Statistical Evaluation of ERG Responses: A New Method to Validate Cycle-by-Cycle Recordings in Advanced Retinal Degenerations.

Purpose: To describe and evaluate a novel method to determine the validity of measurements made using cycle-by-cycle (CxC) recording techniques in patients with advanced retinal degenerations (RD) having low-amplitude flicker electroretinogram (ERG) responses. Methods: The method extends the original CxC recording algorithm introduced by Sieving et al., retaining the original recording setup and the preliminary analysis of raw data. Novel features include extended use of spectrum analysis, reduction of errors due to known sources, and a comprehensive statistical assessment using three different tests. The method was applied to ERG recordings from seven patients with RD and two patients with CNGB3 achromatopsia. Results: The method was implemented as a Windows application to processes raw data obtained from a commercial ERG system, and it features a computational toolkit for statistical assessment of ERG recordings with amplitudes as low as 1 µV, commonly found in advanced RD patients. When recorded using conditions specific for eliciting cone responses, none of the CNGB3 patients had a CxC validated response, indicating that no signal artifacts were present with our recording conditions. A comparison of the presented method with conventional 30 Hz ERG was performed. Bland-Altman plots indicated good agreement (mean difference, -0.045 µV; limits of agreement, 0.193 to -0.282 µV) between the resulting amplitudes. Within-session test-retest variability was 15%, comparing favorably to the variability of standard ERG amplitudes. Conclusions: This novel method extracts highly reliable clinical recordings of low-amplitude flicker ERGs and effectively detects artifactual responses. It has potential value both as a cone outcome variable and planning tool in clinical trials on natural history and treatment of advanced RDs.

Ultraviolet vision in anemonefish improves colour discrimination.

In many animals, ultraviolet (UV) vision guides navigation, foraging, and communication, but few studies have addressed the contribution of UV signals to colour vision, or measured UV discrimination thresholds using behavioural experiments. Here, we tested UV colour vision in an anemonefish (Amphiprion ocellaris) using a five-channel (RGB-V-UV) LED display. We first determined that the maximal sensitivity of the A. ocellaris UV cone was ∼386 nm using microspectrophotometry. Three additional cone spectral sensitivities had maxima at ∼497, 515 and ∼535 nm. We then behaviourally measured colour discrimination thresholds by training anemonefish to distinguish a coloured target pixel from grey distractor pixels of varying intensity. Thresholds were calculated for nine sets of colours with and without UV signals. Using a tetrachromatic vision model, we found that anemonefish were better (i.e. discrimination thresholds were lower) at discriminating colours when target pixels had higher UV chromatic contrast. These colours caused a greater stimulation of the UV cone relative to other cone types. These findings imply that a UV component of colour signals and cues improves their detectability, which likely increases the prominence of anemonefish body patterns for communication and the silhouette of zooplankton prey.

Non-image-forming vision as measured through ipRGC-mediated pupil constriction is not modulated by covert visual attention.

In brightness, the pupil constricts, while in darkness, the pupil dilates; this is known as the pupillary light response (PLR). The PLR is driven by all photoreceptors: rods and cones, which contribute to image-forming vision, and intrinsically photosensitive retinal ganglion cells (ipRGCs), which mainly contribute to non-image-forming vision. Rods and cones cause immediate pupil constriction upon light exposure, whereas ipRGCs cause sustained constriction throughout light exposure. Recent studies have shown that covert attention modulated the initial PLR; however, it remains unclear whether the same holds for the sustained PLR. We tested this by leveraging ipRGCs' responsiveness to blue light, causing the most prominent sustained constriction. While replicating previous studies by showing that pupils constricted more when either directly looking at, or covertly attending to, bright as compared to dim stimuli (with the same color), we also found that the pupil constricted more when directly looking at blue as compared to red stimuli (with the same luminosity). Crucially, however, in two high-powered studies (n = 60), we did not find any pupil-size difference when covertly attending to blue as compared to red stimuli. This suggests that ipRGC-mediated pupil constriction, and possibly non-image-forming vision more generally, is not modulated by covert attention.

