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Purpose The purpose of this study was to investigative the effects of blue light on intrinsically photoreceptive retinal ganglion cells (ipRGCs). Methods Brown Norway rats were used. Nine rats were continuously exposed to blue light (light emitting diodes [LEDs]: 463 nm; 1000 lx) for 2 days (acute exposure [AE]); 9 rats were exposed to 12 hours of blue light and 12 hours of darkness for 10 days (long-term exposure [LTE]); 6 control rats were exposed to 12 hours of white fluorescent light (1000 lx) and 12 hours of darkness for 10 days. Whole-mount retinas were immunolabelled with melanopsin antibodies; melanopsin-positive (MP) ipRGC somas and processes were counted and measured with Neuron J. To detect apoptosis, retinal cryo-sections were stained with terminal deoxynucleotidyl transferase dUTP nick-end labeling. Ultra-thin sections were visualized with transmission electron microscopy. Results The number of MP ipRGC somas was significantly lower in retinas from AE and LTE rats than in those from control rats (P < 0.001 and = 0.002, respectively). The mean length of MP areas of processes was significantly lower in AE rats (P < 0.001). AE rats had severe retinal damage and massive apoptosis in the outer nuclear layer; their mitochondria were damaged in the axons and dendrites of the nerve fiber layer and the inner plexiform layer. Retinal ganglion cells (RGCs) in AE rats appeared to have reduced amounts of free ribosomes and rough endoplasmic reticulum. Conclusions AE to blue light reduces melanopsin expression and damages RGCs, likely including ipRGCs. Changes in the axons and dendrites of RGCs suggest possible disruption of intraretinal and extraretinal signal transmission.

Purpose Human and animal studies suggest that light-mediated dopamine release may underlie the protective effect of time outdoors on myopia development. Melanopsin-containing retinal ganglion cells may be involved in this process by integrating ambient light exposure and regulating retinal dopamine levels. The study evaluates this potential involvement by examining whether melanopsin-driven pupillary responses are associated with adult refractive error. Methods Subjects were 45 young adults (73% female, 24.1 ± 1.8 years) with refractive errors ranging from -6.33 D to +1.70 D. The RAPDx (Konan Medical) pupillometer measured normalized pupillary responses to three forms of square-wave light pulses alternating with darkness at 0.1 Hz: alternating long wavelength (red, peak at 608 nm) and short wavelength (blue, peak at 448 nm), followed by red only and then blue only. Results Non-myopic subjects displayed greater pupillary constriction in the blue-only condition and slower redilation following blue light offset than subjects with myopia (P = 0.011). Pupillary responses were not significantly different between myopic and non-myopic subjects in the red-only condition (P = 0.15). More hyperopic/less myopic refractive error as a continuous variable was linearly related to larger increases in pupillary constriction in response to blue-only stimuli (r = 0.48, P = 0.001). Conclusions Repeated light exposures to blue test stimuli resulted in an adaptation in the pupillary response (more constriction and slower redilation), presumably due to increased melanopsin-mediated input in more hyperopic/less myopic adults. This adaptive property supports a possible role for these ganglion cells in the protective effects of time outdoors on myopia development.

Purpose To investigate how melanopsin-mediated intrinsically photosensitive retinal ganglion cell (ipRGC) signals are integrated binocularly using chromatic pupillometry. We hypothesized that if the melanopsin system is summative, there will be a greater postillumination pupillary response (PIPR) under binocular conditions after viewing bright blue light. Methods Pupillary responses in 10 visually normal participants were recorded with an eye tracker following full-field stimulation of red (long wavelength) and blue (short wavelength) light of equal intensity (dim: 0.1 cd [candela]/m2, bright: 60 cd/m2) and duration (400 ms). Individual monocular (left eye) pupil responses were measured first, followed by binocular responses. Each participant repeated the same protocol on 3 separate days, at similar times of day. PIPR was recorded for bright red and blue conditions only, whereas maximum pupillary constriction (MPC) was measured under both bright and dim conditions during red and blue light stimulation. Results Bright blue light stimulation induced greater PIPR under binocular than monocular viewing conditions (F(1,9) = 79.52, P < 0.001). Bright red light stimulation induced minimal PIPR and showed no significant difference between viewing conditions post Bonferroni correction (F(1,9) = 5.49, P = 0.04). MPC was greater during binocular than monocular viewing conditions for all light stimuli, but was greatest following blue compared to red light stimulation. Conclusions A larger PIPR was induced using a binocular than a monocular full-field stimulus of equal intensity and duration, demonstrating that melanopsin-mediated ipRGC signals are summated binocularly. This study expands our current understanding of the melanopsin system and may be used as an additional marker to stratify diseases according to their etiologies.

Purpose To investigate whether melanopsin-containing ophthalmic trigeminal ganglion cells provide significant input to mediate light-induced discomfort. This is done by studying the effect of ocular topical anesthesia on light-induced discomfort threshold to blue light and red light stimuli using a psychophysical approach. Method Ten visually normal participants completed the experiment consisting of two trials: an anesthesia trial in which light stimuli were presented to both eyes following 0.5% proparacaine eye drops administration, and a placebo trial in which normal saline drops were used. In each trial, a randomized series of 280 blue and red light flashes were presented over seven intensity steps with 20 repetitions for each color and light intensity. Participants were instructed to report whether they perceived each stimulus as either "uncomfortably bright" or "not uncomfortably bright" by pressing a button. The proportion of "uncomfortable" responses was pooled to generate individual psychometric functions, from which 50% discomfort thresholds (defined as the light intensity at which the individuals perceived the stimulus to be uncomfortably bright/unpleasant 50% of the time) were calculated. Results When blue light was presented, there was no significant difference in the light-induced discomfort thresholds between anesthesia and placebo trials (P = 0.44). Similarly, when red light was used, no significant difference in threshold values was found between the anesthesia and placebo trials (P = 0.28). Conclusions Ocular topical anesthesia does not alter the light-induced discomfort thresholds to either blue or red light, suggesting that the melanopsin-containing ophthalmic trigeminal ganglion cells provide little or no significant input in mediating light-induced discomfort under normal physiologic conditions.

Purpose Pupillary light reflex (PLR) is driven by outer retinal photoreceptors and by melanopsin-expressing intrinsically photosensitive retinal ganglion cells of the inner retina. To isolate the melanopic component, we studied patients with severe vision loss due to Leber congenital amaurosis (LCA) caused by gene mutations acting on the outer retina. Methods Direct PLR was recorded in LCA patients (n = 21) with known molecular causation and severe vision loss. Standard stimuli (2.5 log scot-cd.m-2; ∼13 log quanta.cm-2.s-1; achromatic full-field) with 0.1- or 5-second duration were used in all patients. Additional recordings were performed with higher luminance (3.9 log scot-cd.m-2) in a subset of patients. Results The LCA patients showed no detectable PLR to the standard stimulus with short duration. With longer-duration stimuli, a PLR was detectable in the majority (18/21) of patients. The latency of the PLR was 2.8 ± 1.3 seconds, whereas normal latency was 0.19 ± 0.02 seconds. Peak contraction amplitude in patients was 1.1 ± 0.9 mm at 6.2 ± 2.3 seconds, considerably different from normal amplitude of 4.2 ± 0.4 mm at 3.0 ± 0.4 seconds. Recordings with higher luminance demonstrated that PLRs in severe LCA could also be evoked with short-duration stimuli. Conclusions The PLR in severe LCA patients likely represents the activation of the melanopic circuit in isolation from rod and cone input. Knowledge of the properties of the human melanopic PLR allows not only comparison to those in animal models but also serves to define the fidelity of postretinal transmission in clinical trials targeting patients with no outer retinal function.

Purpose: Photo-entrainment of the circadian clock is mediated by melanopsin-expressing retinal ganglion cells (mRGCs) located in the retina. Patients suffering from diabetic retinopathy (DR) show impairment of light regulated circadian activity such as sleep disorders, altered blood pressure, and abnormal melatonin secretion. The aim of this study was to assess the impact of DR on the expression of mRGCs in the human retina.Methods: The expression of mRGCs and RGCs was determined in eye sections containing retinal tissue from patients with DR (n = 6) and respective age-matched controls (n = 8) using immunohistochemistry by costaining with antibodies against RNA binding protein with multiple splicing (RBPMS), which identified RGCs and melanopsin which identified the mRGCs.Results: The expression of RGCs in the retina from patients with severe DR was significantly reduced to a density of 146 ± 76 cells/mm2 compared with controls at 1280 ± 249 cells/mm2. The density of mRGCs was also significantly reduced from 3.12 ± 0.54 cells/mm2 in controls to 0.72 ± 0.18 cells/mm2 in patients with DR, with significant loss of 73.5% and 81.9% in mRGC density in the ganglion cell layer (GCL) and inner nuclear layer (INL), respectively.Conclusions: Our findings show that DR affects the expression of mRGCs in the human retina and could explain the abnormal circadian activity observed in patients with DR.

