Retinal pigment epithelial cells of teleosts contain numerous melanosomes (pigment granules) that exhibit light-dependent motility. In light, melanosomes disperse out of the retinal pigment epithelium (RPE) cell body (CB) into long apical projections that interdigitate with rod photoreceptors, thus shielding the photoreceptors from bleaching. In darkness, melanosomes aggregate through the apical projections back into the CB. Previous research has demonstrated that melanosome motility in the RPE CB requires microtubules, but in the RPE apical projections, actin filaments are necessary and sufficient for motility. We used myosin S1 labeling and platinum replica shadowing of dissociated RPE cells to determine actin filament polarity in apical projections. Actin filament bundles within RPE apical projections are uniformly oriented with barbed ends toward the distal tips. Treatment of RPE cells with the tetravalent lectin, Concanavalin A, which has been shown to suppress cortical actin flow by crosslinking of cell-surface proteins, inhibited melanosome aggregation and stimulated ectopic filopodia formation but did not block melanosome dispersion. The polarity orientation of F-actin in apical projections suggests that a barbed-end directed myosin motor could effect dispersion of melanosomes from the CB into apical projections. Inhibition of aggregation, but not dispersion, by ConA confirms that different actin-dependent mechanisms control these two processes and suggests that melanosome aggregation is sensitive to treatments previously shown to disrupt actin cortical flow.

A moving stimulus paradigm was designed to investigate color contrast encoding in the retina. Recently, this paradigm yielded suggestive evidence for color contrast encoding in zebrafish but the significance and generality remain uncertain since the properties of color coding in the zebrafish inner retina are largely unknown. Here, the question of color contrast is pursued in the goldfish retina where there is much accumulated evidence for retinal mechanisms of color vision and opponent color-coding, in particular. Recordings of a sensitive local field potential of the inner retina, the proximal negative response, were made in the intact, superfused retina in the light-adapted state. Responses to color contrast and achromatic contrast were analyzed by comparing responses to a green moving bar on green versus red backgrounds. The quantitative form of the irradiance/response curves was distinctly different under a range of conditions in 32 retinas, thereby providing robust evidence for red–green color contrast. The color contrast is based on successive contrast, occurs in the absence of overt color opponency, and clearly differs from previous findings in the goldfish retina for simultaneous color contrast mediated by color-opponent neurons. The form of the irradiance/response curves suggests that successive color contrast is particularly important when achromatic contrast is low, as often occurs in natural environments. The present results provide a parallel with the well-known principle of human color vision, first proposed by Kirschmann as the third law of color contrast, and may also have implications for the evolution of vertebrate color vision.

Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (Cm) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging <200 ms. We manipulated Ca2+ influx and the amount of vesicle release by altering the duration and voltage of depolarizing steps. Unlike cones, endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels.

Significant advances have been made over the last decade or two in the elucidation of the molecular pathogenesis of inherited ocular disorders. In particular, remarkable successes have been achieved in exploration of gene-based medicines for these conditions, both in preclinical and in clinical studies. Progress in the development of gene therapies targeted toward correcting the primary genetic defect or focused on modulating secondary effects associated with retinal pathologies are discussed in the review. Likewise, the recent utilization of genes encoding light-sensing molecules to provide new functions to residual retinal cells in the degenerating retina is discussed. While a great deal has been learned over the last two decades, the next decade should result in an increasing number of preclinical studies progressing to human clinical trial, an exciting prospect for patients, those active in research and development and bystanders alike.

S cones expressing the short wavelength-sensitive type 1 (SWS1) class of visual pigment generally form only a minority type of cone photoreceptor within the vertebrate duplex retina. Hence, their primary role is in color vision, not in high acuity vision. In mammals, S cones may be present as a constant fraction of the cones across the retina, may be restricted to certain regions of the retina or may form a gradient across the retina, and in some species, there is coexpression of SWS1 and the long wavelength-sensitive (LWS) class of pigment in many cones. During retinal development, SWS1 opsin expression generally precedes that of LWS opsin, and evidence from genetic studies indicates that the S cone pathway may be the default pathway for cone development. With the notable exception of the cartilaginous fishes, where S cones appear to be absent, they are present in representative species from all other vertebrate classes. S cone loss is not, however, uncommon; they are absent from most aquatic mammals and from some but not all nocturnal terrestrial species. The peak spectral sensitivity of S cones depends on the spectral characteristics of the pigment present. Evidence from the study of agnathans and teleost fishes indicates that the ancestral vertebrate SWS1 pigment was ultraviolet (UV) sensitive with a peak around 360 nm, but this has shifted into the violet region of the spectrum (>380 nm) on many separate occasions during vertebrate evolution. In all cases, the shift was generated by just one or a few replacements in tuning-relevant residues. Only in the avian lineage has tuning moved in the opposite direction, with the reinvention of UV-sensitive pigments.

Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter.

Retinal ganglion cell (RGC) dendritic atrophy is an early feature of many forms of retinal degeneration, providing a challenge to RGC classification. The characterization of these changes is complicated by the possibility that selective labeling of any particular class can confound the estimation of dendritic remodeling. To address this issue we have developed a novel, robust, and quantitative RGC classification based on proximal dendritic features which are resistant to early degeneration. RGCs were labeled through the ballistic delivery of DiO and DiI coated tungsten particles to whole retinal explants of 20 adult Brown Norway rats. RGCs were grouped according to the Sun classification system. A comprehensive set of primary and secondary dendrite features were quantified and a new classification model derived using principal component (PCA) and discriminant analyses, to estimate the likelihood that a cell belonged to any given class. One-hundred and thirty one imaged RGCs were analyzed; according to the Sun classification, 24% (n = 31) were RGCA, 29% (n = 38) RGCB, 32% (n = 42) RGCC, and 15% (n = 20) RGCD. PCA gave a 3 component solution, separating RGCs based on descriptors of soma size and primary dendrite thickness, proximal dendritic field size and dendritic tree asymmetry. The new variables correctly classified 73.3% (n = 74) of RGCs from a training sample and 63.3% (n = 19) from a hold out sample indicating an effective model. Soma and proximal dendritic tree morphological features provide a useful surrogate measurement for the classification of RGCs in disease. While a definitive classification is not possible in every case, the technique provides a useful safeguard against sample bias where the normal criteria for cell classification may not be reliable.

Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologs Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod–cone, cone–cone, and possibly rod–rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals, respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study, we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose–response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus, the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to regulate photoreceptor coupling via a push–pull mechanism. However, the lower concentration of adenosine present in the daytime actually reinforces the dopamine signal through action on the A1 receptor.

Previous studies have shown that multiple sclerosis (MS) affects the visual system, mainly by reducing contrast sensitivity (CS), a function that can be assessed by measuring contrast sensitivity function (CSF). To this end, we measured both the CSF for sine-wave gratings and angular frequency stimuli with 20 participants aged between 21 and 44 years, of both genders, with normal or corrected to normal visual acuity. Of these 20 participants, there were 10 volunteers with clinically defined MS of the relapsing–remitting clinical form, with no history of optic neuritis (ON), as well as 10 healthy volunteers who served as the control group (CG). We used a forced-choice detection paradigm. The results showed reduced CS to both classes of stimuli. Differences were found for sine-wave gratings at spatial frequencies of 0.5, 1.25, and 2.5 cycles per degree (cpd) (P < 0.002) and for angular frequency stimuli of 4, 24, and 48 cycles/360° (P < 0.05). On the one hand, comparing the maxima of the respective CSFs, the CS to angular frequency stimuli (24 cycles/360°) was 1.61-fold higher than that of the CS to vertical sine-wave gratings (4.0 cpd) in the CG; for the MS group, these values were 1.55-fold higher. On the other hand, CS in the MS group attained only 75% for 24 cycles/360° and 78% for 4.0 cpd of the 100% CS estimates found for the CG at the peak frequencies. These findings suggest that MS affects the visual system, mostly at its maximum contrast sensitivities. Also, since angular frequencies and sine-wave gratings operate at distinct levels of contrast in the visual system, MS seems to affect CS at both high and low levels of contrast.