Are ipRGCs involved in human color vision? Hints from physiology, psychophysics, and natural image statistics.

Human photoreceptors consist of cones, rods, and melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs). First studied in circadian regulation and pupillary control, ipRGCs project to a variety of brain centers suggesting a broader involvement beyond non-visual functions. IpRGC responses are stable, long-lasting, and with a particular codification of photoreceptor signals. In comparison with the transient and adaptive nature of cone and rod signals, ipRGCs' signaling might provide an ecological advantage to different attributes of color vision. Previous studies have indicated melanopsin's influence on visual responses yet its contribution to color perception in humans remains debated. We summarized evidence and hypotheses (from physiology, psychophysics, and natural image statistics) about direct and indirect involvement of ipRGCs in human color vision, by first briefly assessing the current knowledge about the role of melanopsin and ipRGCs in vision and codification of spectral signals. We then approached the question about melanopsin activation eliciting a color percept, discussing studies using the silent substitution method. Finally, we explore various avenues through which ipRGCs might impact color perception indirectly, such as through involvement in peripheral color matching, post-receptoral pathways, color constancy, long-term chromatic adaptation, and chromatic induction. While there is consensus about the role of ipRGCs in brightness perception, confirming its direct contribution to human color perception requires further investigation. We proposed potential approaches for future research, emphasizing the need for empirical validation and methodological thoroughness to elucidate the exact role of ipRGCs in human color vision.

Ancestral photoreceptor diversity as the basis of visual behaviour.

Animal colour vision is based on comparing signals from different photoreceptors. It is generally assumed that processing different spectral types of photoreceptor mainly serves colour vision. Here I propose instead that photoreceptors are parallel feature channels that differentially support visual-motor programmes like motion vision behaviours, prey capture and predator evasion. Colour vision may have emerged as a secondary benefit of these circuits, which originally helped aquatic vertebrates to visually navigate and segment their underwater world. Specifically, I suggest that ancestral vertebrate vision was built around three main systems, including a high-resolution general purpose greyscale system based on ancestral red cones and rods to mediate visual body stabilization and navigation, a high-sensitivity specialized foreground system based on ancestral ultraviolet cones to mediate threat detection and prey capture, and a net-suppressive system based on ancestral green and blue cones for regulating red/rod and ultraviolet circuits. This ancestral strategy probably still underpins vision today, and different vertebrate lineages have since adapted their original photoreceptor circuits to suit their diverse visual ecologies.

A case report of retinal dystrophy in patients with PACS1 syndrome.

PACS1 syndrome, also referred to as Schuurs-Hoeijmakers syndrome, is a multisystemic developmental disorder caused by a specific pathogenic variant in the PACS1 (phosphofurin acidic cluster sorting protein 1) gene. Ocular findings in PACS1 syndrome are known to include iris, retina, optic nerve coloboma, myopia, nystagmus, and strabismus. Here, we present the cases of two patients referred to the University of Wisconsin-Madison Department of Ophthalmology and Visual Sciences for ocular evaluation. The first patient is a 14-month-old female who, at 3 months of age, was found to have a depressed rod and cone response on electroretinogram (ERG), consistent with possible retinal dystrophy (RD). This feature has not been previously described in PACS1 syndrome and joins a growing list of calls for expanding the PACS1 phenotype. The second case illustrates a 5-year-old male referred for ocular screening after diagnosing PACS1 syndrome and underwent ERG without abnormal findings. These cases demonstrate the significant variability in the ophthalmic presentation of PACS1 syndrome and the need for early screening. These novel findings may have implications in understanding the mechanism of the PACS1 protein and its role in retinal ciliary phototransduction in photoreceptors.

Cone-driven strong flash electroretinograms in healthy adults: Prevalence of negative waveforms.