Purpose The purpose of this study was to evaluate a chromatic pupillometry protocol for specific functional assessment of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in dogs. Methods Chromatic pupillometry was tested and compared in 37 dogs in different stages of primary loss of rod, cone, and combined rod/cone and optic nerve function, and in 5 wild-type (WT) dogs. Eyes were stimulated with 1-s flashes of dim (1 cd/m2) and bright (400 cd/m2) blue light (for scotopic conditions) or bright red (400 cd/m2) light with 25-cd/m2 blue background (for photopic conditions). Canine retinal melanopsin/Opn4 was cloned, and its expression was evaluated using real-time quantitative reverse transcription-PCR and immunohistochemistry. Results Mean ± SD percentage of pupil constriction amplitudes induced by scotopic dim blue (scDB), scotopic bright blue (scBB), and photopic bright red (phBR) lights in WT dogs were 21.3% ± 10.6%, 50.0% ± 17.5%, and 19.4% ± 7.4%, respectively. Melanopsin-mediated responses to scBB persisted for several minutes (7.7 ± 4.6 min) after stimulus offset. In dogs with inherited retinal degeneration, loss of rod function resulted in absent scDB responses, followed by decreased phBR responses with disease progression and loss of cone function. Primary loss of cone function abolished phBR responses but preserved those responses to blue light (scDB and scBB). Although melanopsin/Opn4 expression was diminished with retinal degeneration, melanopsin-expressing ipRGCs were identified for the first time in both WT and degenerated canine retinas. Conclusions Pupil responses elicited by light stimuli of different colors and intensities allowed differential functional assessment of canine rods, cones, and ipRGCs. Chromatic pupillometry offers an effective tool for diagnosing retinal and optic nerve diseases.

Purpose To analyze the responses of different retinal ganglion cell (RGC) types to acute ocular hypertension (AOH) and intravitreal administration of brain-derived neurotrophic factor (BDNF). Methods In adult albino rats, the anterior chamber of the left eye was cannulated with a needle connected to a saline container elevated 1½ meters above the eye for 75 minutes. Rats received 12 hours before a 5 μl intravitreal injection containing 5 μg BDNF in 1% albumin PBS or vehicle and were analyzed 3, 7, 14, or 45 days later. Both retinas were dissected as wholemounts and immunolabeled for melanopsin (to identify intrinsically photosensitive RGCs) or Brn3a (to identify all RGCs except melanopsin +RGCs). Results During AOH there is ischemic damage and mechanical eye-globe deformation. Acute ocular hypertension results in a progressive loss of Brn3a+RGCs in the vehicle-treated retinas (39%, 35%, 25%, and 13% of the original value, at 3, 7, 14, or 45 days, respectively), whereas BDNF increases their survival to 81%, 73%, 59%, or 57% at the same time periods. In vehicle-treated retinas, 37% or 39% of m+RGCs survive at 14 or 45 days, respectively, whereas BDNF treatment increases their survival to 40% or 78% at the same time points. Conclusions Different types of RGCs respond differently to AOH because Brn3a+RGCs die progressively, but m+RGCs do not. After a transient downregulation of melanopsin expression, their number remains constant and their survival is proportionally higher than that of Brn3a+RGCs. BDNF affords a permanent protection up to 45 days after AOH injury in both types of RGCs.

Purpose The purpose of this study was to investigate the impact of activating melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) on dark-adapted (scotopic) electroretinograms (ERG). Methods We used mice (Opn4Cre/+) expressing cre recombinase in melanopsin-expressing cells for a targeted gene delivery of a chemogenetic Gq-coupled receptor, hM3Dq, to ipRGCs. Intraperitoneal injection of clozapine N-oxide (CNO) at 5 mg/kg was used for acute activation of hM3Dq and thus excitation of ipRGCs in darkness. Dark-adapted flash ERGs were recorded across a 9-fold range of irradiances from hM3Dq Opn4Cre/+ and control Opn4Cre/+ mice before and after intraperitoneal injection of CNO. A- and b-wave amplitudes and implicit times and oscillatory potentials (OPs) were analyzed. Paired-flash stimuli were used to isolate cone-driven responses. Results Clozapine N-oxide application suppressed a- and b-wave amplitudes of the dark-adapted ERG across the flash intensity range in hM3Dq Opn4Cre/+ mice compared to control mice. Examination of the normalized irradiance-response functions revealed a shift in b-wave but not a-wave sensitivity. No changes in a- and b-wave implicit times were detected. Total OP amplitudes were also reduced in hM3Dq Opn4Cre/+ mice compared to controls following CNO administration. The paired-flash method revealed reduction in both the first (rods and cones) and second (cones only) flash response. Conclusions Acute and selective activation of ipRGCs modulates the amplitude of both a- and b-waves of the scotopic ERG, indicating that the influence of this ganglion cell class on the retinal physiology extends to the photoreceptors as well as their downstream pathways.

Purpose: Multiple studies have shown overwhelming evidence supporting the impairment of melanopsin function due to glaucoma. However, few studies have been carried out in humans analyzing the histology of melanopsin-expressing retinal ganglion cells (mRGCs) in retinas with glaucoma. The aim of this study was to analyze the pattern of expression of mRGCs relative to RGCs in the normal retina and retinas harboring varying stages of glaucoma.Methods: Paraffin-embedded human donor eyes with glaucoma (n = 11) and age-matched controls (n = 10) were obtained from Department of Pathology at Rigshospital (Copenhagen, Denmark) for detection of RNA binding protein with multiple splicing (RBPMS) and melanopsin by immunohistochemistry. The density of RBPMS-expressing RGCs and mRGCs in each retina was estimated as the total cell count in the total retinal area analyzed (cell counts/mm2).Results: No significant difference was observed in mRGC expression in the normal retinas and mild-staged retinas with glaucoma; the densities of mRGCs were 3.08 ± 0.47 and 3.00 ± 0.13 cell counts/mm2, respectively. However, the severely staged retinas with glaucoma showed a significant loss in mRGCs density, 1.09 ± 0.35 cell counts/mm2, with 75% of all retained mRGCs occurring in the inner nuclear layer.Conclusions: This is the first report illustrating histologic evidence for reduced mRGC density in the ganglion cell layer of retinas with severely staged glaucoma compared with age-matched controls. This result proposes evaluation of mRGCs integrity as a basis for assessing the pathophysiologic disease progression of glaucoma.

Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones.We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin.Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice14 months. This increased activation occurred in more central and peripheral retinal regions.Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.

Purpose A retinal projection into the dorsal raphe nucleus (DRN), namely, the retino-raphe projection, exists in many species. The rat is one of several species in which a retino-raphe projection has been described; however, the retinal ganglion cell (RGC) types that contribute to this pathway are unknown. Methods Retrograde tracing via cholera toxin subunit B (CTB) was used to reveal DRN-projecting RGCs in rats, combined with intracellular injection in vitro, melanopsin immunostaining in whole-mounted retinas, and serotonin immunostaining to define the DRN. We modified methods of CTB injection into DRN used previously in order to avoid possible contamination with other retinorecipient regions, particularly the superior colliculus (SC). Results The majority of DRN-projecting RGCs showed alpha-like morphology, and some CTB-positive RGCs were colabeled with melanopsin. Approximately 80% of the total population of CTB-labeled DRN-projecting RGCs was alpha-like cells including ON alpha cells and OFF alpha cells; these alpha-like cells were melanopsin immunonegative. Approximately 10% of the remaining DRN-projecting RGCs were melanopsin immunopositive, in which the M1 subtype of intrinsically photosensitive retinal ganglion cell (ipRGC) provided the dominant projection of ipRGCs into DRN, with only few non-M1 ipRGCs involved. The DRN-projecting ipRGCs could be retrogradely labeled following tracer injection into all rostrocaudal aspects of the DRN. Conclusions Both conventional RGCs with alpha-like morphology and melanopsin-expressing ipRGCs project into the rat DRN. Approximately 10% of DRN-projecting RGCs were colabeled with melanopsin, and the majority of these were the M1 subtype of ipRGCs. An ipRGC component of the retino-raphe projection may contribute to a sustained light-mediated modulation of DRN serotonin release.

To determine whether melanopsin-expressing intrinsically photosensitive retinal ganglion cell (ipRGC) inputs to the pupil light reflex (PLR) are affected in early age-related macular degeneration (AMD).The PLR was measured in 40 participants (20 early AMD and 20 age-matched controls) using a custom-built Maxwellian view pupillometer. Sinusoidal stimuli (0.5 Hz, 11.9 seconds duration, 35.6° diameter) were presented to the study eye and the consensual pupil response was measured to lights with high melanopsin excitation (464 nm [blue]) and with low melanopsin excitation (638 nm [red]) that biased activation to the outer retina. Two melanopsin PLR metrics were quantified: the phase amplitude percentage (PAP) during the sinusoidal stimulus presentation and the post-illumination pupil response (PIPR). The PLR during stimulus presentation was analyzed using latency to constriction, the transient pupil response and maximum pupil constriction metrics. Diagnostic accuracy was evaluated using receiver operating characteristic (ROC) curves.The blue PIPR was significantly less sustained in the early AMD group (P0.001). The red PIPR was not significantly different between groups (P0.05). The PAP and blue stimulus constriction amplitude were significantly lower in the early AMD group (P0.05). There was no significant difference between groups in the latency or transient amplitude for both stimuli (P0.05). ROC analysis showed excellent diagnostic accuracy for the blue PIPR metrics (area under the curve0.9).This is the initial report that the melanopsin-controlled PIPR is dysfunctional in early AMD. The noninvasive, objective measurement of the ipRGC controlled PIPR has excellent diagnostic accuracy for early AMD.