Specific variants of human long-wavelength (L) and middle-wavelength (M) cone opsin genes have recently been associated with a variety of vision disorders caused by cone malfunction, including red-green color vision deficiency, blue cone monochromacy, myopia, and cone dystrophy. Strikingly, unlike disease-causing mutations in rhodopsin, most of the cone opsin alleles that are associated with vision disorders do not have deleterious point mutations. Instead, specific combinations of normal polymorphisms that arose by genetic recombination between the genes encoding L and M opsins appear to cause disease. Knockout/knock-in mice promise to make it possible to study how these deleterious cone opsin variants affect the structure, function, and viability of the cone photoreceptors. Ideally, we would like to evaluate different variants that cause vision disorders in humans against a control pigment that is not associated with vision disorders, and each variant should be expressed as the sole photopigment in each mouse cone, as is the case in humans. To evaluate the feasibility of this approach, we created a line of mice to serve as the control in the analysis of disease-causing mutations by replacing exon 2 through 6 of the mouse M-opsin gene with the corresponding cDNA for a human L-opsin variant that is associated with normal vision. Experiments reported here establish that the resulting pigment, which differs from the endogenous mouse M opsin at 35 amino acid positions, functions normally in mouse cones. This pigment was evaluated in mice with and without coexpression of the mouse short wavelength (S) opsin. Here, the creation and validation of two lines of genetically engineered mice that can be used to study disease-causing variants of human L/M-opsins, in vivo, are described.

Anatomical and physiological approaches are beginning to reveal the synaptic origins of parallel ON- and OFF-pathway retinal circuits for the transmission of short (S-) wavelength sensitive cone signals in the primate retina. Anatomical data suggest that synaptic output from S-cones is largely segregated; central elements of synaptic triads arise almost exclusively from the “blue-cone” bipolar cell, a presumed ON bipolar, whereas triad-associated contacts derive primarily from the “flat” midget bipolar cell, a hyperpolarizing, OFF bipolar. Similarly, horizontal cell connectivity is also segregated, with only the H2 cell-type receiving numerous contacts from S-cones. Negative feedback from long (L-) and middle (M-) wavelength sensitive cones via the H2 horizontal cells elicits an antagonistic surround in S-cones demonstrating that S versus L + M or “blue-yellow” opponency is first established in the S-cone. However, the S-cone output utilizes distinct synaptic mechanisms to create color opponency at the ganglion cell level. The blue-cone bipolar cell is presynaptic to the small bistratified, “blue-ON” ganglion cell. S versus L + M cone opponency arises postsynaptically by converging S-ON and LM-OFF excitatory bipolar inputs to the ganglion cell’s bistratified dendritic tree. The common L + M cone surrounds of the parallel S-ON and LM-OFF cone bipolar inputs appear to cancel resulting in “blue-yellow” antagonism without center-surround spatial opponency. By contrast, in midget ganglion cells, opponency arises by the differential weighting of cone inputs to the receptive field center versus surround. In the macula, the “private-line” connection from a midget ganglion cell to a single cone predicts that S versus L + M opponency is transmitted from the S-cone to the S-OFF midget bipolar and ganglion cell. Beyond the macula, OFF-midget ganglion cell dendritic trees enlarge and collect additional input from multiple L and M cones. Thus S-OFF opponency via the midget pathway would be expected to become more complex in the near retinal periphery as L and/or M and S cone inputs sum to the receptive field center. An important goal for further investigation will be to explore the hypothesis that distinct bistratified S-ON versus midget S-OFF retinal circuits are the substrates for human psychophysical detection mechanisms attributed to S-ON versus S-OFF perceptual channels.

Limb remote ischemic conditioning (LRIC) provides a physiologic strategy for harnessing the body’s endogenous protective capabilities against injury induced by ischemia–reperfusion in the central nervous system. The aim of the present study was to determine if LRIC played a role in protecting the retina from ischemia–reperfusion injury. A total of 81 adult male Sprague-Dawley rats were randomly assigned to sham and ischemia/reperfusion with or without remote LRIC arms. The retinal ischemic model was generated through right middle cerebral artery occlusion (MCAO) and pterygopalatine artery occlusion for 60 min followed by 1, 3, and 7 days of subsequent reperfusion. LRIC was conducted immediately following MCAO by tightening a tourniquet around the upper thigh and releasing for three cycles. Paraffin sections were stained with hematoxylin and eosin in order to quantify the number of cells in retinal ganglion cells (RGCs) layer throughout the duration of the study. Cellular expression of glial fibrillary acidic protein (GFAP) was detected and examined through immunohistochemistry. Protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) was also analyzed by Western blot techniques. Our study demonstrated that the loss of cells in RGC layer was attenuated by LRIC treatment at 3 and 7 days following reperfusion (P < 0.05). Immunohistochemistry studies depicted a gradual increase (P < 0.05) in GFAP levels from day 1 through day 7 following ischemia and subsequent reperfusion, whereas LRIC reduced GFAP levels at 1, 3, and 7 days postreperfusion. In addition, LRIC increased the expression of Nrf2 and HO-1 at day 1 and 3 following ischemia/reperfusion. This particular study is the first remote conditioning study applicable to retinal ischemia. Our results strongly support the position that LRIC may be used as a noninvasive neuroprotective strategy, which provides retinal protection from ischemia–reperfusion injury through the upregulation of antioxidative stress proteins, such as Nrf2 and HO-1.