PURPOSE: Both rod and cone-driven signals contribute to the electroretinogram (ERG) elicited by a standard strong flash in the dark. Negative ERGs usually reflect inner retinal dysfunction. However, in diseases where rod photoreceptor function is selectively lost, a negative waveform might represent the response of the dark-adapted cone system. To investigate the dark-adapted cone-driven waveform in healthy individuals, we delivered flashes on a dim blue background, designed to saturate the rods, but minimally adapt the cones. METHODS: ERGs were recorded, using conductive fibre electrodes, in adults from the TwinsUK cohort. Responses to 13 cd m-2 s white xenon flashes (similar to the standard DA 10 flash), delivered on a blue background, were analysed. Photopic and scotopic strengths of the background were 1.3 and 30 cd m-2, respectively; through a dilated pupil, this is expected to largely saturate the rods, but adapt the cones much less than the standard ISCEV background. RESULTS: Mean (SD) participant age was 62.5 (11.3) years (93% female). ERGs from 203 right and 204 left eyes were included, with mean (SD) b/a ratios of 1.22 (0.28) and 1.18 (0.28), respectively (medians, 1.19 and 1.17). Proportions with negative waveforms were 23 and 26%, respectively. Right and left eye b/a ratios were strongly correlated (correlation coefficient 0.74, p < 0.0001). We found no significant correlation of b/a ratio with age. CONCLUSIONS: Over 20% of eyes showed b/a ratios less than 1, consistent with the notion that dark-adapted cone-driven responses to standard bright flashes can have negative waveforms. The majority had ratios greater than 1. Thus, whilst selective loss of rod function can yield a negative waveform (with reduced a-wave) in some, our findings also suggest that loss of rod function can occur without necessarily yielding a negative ERG. One potential limitation is possible mild cone system adaptation by the background.

Carl Bergmann (1814-1865) and the discovery of the anatomical site in the retina where vision is initiated.

A preeminent quest of nineteenth-century visual neuroscience was to identify the anatomical elements of the retina that respond to light. A major breakthrough came in 1854, when Carl Bergmann discovered through direct observation that the human fovea contains only rods and cones. On this basis, he argued that these must necessarily be the light-sensitive elements (i.e., photoreceptors) that initiate vision. Bergmann also argued that Henle's fibers form part of the necessary anatomical link between these distal elements and the proximal ganglion cells, which transmit visual signals to the brain via the optic nerve. However, despite his achievement, Heinrich Müller, not Bergmann, is remembered as the discoverer of human photoreceptors in the literature. This article seeks to correct the record. It situates Bergmann's work alongside that of his contemporaries, sets out his arguments and the critique he received using archival documents, and makes this history more accessible for current readers by comparing what was said to what we know now. We argue that Bergmann's arguments are at least as compelling as those of Müller, and that he should be recognized as a codiscoverer of the anatomic site in the retina where vision is initiated.

A dynamical adaptation model of visual spatiotemporal processing in cones and horizontal cells.

In this work, we introduce a phenomenological model for the cone-horizontal cell assembly, including spatial integration and formation of receptive field-like structures. The model extends our previous dynamical adaptation description with gain control accounting for processes in single cones, valid in severe nonlinear regimes. Here, a spatially extended feedback mechanism is introduced from horizontal cells to cones to account for experimental evidence, contributing thus to the development of a center-surround receptive field in cones and downstream bipolar cells. Feedback gain is defined on different spatial scales by weighting spatial filters: a short scale accounting for cone input to the feedback mechanism and a large scale driven by the syncytium characteristics of horizontal cells. A third spatial scale improves the description, mimicking neighboring cone-cone coupling. This overall spatial integration couples to temporal signal processing, thus obtaining a spatiotemporal model of outer retina responses capable of reproducing nonlinear features in both dimensions (space and time). The model was tested and validated using measurements on horizontal cells from different studies, with excellent performance. By its phenomenological nature, signal processing properties are inferred from model parameters. The model can be used in arrays of processing units with more complex incoming patterns of visual stimuli.