Purpose: To study the population of intrinsically photosensitive retinal ganglion cells (melanopsin-expressing RGCs, m+RGCs) in P23H-1 rats, a rat model of inherited photoreceptor degeneration. Methods: At postnatal (P) times P30, P365, and P540, retinas from P23H dystrophic rats (line 1, rapid degeneration; and line 3, slow degeneration) and Sprague Dawley (SD) rats (control) were dissected as whole-mounts and immunodetected for melanopsin and/or Brn3a. The dendritic arborization of m+RGCs and the numbers of Brn3a+RGCs and m+RGCs were quantified and their retinal distribution and coexpression analyzed. Results: In SD rats, aging did not affect the population of Brn3a+RGCs or m+RGCs or the percentage that showed coexpression (0.27%). Young P23H-1 rats had a significantly lower number of Brn3a+RGCs and showed a further decline with age. The population of m+RGCs in young P23H-1 rats was similar to that found in SD rats and decreased by 22.6% and 28.2% at P365 and P540, respectively, similarly to the decrease of the Brn3a+RGCs. At these ages the m+RGCs showed a decrease of their dendritic arborization parameters, which was similar in both the P23H-1 and P23H-3 lines. The percentage of coexpression of Brn3a was, however, already significantly higher at P30 (3.31%) and increased significantly with age (10.65% at P540). Conclusions: Inherited photoreceptor degeneration was followed by secondary loss of Brn3a+RGCs and m+RGCs. Surviving m+RGCs showed decreased dendritic arborization parameters and increased coexpression of Brn3a and melanopsin, phenotypic and molecular changes that may represent an effort to resist degeneration and/or preferential survival of m+RGCs capable of synthesizing Brn3a. Supported by grants from the Spanish Ministry of Economy and Competitiveness: SAF-2012-38328; ISCIII-FEDER “Una manera de hacer Europa” PI13/00643, PI13/01266, and BFU2012-36845, RETICS: RD12/0034/0014, and RD12/0034/0010.

PURPOSE MYP1 is a locus for X-linked syndromic and nonsyndromic high myopia. Recently, unique haplotypes in OPN1LW were found to be responsible for X-linked syndromic high myopia mapped to MYP1. The current study is to test if such variants in OPN1LW are also responsible for X-linked nonsyndromic high myopia mapped to MYP1. METHODS The proband of the family previously mapped to MYP1 was initially analyzed using whole-exome sequencing and whole-genome sequencing. Additional probands with early-onset high myopia were analyzed using whole-exome sequencing. Variants in OPN1LW were selected and confirmed by Sanger sequencing. Long-range and second PCR were used to determine the haplotype and the first gene of the red-green gene array. Candidate variants were further validated in family members and controls. RESULTS The unique LVAVA haplotype in OPN1LW was detected in the family with X-linked nonsyndromic high myopia mapped to MYP1. In addition, this haplotype and a novel frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW were detected in two other families with X-linked high myopia. The unique haplotype cosegregated with high myopia in the two families, with a maximum LOD score of 3.34 and 2.31 at θ = 0. OPN1LW with the variants in these families was the first gene in the red-green gene array and was not present in 247 male controls. Reevaluation of the clinical data in both families with the unique haplotype suggested nonsyndromic high myopia. CONCLUSIONS Our study confirms the findings that unique variants in OPN1LW are responsible for both syndromic and nonsyndromic X-linked high myopia mapped to MYP1.

Purpose To study the responses of the general population of retinal ganglion cells (Brn3a(+)RGCs) versus the intrinsically photosensitive RGCs (melanopsin-expressing RGCs [m(+)RGCs]) to ocular hypertension (OHT), the effects of brain-derived neurotrophic factor (BDNF) on the survival of axonally intact and axonally nonintact RGCs, and the correlation of vascular integrity with sectorial RGC loss. Methods In Sprague-Dawley rats, 5 μg BDNF or vehicle was intravitreally injected into the left eye followed by laser photocoagulation of the limbal tissues. To identify RGCs with an active retrograde axonal transport, Fluorogold was applied to both superior colliculi 1 week before euthanasia (FG(+)RGCs). Retinas were dissected 12 or 15 days after lasering and immunoreacted against Brn3a (to identify all RGCs except m(+)RGCs), melanopsin, or RECA1 (inner retinal vasculature). Results Ocular hypertension resulted at 12 to 15 days in sectorial loss of FG(+)RGCs (78%-84%, respectively) while Brn3a(+)RGCs were significantly greater, indicating that a substantial proportion (approximately 21%-26%) of RGCs with their retrograde axonal transport impaired survive in the retina. Brain-derived neurotrophic factor increased the survival of Brn3a(+)RGCs to 81% to 67% at 12 to 15 days, respectively. The inner retinal vasculature showed no abnormalities that could account for the sectorial loss of RGCs. At 12 to 15 days, m(+)RGCs decreased to approximately 50% to 51%, but this loss was diffuse across the retina and was not prevented by BDNF. Conclusions The responses of m(+)RGCs against OHT-induced retinal degeneration and neuroprotection differ from those of Brn3a(+)RGCs; while OHT induces similar loss of Brn3a(+)RGCs and m(+)RGCs, Brn3a(+)RGCs are lost in sectors and can be rescued with BDNF, but m(+)RGCs do not respond to BDNF and their loss is diffuse.

Purpose The postillumination pupil response (PIPR) is produced by intrinsically photosensitive retinal ganglion cells (ipRGCs). We aimed to refine the testing conditions for PIPR by investigating whether a greater PIPR can be induced using full-field light stimuli of shorter duration and lower intensity than that produced by existing protocols that use central-field stimuli. Methods Pupil response was recorded with an eye tracker in 10 visually-normal subjects. Red and blue light stimuli were presented using a Ganzfeld system. In Experiment 1 (intensity trials), PIPR was induced using 1-second full-field stimuli of increasing intensities from 0.1 to 400 cd/m(2) (11 steps). For comparison, PIPR also was induced using a 60° × 90° central-field blue stimulus of 400 cd/m(2). In Experiment 2 (duration trials), PIPR was induced using 100 and 400 cd/m(2) full-field stimulus of increasing duration from 4 to 1000 ms (10 steps). Results Results indicated that PIPR increased monotonically with increasing stimulus intensity. Full-field stimulation using blue light at 400 cd/m(2) intensity induced significantly more sustained PIPR than central-field stimulation (P = 0.001). In addition, PIPR increased as the stimulus duration increased from 4 to 200 ms; however, no further increase in PIPR was observed when the duration increased from 400 to 1000 ms. Conclusions Compared to existing central-field protocols, larger PIPR can be induced with a full-field stimulus with lower intensity and shorter duration, indicating that PIPR is a function of stimulus intensity, stimulus duration, and retinal area stimulated. The testing protocol can be refined with this new knowledge to target particular clinical populations.

PURPOSE To investigate the pupillary light reflex (PLR) of patients with severe loss of vision due to Leber's Hereditary Optic Neuropathy (LHON) in the context of a proposed preservation of melanopsin-expressing retinal ganglion cells (mRGCs). METHODS Ten LHON patients (7 males; 51.6 ± 14.1 years), with visual acuities ranging from 20/400 to hand motion perception and severe visual field losses, were tested and compared with 16 healthy subjects (7 males; 42.15 ± 15.4 years) tested as controls. PLR was measured with an eye tracker and the stimuli were controlled with a Ganzfeld system. Pupil responses were measured monocularly, to 1 second of blue (470 nm) and red (640 nm) flashes with 1, 10, 100, and 250 cd/m² luminances. The normalized amplitude of peak of the transient PLR and the amplitude of the sustained PLR at 6 seconds after the flash offset were measured. In addition, optical coherence topography (OCT) scans of the peripapillary retinal nerve fiber layer were obtained. RESULTS The patient's peak PLR responses were on average 15% smaller than controls (P < 0.05), but 5 out of 10 patients had amplitudes within the range of controls. The patients' sustained PLRs were comparable with controls at lower flash intensities, but on average, 27% smaller to the 250 cd/m² blue light, although there was considerable overlap with the PLR amplitudes of control. All patients had severe visual field losses and the retinal nerve fiber layer thickness was reduced to a minimum around the optic disc in 8 of the 10 patients. CONCLUSIONS The PLR is maintained overall in LHON patients despite the severity of optic atrophy. These results are consistent with previous evidence of selective preservation of mRGCs.

To evaluate retinal structure and photoreceptor mosaic integrity in subjects with OPN1LW and OPN1MW mutations.Eleven subjects were recruited, eight of whom have been previously described. Cone and rod density was measured using images of the photoreceptor mosaic obtained from an adaptive optics scanning light ophthalmoscope (AOSLO). Total retinal thickness, inner retinal thickness, and outer nuclear layer plus Henle fiber layer (ONL+HFL) thickness were measured using cross-sectional spectral-domain optical coherence tomography (SD-OCT) images. Molecular genetic analyses were performed to characterize the OPN1LW/OPN1MW gene array.While disruptions in retinal lamination and cone mosaic structure were observed in all subjects, genotype-specific differences were also observed. For example, subjects with "L/M interchange" mutations resulting from intermixing of ancestral OPN1LW and OPN1MW genes had significant residual cone structure in the parafovea (∼25% of normal), despite widespread retinal disruption that included a large foveal lesion and thinning of the parafoveal inner retina. These subjects also reported a later-onset, progressive loss of visual function. In contrast, subjects with the C203R missense mutation presented with congenital blue cone monochromacy, with retinal lamination defects being restricted to the ONL+HFL and the degree of residual cone structure (8% of normal) being consistent with that expected for the S-cone submosaic.The photoreceptor phenotype associated with OPN1LW and OPN1MW mutations is highly variable. These findings have implications for the potential restoration of visual function in subjects with opsin mutations. Our study highlights the importance of high-resolution phenotyping to characterize cellular structure in inherited retinal disease; such information will be critical for selecting patients most likely to respond to therapeutic intervention and for establishing a baseline for evaluating treatment efficacy.

