone density measurements agreed with measurements from previously published histology values. The method presented here allows accurate, quantitative assessment of cone structure in vivo and will be useful for longitudinal studies of the zebrafish cone mosaics.

The cholinergic system is involved in cortical plasticity, attention, and learning. Within the visual cortex the cholinergic system seems to play a role in visual perception. The cholinergic neurons which project into the visual cortex are located in the basal forebrain. It has been shown that mice deficient for the low-affinity neurotrophin receptor p75NTR display increased numbers of cholinergic neurons in the basal forebrain and a denser cholinergic innervation of the hippocampus. This prompted us to analyze whether the cholinergic system is altered in adult p75NTR deficient mice. By analyzing the densities of cholinergic fibers within layer IV as well as within layer V of the visual cortex, we found that adult p75NTR deficient mice display increased cholinergic fiber densities. However, this increase was not accompanied by an increase in the density of local cholinergic neurons within the visual cortex. This indicates that the enhanced cholinergic innervation of the visual cortex is due to alteration of the cholinergic neurons located in the basal forebrain, projecting to the visual cortex. The increased cholinergic innervation of the visual cortex makes the p75NTR deficient mice an attractive model to study the necessity of the cholinergic system for the visual cortex.

Retinal Müller glial cells have been shown to undergo reactive gliosis in a variety of retinal diseases. Upregulation of glial fibrillary acidic protein (GFAP) is a hallmark of Müller cell activation. Reactive gliosis after retinal detachment or ischemia/reperfusion is characterized by hypertrophy and downregulation of inwardly rectifying K+ (Kir) currents. However, this kind of physiological alteration could not be detected in slowly progressing retinal degenerations. The photoreceptor toxin N-methyl-N-nitrosourea (MNU) leads to the rapid loss of cells in the outer nuclear layer and subsequent Müller cell activation. Here, we investigated whether Müller cells from MNU-treated mice exhibit reactive gliosis. We found that Müller cells showed increased GFAP expression and increased membrane capacitance, indicating hypertrophy. Membrane potential and Kir channel-mediated K+ currents were not significantly altered whereas Kir4.1 mRNA expression and Kir-mediated inward current densities were markedly decreased. This suggests that MNU-induced Müller cell gliosis is characterized by plasma membrane increase without alteration in the membrane content of Kir channels. Taken together, our findings show that Müller cells of MNU-treated mice are reactive and respond with a form of gliosis which is characterized by cellular hypertrophy but no changes in Kir current amplitudes.

Recently, two attentional modes have been associated with specific eye movement patterns during scene processing. Ambient mode, characterized by short fixations and long saccades during early scene inspection, is associated with localization of objects. Focal mode, characterized by longer fixations, is associated with more detailed object feature processing during later inspection phase. The aim of the present study was to investigate the development of these attentional modes. More specifically, we examined whether indications of ambient and focal attention modes are similar in infants and adults. Therefore, we measured eye movements in 3- to 12-months-old infants while exploring visual scenes. Our results show that both adults and 12-month-olds had shorter fixation durations within the first 1.5 s of scene viewing compared with later time phases (>2.5 s); indicating that there was a transition from ambient to focal processing during image inspection. In younger infants, fixation durations between two viewing phases did not differ. Our results suggest that at the end of the first year of life, infants have developed an adult-like scene viewing behavior. The evidence for the existence of distinct attentional processing mechanisms during early infancy furthermore underlines the importance of the concept of the two modes.

Recent studies have demonstrated the defocus sign-dependent, bidirectional gene expression regulation of bone morphogenetic proteins, BMP2, 4 and 7 in chick RPE. In this study, we examined the effects of imposed positive (+10 D) and negative (−10 D) lenses on the gene expression of these BMPs and BMP receptors (BMPR1A, BMPR1B, BMPR2) in chick retina and choroid after monocular lens treatment for 2 or 48 h, as indicators of the roles of retinal and choroidal BMPs and receptors in postnatal eye growth regulation. In retina, although all genes were expressed, neither +10 nor −10 D lenses, worn for either 2 or 48 h, significantly altered gene expression. In contrast, treatment-related differential gene expression was detected in the choroid for both BMPs and their receptors, although interestingly, with the +10 D lens, BMP2 was up-regulated by 156.7 ± 19.7% after 2 h, while BMPR1A was down-regulated to 82.3 ± 12.5% only after 48 h. With the −10 D lens, only the gene expression of BMPR1B was significantly altered, being up-regulated by 162.3 ± 21.2% after 48 h. Untreated birds showed no difference in expression between their two eyes, for any of the genes examined. The finding that retinal gene expression for BMP2, 4, 7 and their receptors are not affected by short-term optical defocus contrasts with previous observations of sign-dependent expression changes for the same genes in the RPE. The latter changes were also larger and more consistent in direction than the choroidal gene expression changes reported here. The interrelationship between these various changes and their biological significance for eye growth regulation are yet to be elucidated.

Visual perception is not instantaneous; the perceptual representation of our environment builds up over time. This can strongly affect our responses to visual stimuli. Here, we study the temporal dynamics of visual processing by analyzing the time course of priming effects induced by the well-known Ebbinghaus illusion. In slower responses, Ebbinghaus primes produce effects in accordance with their perceptual appearance. However, in fast responses, these effects are reversed. We argue that this dissociation originates from the difference between early feedforward-mediated gist of the scene processing and later feedback-mediated more elaborate processing. Indeed, our findings are well explained by the differences between low-frequency representations mediated by the fast magnocellular pathway and high-frequency representations mediated by the slower parvocellular pathway. Our results demonstrate the potentially dramatic effect of response speed on the perception of visual illusions specifically and on our actions in response to objects in our visual environment generally.

Amacrine cells are a diverse set of local circuit neurons of the inner retina, and they all release either GABA or glycine, amino acid neurotransmitters that are generally inhibitory. But some types of amacrine cells have another function besides inhibiting other neurons. One glycinergic amacrine cell, the Aii type, excites a subset of bipolar cells via extensive gap junctions while inhibiting others at chemical synapses. Many types of GABAergic amacrine cells also release monoamines, acetylcholine, or neuropeptides. There is now good evidence that another type of amacrine cell releases glycine at some of its synapses and releases the excitatory amino acid glutamate at others. The glutamatergic synapses are made onto a subset of retinal ganglion cells and amacrine cells and have the asymmetric postsynaptic densities characteristic of central excitatory synapses. The glycinergic synapses are made onto other types of ganglion cells and have the symmetric postsynaptic densities characteristic of central inhibitory synapses. These amacrine cells, which contain vesicular glutamate transporter 3, will be the focus of this brief review.