Melanopsin enhances image persistence.

Contributions of the inner retinal photopigment melanopsin to human visual perception are incompletely understood. Here, we use a four-primary display to produce stimuli differing in melanopsin versus cone contrast in psychophysical paradigms in eight subjects with normal color vision. We address two predictions from electrophysiological recordings of the melanopsin system in non-human mammals: melanopsin influences color and/or supports image persistence under visual fixation. We first construct chromatic contrast sensitivity contours for stimuli differing in melanopsin excitation presented as a central annulus (10°) or peripheral (22.5°) spot. We find that although including melanopsin contrast produces modest changes in the average chromatic coordinates in both eccentricities, this occurs equally at low (0.5 Hz) and higher (3.75 Hz) temporal frequencies, arguing that it reflects divergence in cone spectral sensitivity in our participants from that captured in standardized cone fundamentals rather than a melanopsin contribution to color. We continue to ask whether the established ability of melanopsin to sustain firing of visual neurons under extended light exposure has a visual correlate, using the optical illusion of Troxler fading in which blurred spots in periphery disappear during visual fixation. We find that introducing additional melanopsin contrast (+28% Michelson contrast) to either bright or dark spots increases fading latency by 35% ± 8.8% and 41% ± 13.6%, respectively. Our data argue that the primary contribution of melanopsin to perception under these conditions is not to provide a color percept but rather to enhance persistence of low spatial frequency patterns during visual fixation.

Losing, preserving, and restoring vision from neurodegeneration in the eye.

The retina is a part of the brain that sits at the back of the eye, looking out onto the world. The first neurons of the retina are the rod and cone photoreceptors, which convert changes in photon flux into electrical signals that are the basis of vision. Rods and cones are frequent targets of heritable neurodegenerative diseases that cause visual impairment, including blindness, in millions of people worldwide. This review summarizes the diverse genetic causes of inherited retinal degenerations (IRDs) and their convergence onto common pathogenic mechanisms of vision loss. Currently, there are few effective treatments for IRDs, but recent advances in disparate areas of biology and technology (e.g., genome editing, viral engineering, 3D organoids, optogenetics, semiconductor arrays) discussed here enable promising efforts to preserve and restore vision in IRD patients with implications for neurodegeneration in less approachable brain areas.

Ultrafast Transient Absorption Spectra and Kinetics of Rod and Cone Visual Pigments.

Rods and cones are the photoreceptor cells containing the visual pigment proteins that initiate visual phototransduction following the absorption of a photon. Photon absorption induces the photochemical transformation of a visual pigment, which results in the sequential formation of distinct photo-intermediate species on the femtosecond to millisecond timescales, whereupon a visual electrical signal is generated and transmitted to the brain. Time-resolved spectroscopic studies of the rod and cone photo-intermediaries enable the detailed understanding of initial events in vision, namely the key differences that underlie the functionally distinct scotopic (rod) and photopic (cone) visual systems. In this paper, we review our recent ultrafast (picoseconds to milliseconds) transient absorption studies of rod and cone visual pigments with a detailed comparison of the transient molecular spectra and kinetics of their respective photo-intermediaries. Key results include the characterization of the porphyropsin (carp fish rhodopsin) and human green-cone opsin photobleaching sequences, which show significant spectral and kinetic differences when compared against that of bovine rhodopsin. These results altogether reveal a rather strong interplay between the visual pigment structure and its corresponding photobleaching sequence, and relevant outstanding questions that will be further investigated through a forthcoming study of the human blue-cone visual pigment are discussed.

Rod and Cone Function Measured Objectively by Chromatic Pupil Campimetry Show a Different Preservation Between Distinct Genotypes in Retinitis Pigmentosa.