PURPOSE. Nonvisual light-dependent functions in humans are conveyed mainly by intrinsically photosensitive retinal ganglion cells, which express melanopsin as photopigment. We aimed to identify the effects of circadian phase and sleepiness across 24 hours on various aspects of the pupil response to light stimulation. METHODS. We tested 10 healthy adults hourly in two 12-hour sessions covering a 24-hour period. Pupil responses to narrow bandwidth red (635 +/- 18 nm) and blue (463 +/- 24 nm) light (duration of 1 and 30 seconds) at equal photon fluxes were recorded, and correlated with salivary melatonin concentrations at the same circadian phases and to subjective sleepiness ratings. The magnitude of pupil constriction was determined from minimal pupil size. The post-stimulus pupil response was assessed from the pupil size at 6 seconds following light offset, the area within the redilation curve, and the exponential rate of redilation. RESULTS. Among the measured parameters, the pupil size 6 seconds after light offset correlated with melatonin concentrations (P < 0.05) and showed a significant modulation over 24 hours with maximal values after the nocturnal peak of melatonin secretion. In contrast, the post-stimulus pupil response following red light stimulation correlated with subjective sleepiness (P < 0.05) without significant changes over 24 hours. CONCLUSIONS. The post-stimulus pupil response to blue light as a marker of intrinsic melanopsin activity demonstrated a circadian modulation. In contrast, the effect of sleepiness was more apparent in the cone contribution to the pupil response. Thus, pupillary responsiveness to light is under influence of the endogenous circadian clock and subjective sleepiness. (Invest Ophthalmol Vis Sci. 2012;53:4546-4555) DOI: 10.1167/iovs.12-9494

PURPOSE Retinal ganglion cells (RGCs) expressing the photopigment melanopsin (Opn4) display intrinsic photosensitivity. In this study, the presence of nonvisual phototransduction cascade components in the developing chicken retina and primary RGCs cultures was investigated, focusing on the two Opn4 genes: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. METHODS Retinas were dissected at different embryonic (E) and postnatal (P) days, and primary RGC cultures were obtained at E8 and kept for 1 hour to 5 days. Samples were processed for RT-PCR and immunochemistry. RESULTS Embryonic retinas expressed the master eye gene Pax6, the prospective RGC specification gene Brn3, and components of the nonvisual phototransduction cascade, such as Opn4m and the G protein q (Gq) mRNAs at very early stages (E4-E5). By contrast, expression of photoreceptor cell markers (CRX, red-opsin, rhodopsin, and α-transducin) was observed from E7 to E12. Opn4m protein was visualized in the whole retina as early as E4 and remained elevated from E6 to the postnatal days, whereas Opn4x was weakly detected at E8 and highly expressed after E11. RGC cultures expressed Gq mRNA, as well as both Opn4 mRNAs and proteins. Opn4m was restricted exclusively to the GC layer at all ages, whereas Opn4x was limited to the forming GC layer and optic nerve at E8, but by E15, its expression was mostly in Prox1(+) horizontal cells. CONCLUSIONS The early expression onset of nonvisual phototransduction molecules could confer premature photosensitivity to RGCs, while the appearance of Opn4x expression in horizontal cells suggests the identification of a novel type of photosensitive cell in birds.

Purpose: In vertebrates, intrinsically photosensitive retinal ganglion cells (ipRGCs) acting as nonvisual photoreceptors transmit environmental illumination information to the brain, regulating diverse non–image-forming tasks. The phototransduction cascade in chicken ipRGCs has been shown to resemble that of rhabdomeric photoreceptors and involves phospholipase C (PLC) activation. The current work was an investigation of the participation of the phosphoinositide (PIP) cycle in this mechanism and of whether changes in activities of inositol 1,4,5-trisphosphate (IP3) and PIP kinase are triggered by light. Conclusions.: The results indicate for the first time that light stimulation of chicken RGC cultures activates the PIP cycle, causing an increase in intracellular levels of IP3, changes in levels of phosphatidic acid, PIP, and PIP2; and mobilization of Ca2+. Methods.: Primary cultures of Thy-1 immunopurified chicken embryonic RGCs were exposed to bright light pulses or kept in the dark, to assess intracellular Ca2+ mobilization by Fluo-3 AM fluorescence microscopy, IP3 levels, and enzymatic activities of diacylglycerol, phosphatidylinositol, and phosphatidylinositol phosphate kinases (DAGK, PIK, and PIPK, respectively), by radioactive assays. The presence of different melanopsins (Opn4m and Opn4x) and other photopigments was determined by RT-PCR and immunochemistry. Results.: Cultured RGCs expressing different nonvisual photopigments displayed a significant and rapid increase in IP3 levels (1.3-fold) and Ca2+ mobilization by light, which was reversed by administration of the PLC inhibitor U73122 (5 μM). Brief light pulses also caused a very rapid and transient activation of DAGK, PIK, and PIPK compared with that in the dark control. Fil: Contin, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Verra, Daniela Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina Fil: Salvador, Gabriela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Ilincheta, Monica Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Giusto, Norma Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Guido, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina

PURPOSE. Organ cultures of the rodent retina could provide a powerful tool in the study of cone development and differentiation. Previous attempts, however, have failed to show Mcone development in organ cultures of the mouse and rat retina. This study mimicked the in vivo dynamics of S- and M-cone development in a culturing approach for the postnatal rat retina. METHODS. Retinas of Brown Norway rats were collected at different developmental ages (postnatal day [P]0 –P270) to study cone development in vivo. For culturing, the retinas were prepared from P0 to P2 animals and allowed to develop in organ culture for 2 to 15 days. Subsequently, opsin expression was analyzed immunohistochemically and morphometrically. RESULTS. In control retinas, S-opsin was already expressed at birth, whereas M-opsin was detected after P4. The maximum density of S-opsin–positive cones was reached at P10 (17,000 cells/mm 2 ) and of M-opsin–positive cones, at P12 (14,000 cells/mm 2 ). The number of both cone types decreased gradually thereafter to 1,000 S-opsin cones/mm 2 and 4,000 Mopsin cones/mm 2 in the adult. In culture, both cone types developed with dynamics of appearance comparable to those in vivo, with a peak density of 12,300 cones/mm 2 for S-opsin and 7,500 cones/mm 2 for M-opsin labeling. CONCLUSIONS. These results in rat retina showed for the first time that cone development and expression dynamics can be mimicked in organ culture. With this experimental approach, it will be possible to evaluate aspects of cone development under controlled experimental conditions and to elucidate factors crucial for proper cone differentiation. (Invest Ophthalmol Vis Sci. 2010;51:5320 –5327) DOI:10.1167/iovs.09-4741

Purpose To quantify the whole population of S- and L-cones in the albino (Sprague-Dawley, SD) and pigmented (Piebald Virol Glaxo, PVG) rats and to study their topographical distribution within the retina. Methods Retinal radial sections and whole-mounted retinas were double immunodetected with antibodies against UV-sensitive and L-opsins to detect the S- and L-cones, respectively. Two automated routines were developed to quantify the whole population of S- and L-cones. Detailed isodensity maps of each cone type were generated. In both strains, the presence of dual cones was detected, these were semiautomatically quantified and their distribution determined. The matching distribution of retinal ganglion cells (RGCs) and L-cones was attained by double immunodetection of Brn3a and L-opsin, respectively. Results The mean number +/- SEM of L- or S-cones in SD and PVG retinas was 231,736 +/- 14,517 and 239,939 +/- 6,494 or 41,028 +/- 5,074, and 27,316 +/- 2,235, respectively. There was an increasing gradient of S-cone density along the inferonasal quadrant, although the highest densities were found in the retinal rims. The distribution of L-cones seemed to be complementary to the S-cones. The highest densities were observed in the superior nasotemporal axis, paralleling the distribution of Brn3a-positive RGCs. Conclusions These data establish, for the first time, the total number and the topographical distribution of S- and L-cones in two rat strains and demonstrate the correlation of L-cones and RGC spatial distribution.

Purpose To examine the molecular profile of fetal human retinal progenitor cells (hRPCs) expanded in vitro and those grown in a co-culture system with mouse retina through the analysis of protein and gene expression and neurotransmitter-stimulated calcium dynamics. Methods hRPCS were isolated from human retina of 14 to 18 weeks gestational age (GA) and expanded in vitro. Immunoblot, microarray, and immunocytochemistry (ICC) assays were performed on undifferentiated hRPCs and those co-cultured with mouse retinas for 2 weeks. Cell function was assessed by using calcium imaging. Results The ICC results showed a gradual decrease in the percentages of KI67-, SOX2-, and vimentin-positive cells from passages (P) 1 to P6, whereas a sustained expression of nestin and PAX6 was observed through P6. Microarray analysis of P1 hRPCs showed the expression of early retinal developmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2, HES5, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). At P6, hRPCs continued to express VIM, KI67, NES, PAX6, SOX2, GNL3, and SIX6. On co-culture, there was a significant increase in the expression of MKI67, PAX6, SOX2, GNL3, SIX3, and RHO (rhodopsin). Calcium imaging showed a functional response to excitatory neurotransmitters. Conclusions Fetal-derived hRPCs show molecular characteristics indicative of a retinal progenitor state up to P6 (latest passage studied). They show a progressive decrease in the expression of immature markers as they reach P6. These cells are functional, respond to excitatory neurotransmitters, and exhibit changes in expression patterns in response to co-culture with mouse retina.