The eye has become an excellent target for gene therapy, and gene augmentation therapy of inherited retinal disorders has made major progress in recent years. Nevertheless, a recent study indicated that gene augmentation intervention might not stop the progression of retinal degeneration in patients. In addition, for many genes, viral-mediated gene augmentation is currently not feasible due to gene size and limited packaging capacity of viral vectors as well as expression of various heterogeneous isoforms of the target gene. Thus, alternative gene-based strategies to stop or delay the retinal degeneration are necessary. This review focuses on an alternative pharmacologic treatment strategy based on the usage of translational read-through inducing drugs (TRIDs) such as PTC124, aminoglycoside antibiotics, and designer aminoglycosides for overreading in-frame nonsense mutations. This strategy has emerged as an option for up to 30–50% of all cases of recessive hereditary retinal dystrophies. In-frame nonsense mutations are single-nucleotide alterations within the gene coding sequence resulting in a premature stop codon. Consequently, translation of such mutated genes leads to the synthesis of truncated proteins, which are unable to fulfill their physiologic functions. In this context, application of TRIDs facilitates the recoding of the premature termination codon into a sense codon, thus restoring syntheses of full-length proteins. So far, clinical trials for non-ocular diseases have been initiated for diverse TRIDs. Although the clinical outcome is not analyzed in detail, an excellent safety profile, namely for PTC124, was clearly demonstrated. Moreover, recent data demonstrated sustained read-through efficacies of nonsense mutations causing retinal degeneration, as manifested in the human Usher syndrome. In addition, a strong retinal biocompatibility for PTC124 and designer aminoglycosides has been demonstrated. In conclusion, recent progress emphasizes the potential of TRIDs as an alternative pharmacologic treatment strategy for treating nonsense mutation-based retinal disorders.

Blindness represents an increasing global problem with significant social and economic impact upon affected patients and society as a whole. In Europe, approximately one in 30 individuals experience sight loss and 75% of those are unemployed, a social burden which is very likely to increase as the population of Europe ages. Diseases affecting the retina account for approximately 26% of blindness globally and 70% of blindness in the United Kingdom. To date, there are no treatments to restore lost retinal cells and improve visual function, highlighting an urgent need for new therapeutic approaches. A pioneering breakthrough has demonstrated the ability to generate synthetic retina from pluripotent stem cells under laboratory conditions, a finding with immense relevance for basic research, in vitro disease modeling, drug discovery, and cell replacement therapies. This review summarizes the current achievements in pluripotent stem cell differentiation toward retinal cells and highlights the steps that need to be completed in order to generate human synthetic retinae with high efficiency and reproducibly from patient-specific pluripotent stem cells.

The parallel processing of visual features by distinct neuron populations is a central characteristic of the mammalian visual system. In the A laminae of the cat dorsal lateral geniculate nucleus (dLGN), parallel processing streams originate from two principal neuron types, called X and Y cells. Disruption of visual experience early in life by monocular deprivation has been shown to alter the structure and function of Y cells, but the extent to which deprivation influences X cells remains less clear. A transcription factor, FoxP2, has recently been shown to selectively label X cells in the ferret dLGN and thus provides an opportunity to examine whether monocular deprivation alters the soma size of X cells. In this study, FoxP2 labeling was examined in the dLGN of normal and monocularly deprived cats. The characteristics of neurons labeled for FoxP2 were consistent with FoxP2 being a marker for X cells in the cat dLGN. Monocular deprivation for either a short (7 days) or long (7 weeks) duration did not alter the density of FoxP2-positive neurons between nondeprived and deprived dLGN layers. However, for each deprived animal examined, measurement of the cross-sectional area of FoxP2-positive neurons (X cells) revealed that within deprived layers, X cells were smaller by approximately 20% after 7 days of deprivation, and by approximately 28% after 7 weeks of deprivation. The observed alteration to the cross-sectional area of X cells indicates that perturbation of this major pathway contributes to the functional impairments that develop from monocular deprivation.