Purpose: Verifying whether specific genotypes causing retinitis pigmentosa (RP) show differences in the preservation of rod and cone function measured by chromatic pupil campimetry (CPC). Methods: Sixty-three RP eyes (37 male, 14-58 years) were measured using CPC with specific photopic and scotopic protocols, and the relative maximal constriction amplitudes and latencies to constriction onset were analyzed per genotype (RP due to variants in EYS, n = 14; PDE6A, n = 10; RPE65, n = 15; USH2A, n = 10; and RPGR, n = 14). Correlation analyses between the pupillary responses were performed with age, full-field stimulus threshold (FST), and optical coherence tomography (OCT) for cones and rods, respectively, to the genotype. Results: Pupillary responses were most severely reduced in RPE65-RP. Patients with disease-associated variants in EYS and USH2A were accompanied with better-preserved rod function compared with the other subgroups, reaching statistical significance between EYS and RPE65. Cone function was statistically significantly correlated with age in USH2A-RP with an annual decline of 2.4%. Correlations of pupillary responses were found with FST but barely with the ellipsoid zone area in OCT. Latency was significantly more prolonged in RPE65-RP compared with the other genotypes for cones. Conclusions: Rod and cone function measured objectively by CPC showed a different preservation between genotypes in RP. However, heterogeneity inside the same genotype was present. CPC data correlated with FST, but structural OCT parameters seem to be limited indicators for photoreceptor function in RP. Prolonged time dynamics for cones in RPE65 mutations suggest an impact on cone processing and might provide additional information in the evaluation of therapy effects.

Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.

PySilSub: An open-source Python toolbox for implementing the method of silent substitution in vision and nonvisual photoreception research.

The normal human retina contains several classes of photosensitive cell-rods for low-light vision, three cone classes for daylight vision, and intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing melanopsin for non-image-forming functions, including pupil control, melatonin suppression, and circadian photoentrainment. The spectral sensitivities of the photoreceptors overlap significantly, which means that most lights will stimulate all photoreceptors to varying degrees. The method of silent substitution is a powerful tool for stimulating individual photoreceptor classes selectively and has found much use in research and clinical settings. The main hardware requirement for silent substitution is a spectrally calibrated light stimulation system with at least as many primaries as there are photoreceptors under consideration. Device settings that will produce lights to selectively stimulate the photoreceptor(s) of interest can be found using a variety of analytic and algorithmic approaches. Here we present PySilSub (https://github.com/PySilentSubstitution/pysilsub), a novel Python package for silent substitution featuring flexible support for individual colorimetric observer models (including human and mouse observers), multiprimary stimulation devices, and solving silent substitution problems with linear algebra and constrained numerical optimization. The toolbox is registered with the Python Package Index and includes example data sets from various multiprimary systems. We hope that PySilSub will facilitate the application of silent substitution in research and clinical settings.

Multimodal high-resolution retinal imaging using a camera-based DMD-integrated adaptive optics flood-illumination ophthalmoscope.

We demonstrate the feasibility of a multimodal adaptive optics flood-illumination ophthalmoscope, able to provide both bright-field and dark-field images (such as phase contrast). The multimodality was made possible by integrating a digital micromirror device (DMD) at the illumination path to project a sequence of complementary high-resolution patterns into the retina. Through a versatile post-processing method that digitally selects backscattered or multiply scattered photons, we were able: (1) to achieve up to four-fold contrast increase of bright-field images when imaging the photoreceptor mosaic and nerve fibers; and (2) to visualize translucent retinal features such as capillaries, red blood cells, vessel walls, ganglion cells, and photoreceptor inner segments through phase contrast.

Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3): Role in Retinal Development and Disease.