Purpose To characterize the clinical, psychophysical, and electrophysiological phenotype of 19 patients with enhanced S-cone syndrome (ESCS) and relate the phenotype to the underlying genetic mutation. Methods Patients underwent ophthalmic examination and functional testing including pattern ERG, full-field ERG, and long-duration and short-wavelength stimulation. Further tests were performed in some patients, including color contrast sensitivity (CCS), multifocal ERG, fundus autofluorescence imaging (FAI), optical coherence tomography (OCT), and fundus fluorescein angiography (FFA). Mutational screening of NR2E3 was undertaken in 13 patients. Results The fundus appearance was variable, from normal to typical nummular pigment clumping at the level of the retinal pigment epithelium in older patients. Nine patients had foveal schisis, and one had peripheral schisis. Pattern ERG was abnormal in all patients. In all patients, ISCEV Standard photopic and scotopic responses had a similar waveform, the rod-specific-ERG was undetectable and the 30-Hz flicker ERG was markedly delayed with an amplitude lower than the photopic a-wave. Most ERG responses arose from short-wavelength-sensitive mechanisms, and a majority of patients showed possible OFF-related activity. Multifocal ERG showed relative preservation of central function, but reduced responses with increased eccentricity. Mutations were identified in NR2E3 in 12 of 13 patients including four novel variants. Conclusions The phenotype in ESCS is variable, both in fundus appearance and in the severity of the electrophysiological abnormalities. The ERGs are dominated by short-wavelength-sensitive mechanisms. The presence, in most of the patients, of possible OFF-related ERG activity is a finding not usually associated with S-cones.

Retinitis pigmentosa (RP) is a progressive neurodegenerative disease resulting in blindness for which there is no current treatment. Although the members of the family of RP diseases differ in etiology, their outcomes are the same: apoptosis of rods and then by cones. Recently, the bile acid tauroursodeoxycholic acid (TUDCA) has been shown to have antiapoptotic properties in neurodegenerative diseases, including those of the retina. In this study the authors examined the efficacy of TUDCA on preserving rod and cone function and morphology at postnatal day 30 (P30) in the rd10 mouse, a model of RP.Wild-type C57BL/6J and rd10 mice were systemically injected with TUDCA (500 mg/kg) every 3 days from P6 to P30 and were compared with vehicle (0.15 M NaHCO(3)). At P30, retinal function was measured with electroretinography, and morphologic preservation of the rods and cones was assessed with immunohistochemistry.Dark-adapted electroretinographic (ERG) responses were twofold greater in rd10 mice treated with TUDCA than with vehicle, likewise light-adapted responses were twofold larger in TUDCA-treated mice than in controls at the brightest ERG flash intensities. TUDCA-treated rd10 retinas had fivefold more photoreceptors than vehicle-treated retinas. TUDCA treatments did not alter retinal function or morphology of wild-type mice when administered to age-matched mice.TUDCA is efficacious and safe in preserving vision in the rd10 mouse model of RP when treated between P6 and P30. At P30, a developmental stage at which nearly all rods are absent in the rd10 mouse model of RP, TUDCA treatment preserved rod and cone function and greatly preserved overall photoreceptor numbers.

PURPOSE. To describe and classify the morphologic changes in a naturally occurring dog model of early-onset cone-rod dystrophy (CRD) and to correlate these with earlier described clinical characteristics of the disease in dogs. METHODS. Purpose-bred Standard Wire-Haired Dachshunds (SWHDs) derived from a large pedigree of dogs with earlyonset CRD were euthanatized at defined ages to characterize morphologic changes in the disease process. Specimens were examined by light microscopy, including morphometric studies, electron microscopy, and immunohistochemistry. Peanut agglutinin (PNA), protein kinase C (PKC), synaptophysin (Syn), rhodopsin (Rho)-63, glial fibrillary acidic protein (GFAP), and short-wavelength cone opsin (OS) were used for immunohistochemical characterization. RESULTS. The photopic cone-system-derived ERG amplitudes were already significantly reduced or nonrecordable in CRD-affected dogs at 5 weeks, the earliest age studied. The outer retina was morphologically most severely affected initially, with a subsequent degeneration of the inner retina. Cone degeneration was more pronounced than rod degeneration in young CRD-affected dogs. There was a marked phenotypic variation based on morphologic findings in the affected dogs. At the earliest time point studied (5-8 weeks) cone photoreceptor and glial cell abnormalities were observed, in accordance with earlier studies based on electrophysiological and clinical findings in which day blindness and abnormal cone ERGs were observed in young affected SWHD puppies. Preliminary genetic studies have indicated an autosomal recessive mode of inheritance for the defect. CONCLUSIONS. Through functional and structural characterization, early-onset cone abnormalities were found, consistent with a cone dysplasia at an age when rod structure was normal. Further studies are in progress to identify the gene(s) involved in this retinal disease process. The presently described natural animal model of primary cone dysplasia followed by rod degeneration may provide further insight into the human counterpart. Further studies are needed to ascertain an autosomal recessive mode of inheritance for CRD in the SWHD. (Less)

PURPOSE. Gene therapy for retinal degeneration requires well-defined promoters that drive expression in rod and cone photoreceptors. This study was undertaken to develop short, active derivatives of the human rhodopsin kinase (RK) gene promoter for targeting transgene expression in rods and cones. RK, also known as G protein-coupled receptor kinase 1 (GRK1), is a component of the light adaptation pathway expressed in rods and cones.METHODS. Human RK (hRK) promoter and its concatemers or derivatives extending into the conserved 5' untranslated region (5'-UTR) were assayed for promoter activity in WERI retinoblastoma or Crx/Sp1-supplemented HEK-293 cells. The derivative displaying the highest activity was linked to a GFP reporter and packaged in a pseudotyped adenoassociated viral vector (AAV2/5). The AAV vector was tested in vivo by sub-retinal injections in wild-type mice, in the all-cone Nrl(-/-) mice, and in the cone-rich diurnal Nile grass rat (Arvicanthis niloticus). Control eyes received a similar AAV2/5 vector carrying a mouse rod opsin (mOps) promoter-controlled GFP reporter.RESULTS. The hRK promoter with the full 5' untranslated sequence (-112 to +180) was the most active in cell culture. Delivered by the AAV2/5 vector, RK promoter drove GFP expression specifically in photoreceptors. In rods, hRK promoter-mediated expression was as efficient as, but appeared more uniform than, mOps promoter-mediated expression. In cones, the hRK promoter drove expression, whereas the mOps promoter did not.CONCLUSIONS. The hRK promoter is active and specific for rod and cone photoreceptors. Because of its small size and proven activity in cones, it is a promoter of choice for somatic gene transfer and gene therapy targeting rods and cones.

Evidence indicates that the melanopsin-containing intrinsically photosensitive retinal ganglion cells (ipRGCs) receive input from rods and cones, which are thought to modulate the irradiance detecting system driving entrainment of the circadian system and pupillomotor control. This study was performed to identify retinal cells that have synaptic contact with ipRGCs.Immunohistochemistry and high-power confocal microscopy were used to generate stacks of digital images of sections stained with antibodies against melanopsin, protein kinase C (PKCalpha), tyrosine hydroxylase (TH), presynaptic terminal markers (C-terminal binding protein 2 [CtBP2], vesicular monoamine transporter 2 [VMAT2] and postsynaptic marker (glutamate receptor subunit 4 [GluR4]). Results were analyzed in a computer-based three-dimensional reconstruction program for cellular contacts.Markers and melanopsin rod bipolar processes were found to have axosomatic and axodendritic contact with melanopsin-containing RGCs. Typically, three to four contacts were found on the soma of the melanopsin-containing RGCs, together with contacts on proximal dendrites. Contacts visualized by only CtBP2 immunoreactivity could also be demonstrated on melanopsin cell bodies and processes representing contacts with other types of bipolar cells. At the border of the inner plexiform layer (IPL) and inner nuclear layer (INL), where melanopsin processes stratify, contacts between melanopsin and TH or VMAT2 immunoreactivity processes were observed.Through confocal microscopy and computer-based three-dimensional analyses, this study demonstrates that melanopsin-containing RGCs have synaptic contact with PKC/CtBP2-containing rod bipolar cells and TH/VMAT2-immunoreactive amacrine cells through axodendritic and axosomatic contact, supporting electrophysiological observations that rods and cones signal to the melanopsin-containing intrinsically photosensitive RGCs.

Purpose Folds or pseudorosettes in the outer retina are commonly observed in animals with genetic, viral, or chemically induced retinal degeneration. This study examined the effect of genetic ablation of cone photoreceptors on the production of retinal folds in two mouse retinopathy models. One is the rd7/rd7 retina, a model of enhanced S-cone syndrome, Goldman-Favre syndrome, and clumped pigmentary retinopathy that is also associated with an approximately twofold excess of S-cones. The other is postnatal day (P)0 N-methyl-N-nitrosourea (MNU) treatment. Methods A transgene that directs cone-specific expression of the diphtheria toxin A chain was used to ablate cone photoreceptors. Retinal folds, numbers of photoreceptors, and numbers of cones were quantified in retina flatmounts or transverse sections, and photoreceptor apoptosis was quantified by immunostaining for activated caspase 3. Results Cone ablation by the cone-DTA transgene eliminated folds in the rd7/rd7 retina, whereas chemical ablation of up to 30% of rods (by exposure to MNU at P13) had little or no effect on folds in the rd7/rd7 retina. Cone ablation by the cone-DTA transgene had no effect on retinal folds produced by P0 MNU treatment or on the progressive loss of rod photoreceptors in the rd7/rd7 retina. Conclusions Despite their relatively low abundance, cones play a critical role in retinal folding in the rd7/rd7 retina. The relevant molecular and cellular mechanisms remain to be determined.