The appearance of colors can be affected by their spatiotemporal context. The shift in color appearance according to the surrounding colors is called color induction or chromatic induction; in particular, the shift in opponent color of the surround is called chromatic contrast. To investigate whether chromatic induction occurs even when the chromatic surround is imperceptible, we measured chromatic induction during interocular suppression. A multicolor or uniform color field was presented as the surround stimulus, and a colored continuous flash suppression (CFS) stimulus was presented to the dominant eye of each subject. The subjects were asked to report the appearance of the test field only when the stationary surround stimulus is invisible by interocular suppression with CFS. The resulting shifts in color appearance due to chromatic induction were significant even under the conditions of interocular suppression for all surround stimuli. The magnitude of chromatic induction differed with the surround conditions, and this difference was preserved regardless of the viewing conditions. The chromatic induction effect was reduced by CFS, in proportion to the magnitude of chromatic induction under natural (i.e., no-CFS) viewing conditions. According to an analysis with linear model fitting, we revealed the presence of at least two kinds of subprocesses for chromatic induction that reside at higher and lower levels than the site of interocular suppression. One mechanism yields different degrees of chromatic induction based on the complexity of the surround, which is unaffected by interocular suppression, while the other mechanism changes its output with interocular suppression acting as a gain control. Our results imply that the total chromatic induction effect is achieved via a linear summation of outputs from mechanisms that reside at different levels of visual processing.

Color information is encoded by two parallel pathways in the mammalian retina. One pathway compares signals from long- and middle-wavelength sensitive cones and generates red-green opponency. The other compares signals from short- and middle-/long-wavelength sensitive cones and generates blue-green (yellow) opponency. Whereas both pathways operate in trichromatic primates (including humans), the fundamental, phylogenetically ancient color mechanism shared among most mammals is blue-green opponency. In this review, we summarize the current understanding of how signals from short-wavelength sensitive cones are processed in the primate and nonprimate mammalian retina, with a focus on the inner plexiform layer where bipolar, amacrine, and ganglion cell processes interact to facilitate the generation of blue-green opponency.

This study examined the effects of metabotropic glutamate receptor 6 (mGluR6) deficiency on ribbon synapse formation in rod spherules and cone pedicles using serial-section electron microscopy. In a wild-type (WT) mouse, only 3% of spherules had one invaginating bipolar dendrite (1B-type) and 97% of spherules were 2B-type. In contrast, in an mGluR6-knockout (KO) mouse, 29% of spherules were 1B-type and 71% of spherules were 2B-type. Spherules without bipolar invagination were not observed in either genotype. The single invaginating dendrites in 1B-type spherules were larger and the surface areas of synaptic ribbons were 23% smaller in the mGluR6-KO mouse than in the WT mouse. In cones, the number of invaginating bipolar dendrites decreased from 12 in the WT mouse to 9.5 in the mGluR6-KO mouse. This decrease correlated with a decrease in the number of cone synaptic ribbons from 10 in the WT mouse to 8 in the mGluR6-KO mouse. The mGluR6-KO phenotype showed negative effects on ribbon synapse formation. This negativity was similar to those in mGluR6-nob4, Gβ3-KO, Gβ5-KO, and RGS-7:RGS-11 double-KO mice, but the detailed manners and degrees of alterations appeared to vary depending on different missing components. Two published morphological assessments of the RGS-7:RGS-11 double-KO phenotype reported conflicting data; therefore, we tested the statistical techniques used in the two analyses. One statistical evaluation measure was effective in identifying a significant difference in structure between the mutant and WT phenotypes, whereas the other measure was ineffective. Conventional random section analysis using the effective measure provided sufficient data for a statistical test of the occurrence of structural changes. However, serial section analysis was required to determine the absolute numbers of ribbons and invaginating dendrites and to estimate structural parameters such as ribbon surface area.