NR2E3 is a nuclear hormone receptor gene required for the correct development of the retinal rod photoreceptors. Expression of NR2E3 protein in rod cell precursors suppresses cone-specific gene expression and, in concert with other transcription factors including NRL, activates the expression of rod-specific genes. Pathogenic variants involving NR2E3 cause a spectrum of retinopathies, including enhanced S-cone syndrome, Goldmann-Favre syndrome, retinitis pigmentosa, and clumped pigmentary retinal degeneration, with limited evidence of genotype-phenotype correlations. A common feature of NR2E3-related disease is an abnormally high number of cone photoreceptors that are sensitive to short wavelength light, the S-cones. This characteristic has been supported by mouse studies, which have also revealed that loss of Nr2e3 function causes photoreceptors to develop as cells that are intermediate between rods and cones. While there is currently no available cure for NR2E3-related retinopathies, there are a number of emerging therapeutic strategies under investigation, including the use of viral gene therapy and gene editing, that have shown promise for the future treatment of patients with NR2E3 variants and other inherited retinal diseases. This review provides a detailed overview of the current understanding of the role of NR2E3 in normal development and disease, and the associated clinical phenotypes, animal models, and therapeutic studies.

Short-wavelength artificial light affects visual neural pathway development in mice.

Nearly all modern life depends on artificial light; however, it does cause health problems. With certain restrictions of artificial light emitting technology, the influence of the light spectrum is inevitable. The most remarkable problem is its overload in the short wavelength component. Short wavelength artificial light has a wide range of influences from ocular development to mental problems. The visual neuronal pathway, as the primary light-sensing structure, may contain the fundamental mechanism of all light-induced abnormalities. However, how the artificial light spectrum shapes the visual neuronal pathway during development in mammals is poorly understood. We placed C57BL/6 mice in three different spectrum environments (full-spectrum white light: 400-750 nm; violet light: 400 ± 20 nm; green light: 510 ± 20 nm) beginning at eye opening, with a fixed light time of 7:00-19:00. During development, we assessed the ocular axial dimension, visual function and retinal neurons. After two weeks under short wavelength conditions, the ocular axial length (AL), anterior chamber depth (ACD) and length of lens thickness, real vitreous chamber depth and retinal thickness (LLVR) were shorter, visual acuity (VA) decreased, and retinal electrical activity was impaired. The density of S-cones in the dorsal and ventral retinas both decreased after one week under short wavelength conditions. In the ventral retina, it increased after three weeks. Retinal ganglion cell (RGC) density and axon thickness were not influenced; however, the axonal terminals in the lateral geniculate nucleus (LGN) were less clustered and sparse. Amacrine cells (ACs) were significantly more activated. Green light has few effects. The KEGG and GO enrichment analyses showed that many genes related to neural circuitry, synaptic formation and neurotransmitter function were differentially expressed in the short wavelength light group. In conclusion, exposure to short wavelength artificial light in the early stage of vision-dependent development in mice delayed the development of the visual pathway. The axon terminus structure and neurotransmitter function may be the major suffering.

Maternal diabetes affects rat offspring retinal structure and function: Sex-specific vulnerabilities at infancy.

AIMS: Maternal diabetes negatively impacts the offspring's brain, but little is known about its effects on the retina, which is also part of the central nervous system. We hypothesized that maternal diabetes adversely influences offspring retina development leading to structural and functional deficits. MAIN METHODS: Retinal structure and function were evaluated at infancy, by optical coherence tomography and electroretinography, in male and female offspring of control, diabetic and diabetic-treated with insulin Wistar rats. KEY FINDINGS: Maternal diabetes induced a delay in male and female offspring eye-opening, while insulin treatment expedited it. Structural analysis showed that maternal diabetes decreased the thickness of the inner and outer segment layer of photoreceptors in male offspring. Electroretinography also revealed that maternal diabetes decreased the amplitude of scotopic b-wave and flicker response in males, suggesting bipolar cells and cone photoreceptor dysfunction, an effect not observed in females. Conversely, maternal diabetes decreased cone arrestin protein levels in female retinas, while not affecting cone photoreceptor number. Dam insulin therapy was efficient in preventing the offspring photoreceptor changes. SIGNIFICANCE: Our results suggest that photoreceptors are affected by maternal diabetes, which may account for visual impairments at infancy. Notably, both male and female offspring presented specific vulnerabilities to hyperglycemia in this sensitive period of development.