PURPOSE. To determine the relative contributions of inner and outer retinal photoreception to the pupillary light response. METHODS. Wild-type, retinal degenerate (rd/rd), and melanopsin mutant (opn4-/-) mice were tested for pupillary light responsiveness by video pupillometry before, during, and after exposure to supersaturating light intensities. Similar lighting protocols were used to probe responses of intrinsically photosensitive retinal ganglion cells (ipRGCs) recorded with multielectrode arrays ex vivo. RESULTS. Both outer retinal photoreceptors (rods and cones) and inner retinal photoreceptors (intrinsically photosensitive retinal ganglion cells [ipRGCs]) are sufficient to drive the pupillary light response in mice. After supersaturating light exposure, rather than bleaching or adapting, rd/rd mice showed paradoxical potentiation of responses to subsaturating light exposure. opn4 -/- mice, in contrast, could not sustain pupillary constriction under continuous bright illumination, and showed desensitization after bright-light exposure. Both the intensity of light necessary to induce potentiation and the spectral sensitivity for sustained and potentiated responses differed from that necessary to trigger pupillary constriction, suggesting that photopotentiation is dependent on a pigment-state distinct from that triggering the pupillary light response itself. Multielectrode array recordings of ipRGCs from rd/rd retinas demonstrated persistent cell firing under continuous light exposure but did not show potentiation. CONCLUSIONS. Unique photoreceptive properties of intrinsically photosensitive RGCs confer resistance to bleaching and/or adaptation under continuous bright illumination to the pupillary light response and suggest the presence of a photopigment with multiple absorption states.

Purpose—To study the possibility of generating photoreceptors through programming RPE transdifferentiation by examining cell differentiation after transplantation into the developing chick eye. Methods—RPE was isolated, and the cells were dissociated, cultured, and guided to transdifferentiate by infection with retrovirus expressing neuroD (RCAS-neuroD), using RCAS– green fluorescence protein (GFP) as a control. The cells were then harvested and microinjected into the developing eyes of day 5 to day 7 chick embryos, and their development and integration were analyzed. Results—Cells from the control culture integrated into the host RPE. When grafted cells were present in large number, multilayered RPE-like tissues were formed, and the extra tissues consisted of grafted cells and host cells. None of the cells from the control culture expressed photoreceptorspecific genes. In contrast, most cells from RCAS-neuroD–infected culture remained depigmented. A large number of them expressed photoreceptor-specific genes, such as visinin and opsins. Antibodies against red opsin decorated the apical tips and the cell bodies of the grafted, transdifferentiating cells. In the subretinal space, visinin + cells aligned along the RPE or an RPElike structure. When integrated into the host outer nuclear layer, grafted cells emanated elaborate, axonal arborization into the outer plexiform layer of the host retina. Conclusions—Cultured RPE cells retained their remarkable regenerative capabilities. Cells guided to transdifferentiate along the photoreceptor pathway by neuroD developed a highly ordered cellular structure and could integrate into the outer nuclear layer. These data suggest that, through genetic programming, RPE cells could be a potential source of photoreceptor cells. Photoreceptors are the primary sensory neurons residing in the outer nuclear layer (ONL) of the vertebrate retina. Photoreceptor degeneration is a common cause of human visual impairments resulting from light damage, genetic changes, and aging. The low quality of life

PURPOSE. To examine the relationship between short-wavelength–sensitive (SWS) resolution acuity and epidemiologically defined stages of early age-related maculopathy (ARM). METHODS. Subjects consisted of 88 adults aged 51 to 87 years. Psychophysical testing was undertaken in only one eye of each subject (the study eye). All study eyes had a LogMAR acuity of 0.30 (20/40 Snellen) or better. SWS and achromatic grating resolution acuity were measured at 6° eccentricity from the fovea. Stereoscopic color fundus photographs centered on the macula were taken on both eyes of each subject and were graded using the Wisconsin Age-Related Maculopathy Grading System (WARMGS). After grading, features of ARM were combined to assign a severity stage from 0 to 5 using the methods described by the Rotterdam Eye Study. Relationships between visual function, study eye ARM stage, and fellow eye status were examined with the use of standard statistical analysis. RESULTS. Although SWS resolution acuity was significantly reduced in eyes classified as having any ARM compared with eyes classified as having no ARM (P 0.002), there was no relationship between the severity of functional deficits and the morphologic severity from stage 1 to stage 4. On reassigning subject eyes to a revised severity staging (stage 0, stages 1 to 4 combined, and stage 5), SWS acuity was significantly different among these three groups (P 0.001). No significant relationship was found between achromatic resolution acuity and ARM staging. The status of the fellow eye (advanced macular degeneration present or absent) was not significantly related to visual function in the study eye. CONCLUSIONS. Significant functional deficits in SWS resolution acuity were found in eyes with ARM features, but the severity of functional loss did not correlate well with the currently accepted method of assigning a morphologic severity stage. Longitudinal studies may reveal further information on the relationships between functional deficits, ARM status, disease progression, and outcome. (Invest Ophthalmol Vis Sci. 2006; 47:4624 – 4631) DOI:10.1167/iovs.05-1227

Purpose To investigate the survival of melanopsin-expressing retinal ganglion cells (mRGCs) after the induction of chronic ocular hypertension. Methods Intraocular pressure (IOP) was elevated in adult Sprague-Dawley rats using an argon laser to photocoagulate the episcleral and limbal veins. IOP was measured with a calibrated tonometer and monitored for a period. Seven days before the animals were killed, a piece of sterile foam soaked with gold fluorescent dye was placed onto the superior colliculus (SC) to label the SC-projecting retinal ganglion cells (scRGCs) retrogradely. mRGCs were visualized by free floating immunohistochemistry on whole-mounted retinas. The number of surviving scRGCs and mRGCs were counted on flatmounted retinas. The branching pattern of dendrites and soma size of mRGCs were examined. Results An approximately 1.7-fold increase of IOP and a significant loss of scRGCs were found in experimental eyes after laser photocoagulation. However, no significant cell loss or morphologic changes on mRGCs and their dendrites after the induction of chronic ocular hypertension are noticed over a 12-week period. Conclusions Although the degeneration of retinal ganglion cells (RGCs) is a major concern in glaucomatous damage, the findings show that mRGCs are less susceptible to death after the induction of chronic ocular hypertension. This result indicates that mRGCs carry some unique properties that are different from those of other subpopulations of RGCs. The immunohistochemistry approach can be used to distinguish easily these mRGCs from other subtypes. This method provides a useful tool to investigate their injury-resistant properties that are informative for the development of effective neuroprotective treatment for glaucoma.

PURPOSE. Melanopsin, expressed in a subset of intrinsically photosensitive ganglion cells that project to the suprachiasmatic nucleus (SCN), is involved in the photic entrainment of circadian rhythms and other non–image-forming functions (pupil light reflex, masking, acute heart rate response, and alertness). Melanopsin has recently been shown to be a “bireactive” photopigment that functions as a photosensory opsin using 11-cis retinaldehyde as a chromophore and has intrinsic photoisomerase activity. Melanopsin is widely distributed in the retina of vertebrates and, depending on the species, is expressed in ganglion, amacrine, horizontal, and photoreceptor cells. The present study was conducted to determine the distribution of this opsin in the human retina. METHODS. Human donor eyes were obtained from donors and fixed shortly after death. Immunohistochemistry was used to determine melanopsin expression in the retinas of three donors. The possible coexpression of this photopigment with other opsins was studied by double-labeling immunocytochemistry and confocal analysis. RESULTS. In addition to the expected labeling in ganglion cells of the inner retinal layers, an unexpected finding showed melanopsin-immunopositive label in the outer segments of cones that did not coexpress other known opsins. These melanopsin-expressing cones are extremely sparse (5–25 cones/ mm 2 ; 0.1%– 0.5% of the entire cone population) and are located in the peripheral retina. CONCLUSIONS. The presence of melanopsin in human cones suggests image and non–image-forming roles in visual responses at both the cone input and ganglion cell output stages and their involvement in a broad spectrum of irradiance detection functions in the visual system. (Invest Ophthalmol Vis Sci. 2006;47:1636 –1641) DOI:10.1167/iovs.05-1459

PURPOSE. To quantify optical coherence tomography (OCT) images of the central retina in patients with blue-cone monochromatism (BCM) and achromatopsia (ACH) compared with healthy control individuals. METHODS. The study included 15 patients with ACH, 6 with BCM, and 20 control subjects. Diagnosis of BCM and ACH was established by visual acuity testing, morphologic examination, color vision testing, and Ganzfeld ERG recording. OCT images were acquired with the Stratus OCT 3 (Carl Zeiss Meditec AG, Oberkochen, Germany). Foveal OCT images were analyzed by calculating longitudinal reflectivity profiles (LRPs) from scan lines. Profiles were analyzed quantitatively to determine foveal thickness and distances between reflectivity layers. RESULTS. Patients with ACH and BCM had a mean visual acuity of 20/200 and 20/60, respectively. Color vision testing results were characteristic of the diseases. The LRPs of control subjects yielded four peaks (P1‐P4), presumably representing the RPE (P1), the ovoid region of the photoreceptors (P2), the external limiting membrane (ELM) (P3), and the internal limiting membrane (P4). In patients with ACH, P2 was absent, but foveal thickness (P1‐P4) did not differ significantly from that in the control subjects (187 20 vs. 192 14 m, respectively). The distance from P1 to P3 did not differ significantly (78 10 vs. 82 5 m) between ACH and controls subjects. In patients with BCM, P3 was lacking, and P2 advanced toward P1 compared with the control subjects (32 6 vs. 48 4 m). Foveal thickness (153 16 m) was significantly reduced compared with that in control subjects and patients with ACH. CONCLUSIONS. Quantitative OCT image analysis reveals distinct patterns for controls subjects and patients with ACH and BCM, respectively. Quantitative analysis of OCT imaging can be useful in differentiating retinal diseases affecting photoreceptors. Foveal thickness is similar in both normal subjects and patients with ACH but is decreased in patients with BCM. (Invest Ophthalmol Vis Sci. 2006;47:1161‐1166) DOI:10.1167/iovs.05-0783

PURPOSE. Recent in vitro evidence has shown that RPE65 is the isomerohydrolase that converts all-trans retinyl ester to 11-cis retinal, the chromophore for visual pigments in vertebrates. Homozygous RPE65 knockout (Rpe65 -/- ) mice lack 11-cis retinoids and have early cone degeneration. The purpose of this study is to determine whether RPE65 gene delivery restores the isomerohydrolase activity and normal profile of endogenous retinoids in Rpe65 -/- mice. METHODS. Adenovirus-expressing RPE65 (Ad-RPE65) was injected into the subretinal space of Rpe65 -/- mice. The expression of RPE65 was determined by immunohistochemistry and Western blot analysis. The isomerohydrolase activity was measured in vitro in eyecup homogenates. Endogenous retinoid profile in the eyecups was analyzed by high-performance liquid chromatography (HPLC). Photoreceptor-specific gene expression was determined with real-time RT-PCR. Cone degeneration was determined by cone-specific staining and counting cones in flatmounted retina. RESULTS. High levels of RPE65 expression from the Ad-RPE65 injection generated robust isomerohydrolase activity in the eyecup of Rpe65 -/- mice, at levels comparable to those in wild-type (wt) mice. Consequently, the RPE65 gene delivery resulted in substantial amounts of 11-cis retinal in Rpe65 -/- mice. The RPE65 gene delivery prevented the downregulation of cone-specific genes, including both cone opsins and cone tranducin a subunit in Rpe65 -/- mice. Moreover, the Ad-RPE65 injection also prevented massive cone degeneration at early ages of Rpe65 -/- mice. CONCLUSIONS. RPE65 gene delivery generates isomerohydrolase activity and restores retinoid profile in Rpe65 -/- mice. Regeneration of 11-cis retinal is essential for survival of cone photoreceptors.

Purpose Retinitis pigmentosa GTPase regulator (RPGR) is a photoreceptor protein anchored in the connecting cilia by an RPGR-interacting protein (RPGRIP). Loss of RPGRIP causes Leber congenital amaurosis (LCA), a severe form of photoreceptor degeneration. The current study was an investigation of whether somatic gene replacement could rescue degenerating photoreceptors in a murine model of LCA due to a defect in RPGRIP. Methods An RPGRIP expression cassette, driven by a mouse opsin promoter, was packaged into recombinant adeno-associated virus (AAV). The AAV vector was delivered into the right eyes of RPGRIP(-/-) mice by a single subretinal injection into the superior hemisphere. The left eyes received a saline injection as a control. Full-field electroretinograms (ERGs) were recorded from both eyes at 2, 3, 4, and 5 months after injection. After the final follow-up, retinas were analyzed by immunostaining or by light and electron microscopy. Results Delivery of the AAV vector led to RPGRIP expression and restoration of normal RPGR localization at the connecting cilia. Photoreceptor preservation was evident by a thicker cell layer and well-developed outer segments in the treated eyes. Rescue was more pronounced in the superior hemisphere coincident with the site of delivery. Functional preservation was demonstrated by ERG. Conclusions AAV-mediated RPGRIP gene replacement preserves photoreceptor structure and function in a mouse model of LCA, despite ongoing cell loss at the time of intervention. These results indicate that gene replacement therapy may be effective in patients with LCA due to a defect in RPGRIP and suggest that further preclinical development of gene therapy for this disorder is warranted.

PURPOSE The retinoid X receptors (RXRs) are members of the family of ligand-dependent nuclear hormone receptors. One of these genes, RXRgamma, is expressed in highly restricted regions of the developing central nervous system (CNS), including the retina. Although previous studies have localized RXRgamma to developing cone photoreceptors in several species, its function in these cells is unknown. A prior study showed that thyroid hormone receptor beta2 (TRbeta2) is necessary to establish proper cone patterning in mice by activating medium-wavelength (M) cone opsin and suppressing short-wavelength (S) cone opsin. Thyroid hormone receptors often regulate gene transcription as heterodimeric complexes with RXRs. METHODS To determine whether RXRgamma cooperates with TRbeta2 to regulate cone opsin patterning, the developmental expression of RXRgamma was examined, and cone opsin expression in RXRgamma-null mice was analyzed. RESULTS RXRgamma was expressed in postmitotic cones and was transiently downregulated at the time of S-opsin onset in both mouse and human cones. RXRgamma-null mice expressed S-opsin in all cones, similar to the TRbeta2-null mice. Unlike TRbeta2-null mice, which did not express M-opsin, RXRgamma-null mice had a normal pattern of M-opsin expression. CONCLUSIONS RXRgamma is essential (along with TRbeta2) for suppressing S-opsin in all immature cones and in dorsal cones in the mature retina, but it is not necessary for M-opsin regulation. These results demonstrate a critical role for RXRs in regulating cell differentiation in the CNS and highlight a remarkable conservation of opsin regulation from Drosophila to mammals.

Purpose Activating ligands of gp130, including leukemia inhibitory factor (LIF), can block differentiation and function of retinal neurons. This study focused on determining whether LIF inhibits differentiation of photoreceptors by altering cell fate or by blocking the expression of essential transcription factors in vivo. Methods Transgenic mice were generated that had lens-specific expression of the secreted human LIF protein. Retinal differentiation was assessed by histology and by gene expression analysis, with in situ hybridization, immunohistochemistry, and real-time qRT-PCR. Electroretinograms were used to assess retinal function. Results LIF did not prevent or alter the timing of outer and inner nuclear layer separation, but it inhibited phototransduction gene expression in both rods and cones, thereby blocking functional maturation of photoreceptors. LIF also reduced the expression of Crx, Nrl, and Nr2e3, and upregulated the expression of transcription inhibitors Baf and Fiz1. Conclusions LIF expression did not appear to alter photoreceptor cell fate specification, but it inhibited subsequent differentiation. These results suggest that gp130 ligands can inhibit photoreceptor functional differentiation by reducing Crx- and Nrl-dependent transcription.

Purpose The retinitis pigmentosa GTPase regulator (RPGR) is essential for the maintenance of photoreceptor viability. RPGR is expressed as constitutive and ORF15 variants because of alternative splicing. This study was designed to examine whether the retina-specific ORF15 variant alone could substantially substitute for RPGR function. A further objective was to test whether the highly repetitive purine-rich region of ORF15 could be abbreviated without ablating the function, so as to accommodate RPGR replacement genes in adenoassociated virus (AAV) vectors. Methods A cDNA representing RPGR-ORF15 but shortened by 654 bp in the repetitive region was placed under the control of a chicken beta-actin (CBA) hybrid promoter. The resultant construct was transfected into mouse embryonic stem cells. Clones expressing the transgene were selected and injected into mouse blastocysts. Transgenic chimeras were crossed with RPGR knockout (KO) mice. Mice expressing the transgene but null for endogenous RPGR (Tg/KO) were studied from 1 month to 18 months of age by light and electron microscopy, immunofluorescence, and electroretinography (ERG). The results were compared with those of wild-type (WT) and RPGR-null control mice. Results Transgenic RPGR-ORF15 was found in the connecting cilia of rod and cone photoreceptors, at approximately 20% of the WT level. Photoreceptor morphology, cone opsin localization, expression of GFAP (a marker for retinal degeneration) and ERGs were consistent with the transgene exerting substantial rescue of retinal degeneration due to loss of endogenous RPGR. Conclusions RPGR-ORF15 is the functionally significant variant in photoreceptors. The length of its repetitive region can be reduced while preserving its function. The current findings should facilitate the design of gene replacement therapy for RPGR-null mutations.

PURPOSE. The putative circadian photoreceptor melanopsin is found in rodents in a subpopulation of intrinsic light-sensitive retinal ganglion cells (RGCs) constituting the retinohypothalamic tract (RHT). The study was conducted to determine whether melanopsin is expressed in the human retina and costored with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), a marker for the RHT, projecting to the suprachiasmatic nucleus (SCN). Furthermore, whether melanopsin expression is conserved in retinas of blind patients with severe retinal degeneration was investigated. METHODS. In situ hybridization and immunohistochemistry was used to demonstrate melanopsin synthesis in human eyes of 17 donors and two postmortem hypothalami containing the SCN. The coexistence of melanopsin and PACAP in elements of the retinohypothalamic tract was studied by dual-labeling immunocytochemistry. RESULTS. Melanopsin expression was found in a subpopulation of RGCs located in the ganglion cell layer and displaced in the inner nuclear cell layer. Melanopsin-containing cells comprised approximately 0. 8% of all RGCs, with a distinct morphology characterized by two to four dendritic processes constituting a panretinal network. Melanopsin immunoreactivity was primary present at perikaryal boundaries and neuronal processes and to some extent also in the cytoplasm. PACAP and melanopsin were colocalized in the RGCs and PACAP-containing nerve fibers, seemingly innervating the retinorecipient part of the SCN. Melanopsin-expressing RGCs were conserved in retinas of blind patients with severe degeneration of the outer and/or inner layers. CONCLUSIONS. Given the expression of melanopsin in PACAPcontaining RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people. (Invest Ophthalmol Vis Sci. 2004;45:4202‐4209) DOI:10.1167/iovs.04-0313

Purpose To use progenitor cells isolated from the neural retina for transplantation studies in mice with retinal degeneration. Methods Retinal progenitor cells from postnatal day 1 green fluorescent protein-transgenic mice were isolated and characterized. These cells can be expanded greatly in culture and express markers characteristic of neural progenitor cells and/or retinal development. Results After they were grafted to the degenerating retina of mature mice, a subset of the retinal progenitor cells developed into mature neurons, including presumptive photoreceptors expressing recoverin, rhodopsin, or cone opsin. In rho-/- hosts, there was rescue of cells in the outer nuclear layer (ONL), along with widespread integration of donor cells into the inner retina, and recipient mice showed improved light-mediated behavior compared with control animals. Conclusions These findings have implications for the treatment of retinal degeneration, in which neuronal replacement and photoreceptor rescue are major therapeutic goals.

PURPOSE. The chick retina and pineal gland exhibit circadian oscillations in biochemical and physiological processes in vivo and in vitro, which entrain to 24-hour light- dark cycles. However, the phototransduction mechanisms responsible for entrainment are largely unknown. The present study characterizes two candidate opsinlike genes that may play a role in entrainment of the clocks in these tissues. METHODS. Bioinformatics, cladistic techniques, and in situ hybridization and Northern blot analyses were conducted to characterize, localize, and determine the circadian expression of the candidate opsinlike genes in the retina and pineal gland. RESULTS. Two candidate photosensors and/or photoisomerases were predominantly distributed within the pineal gland and retina: the retinal pigmented epithelium-derived rhodopsin homologue (peropsin, gRrh) and retinal G-protein- coupled receptor opsin (RGR opsin, gRgr). Northern blot and in situ analyses revealed mRNA expression for both opsins in the pineal gland, retina, and brain tissue. The mRNA for both genes within the pineal gland and retina is regulated on a circadian basis such that they are highest in late subjective day. Digoxigenin in situ analyses showed retinal gRgr message within the inner nuclear layer (INL) and retinal ganglion cell layer (RGL), whereas gRrh message was distributed predominantly in the RGL. In the pineal gland, gRgr message was sparsely distributed among pinealocytes in follicles, but not within the follicles themselves, whereas gRrh was localized in interstitial areas indicative of astrocytic and/or endothelial origin. CONCLUSIONS. The presence of two putative photoisomerases within the pineal gland and in retinal layers associated with biological clock function provides two candidate opsinlike genes that may serve in the visual cycle regulation of the circadian clock.

PURPOSE. The Chx10 homeobox gene is expressed in neural progenitor cells during retinal development. The absence of Chx10 causes microphthalmia in humans and in the mouse mutant ocular retardation. The purpose of this study was to examine how neuronal development is affected by absence of the Chx10 transcription factor in the mouse retina.METHODS. Expression of transcription factor genes, Crx, Pou4f2, and Pax6, that mark specific cell types as they begin to differentiate was analyzed by RNA in situ hybridization of retina from wild-type and Chx10-null ocular retardation mice (Chx10(or-J/or-J)). RT-PCR analysis was used to compare expression of these genes and putative targets of Crx regulation. Photoreceptor development was analyzed by using peanut agglutinin (PNA)-rhodamine and blue cone opsin antibody to label cones and rhodopsin antibody to label rods.RESULTS. The photoreceptor gene Crx, normally expressed during embryonic retinal development, was not detected in the embryonic mutant retina, but was expressed after birth. Expression of the targets of Crx regulation, rhodopsin, peripherin, rod phosphodiesterase beta (Pdeb), and arrestin, with the exception of interphotoreceptor retinoid binding protein (Irbp), was delayed in the Chx10(or-J/or-J) retina. Rhodopsin localization in rod outer segments was also delayed. By contrast, temporal and spatial expression of Pou4f2 and Pax6 in developing ganglion and amacrine cells and PNA and blue opsin in developing cone cells was relatively normal in the mutant.CONCLUSIONS. Delay of the normal temporal expression of genes essential for photoreceptor disc morphogenesis leads to failure of correct rod and cone outer segment formation in the Chx10(or-J/or-J) mutant retina. In addition, the absence of Chx10 appears to affect the development of late-born cells more than that of early-born cells, in that a low number of rods develops, whereas formation of ganglion, amacrine, and cone cells is relatively unaffected.

PURPOSE. To evaluate the physiological characteristics of the macula in patients with Vogt-Koyanagi-Harada disease during the convalescent stage with specific reference to the kinetics of foveal cone photopigment regeneration. Moons. Six eyes of three patients at the convalescent stage of Vogt-Koyanagi-Harada disease were studied. All the eyes had best corrected visual acuity of 1.0 or better and had had no recurrence of inflammation for at least 12 months after the last episode. Foveal cone densitometry (FCD), focal macular electroretinograms, color vision tests, two-color perimetry, and optical coherence tomography (OCT) were performed. RESULTS. No regeneration of cone photopigments was detected within the 7-minute testing time by FCD in all eyes at the first examination after the last episode. However, the other functional tests were normal, and the OCT-determined macular morphology was also normal. The regeneration kinetics of the foveal cone photopigment improved in three of six eyes at 36, 37, and 19 months after the last episode, whereas the other three remained delayed at 18, 18, and 49 months. CONCLUSIONS. These findings suggest that a disorder of the foveal cone photopigment regeneration, and its recovery, requires a significantly longer time than that of other macular functions in some patients with Vogt-Koyanagi-Harada disease.

PURPOSE Drusen are variably sized extracellular deposits that form between the retinal pigmented epithelium (RPE) and Bruch's membrane. They are commonly found in aged eyes, however, numerous and/or confluent drusen are a significant risk factor for age-related macular degeneration. The purpose of this study was to investigate the impact of drusen on overlying cells of the retina. METHODS Tissue containing retina and RPE/choroid was dissected from human donor eyes, embedded in agarose, and sectioned at 100 micro m using a vibratome. Sections were immunostained with a panel of antibodies that labeled glial cells, first-, second-, and third-order retinal neurons and processed for confocal microscopy. RESULTS Retinal cells that overlie both soft and hard drusen exhibited numerous structural and molecular abnormalities. Normally detectable only in the outer segments of rod photoreceptors, rod opsin immunolabeling was also observed in the inner segment, cell body, axon, and axon terminal of photoreceptors that overlie drusen. Labeling with this antibody also revealed the deflection and shortening of rod inner and outer segments. Cone photoreceptors displayed similar structural abnormalities, as well as a decrease in cone opsin immunoreactivity. Drusen-associated abnormalities in the synaptic terminals of photoreceptor cells were also observed. In addition, an increase in intermediate filament protein immunoreactivity (vimentin and glial fibrillary acidic protein) was observed within Muller glial cells in areas of retina overlying drusen. Both soft and hard drusen were associated with a similar spectrum of effects in both macular and extramacular regions. Second- and third-order neurons, including bipolar, horizontal, amacrine, and ganglion cells all appeared unaffected. The structural and molecular abnormalities observed in photoreceptors and Muller glial cells were confined to retinal regions directly overlying and immediately adjacent to drusen; more distant retinal regions appeared unperturbed. Remarkably, significant abnormalities were observed over small subclinical drusen. CONCLUSIONS Retinal cells overlying both soft and hard drusen exhibit structural and molecular abnormalities indicative of photoreceptor degeneration and Muller glial activation. These abnormalities resemble the degenerative effects common to many forms of retinal degeneration, but are confined to areas directly overlying drusen. This suggests that photoreceptor cell function is compromised as a consequence of drusen formation.

PURPOSE. Anterior ischemic optic neuropathy (AION) is caused by sudden loss of vascular supply to retinal ganglion cell (RGC) axons in the anterior portion of the optic nerve and is a major cause of optic nerve dysfunction. There has been no easily obtainable animal model of this disorder. The current study was conducted to design a novel model of rodent AION (rAION), to enable more detailed study of this disease. METHODS. A novel rodent photoembolic stroke model was developed that is directly analogous to human AION. Using histologic, electrophysiological, molecular- and cell biological methods, the early changes associated with isolated RGC axonal ischemia were characterized. RESULTS. Functional (electrophysiological) changes occurred in RGCs within 1 day after rAION, with a loss of visual evoked potential (VEP) amplitude that persisted in the long term. The retinal gene expression pattern rapidly changed after rAION induction, with an early (