Reaction time measures of non-chromatic contamination in opponent stimuli.

Simple visual reaction times (RTs) are highly sensitive to the presence of transient activity. Transient and sustained visual mechanisms generate different RT versus contrast functions because they have different gains. To identify non-chromatic (transient) activity, we can compare RT versus contrast functions obtained with either fast or slow onset stimuli. To test this, the stimulus adopted was a temporal modulation along the red-green axis, introducing non-chromatic components by varying the red-green ratio. The technique was sensitive to departures from isoluminance for all observers; therefore, we present this method as a way to detect transient contamination in a chromatic stimulus.

Comparison of macaque and human L- and M-cone driven electroretinograms.

PURPOSE: The macaque retina is often used as a model for the human retina. However, there are only a handful of direct in vivo comparisons of the retinal physiology in humans and macaques. In the current study, ERG responses to luminance, L-cone isolating and M-cone isolating stimuli with sinusoidal, sawtooth and square wave temporal profiles were measured. The results were compared with those obtained from human observers. METHODS: The responses from five anesthetized adult macaques were measured. Full field stimuli were created. L- and M-cone isolating stimuli were based on the triple silent substitution technique. Sinusoidal stimuli had temporal frequencies between 4 and 56 Hz in 4 Hz steps. Sawtooth stimuli with rapid-on ramp-off and with rapid-off ramp-on excitation profiles had a frequency of 4 Hz. Square stimuli were presented at 2 Hz. RESULTS: Macaque and human ERGs in response to L- and M-cone isolating stimuli reflect L/M opponency and luminance activity. In responses to sine waves, cone opponency dominates at low temporal frequencies (4-12 Hz); luminance dominates at high temporal frequencies. The responses to sawtooth and square wave stimuli reflect a mixture of chromatic and luminance activity. L:M response ratios vary between individuals both in macaques and humans. Macaques show more complex responses, including greater second harmonic contributions than those in humans. CONCLUSIONS: Macaque and human ERGs share basic underlying mechanisms reflecting L/M opponency and luminance activity. There may be quantitative differences possibly reflecting differences in contributions of inner retinal mechanisms to the ERGs.

Anomalous pupillary responses to M-cone onsets are linked to ${\rm L}{:}{\rm M}$L:M ratio.

M-cone stimulation induces a pupil constriction to stimulus offset, whereas, with L cones, the pupil responds conventionally with a constriction to onset. To test the possibility that this paradox is linked to the ${\rm L}{:}{\rm M}$L:M ratio, we measured the strength of the effect by injecting a variable amount of positive or negative luminance contamination on either side of M-cone isolation and identifying a balance point at which the pupil responded equally to onset and offset. Nineteen individuals were recruited. In observers with low ${\rm L}{:}{\rm M}$L:M ratio, the paradoxical effect was weak. There was a significant relationship (${{r}^2} = {0.561}$r2=0.561) between the balance point and ${\rm L}{:}{\rm M}$L:M ratio. The effect is likely to be linked to strong inhibitory signals associated with cone-opponent pathways.

Rod- versus cone-driven ERGs at different stimulus sizes in normal subjects and retinitis pigmentosa patients.

PURPOSE: To study how rod- and cone-driven responses depend on stimulus size in normal subjects and patients with retinitis pigmentosa (RP), and to show that comparisons between responses to full-field (FF) and smaller stimuli can be useful in diagnosing and monitoring disorders of the peripheral retina without the need for lengthy dark adaptation periods. METHOD: The triple silent substitution technique was used to isolate L-cone-, M-cone- and rod-driven ERGs with 19, 18 and 33% photoreceptor contrasts, respectively, under identical mean luminance conditions. Experiments were conducted on five normal subjects and three RP patients. ERGs on control subjects were recorded at nine different temporal frequencies (between 2 and 60 Hz) for five different stimulus sizes: FF, 70°, 60°, 50° and 40° diameter circular stimuli. Experiments on RP patients involved rod- and L-cone-driven ERG measurements with FF and 40° stimuli at 8 and 48 Hz. Response amplitudes were defined as those of the first harmonic component after Fourier analysis. RESULTS: In normal subjects, rod-driven responses displayed a fundamentally different behavior than cone-driven responses, particularly at low temporal frequencies. At low and intermediate temporal frequencies (≤ 12 Hz), rod-driven signals increased by a factor of about four when measured with smaller stimuli. In contrast, L- and M-cone-driven responses in this frequency region did not change substantially with stimulus size. At high temporal frequencies (≥ 24 Hz), both rod- and cone-driven response amplitudes decreased with decreasing stimulus size. Signals obtained from rod-isolating stimuli under these conditions are likely artefactual. Interestingly, in RP patients, both rod-driven and L-cone-driven ERGs were similar using 40° and FF stimuli. CONCLUSION: The increased responses with smaller stimuli in normal subjects to rod-isolating stimuli indicate that a fundamentally different mechanism drives the ERGs in comparison with the cone-driven responses. We propose that the increased responses are caused by stray light stimulating the peripheral retina, thereby allowing peripheral rod-driven function to be studied using the triple silent substitution technique at photopic luminances. The method is effective in studying impaired peripheral rod- and cone- function in RP patients.

Paradoxical pupil responses to isolated M-cone increments.

M-cone onsets appear dimmer than the background and elicit electroretinograms (ERGs) resembling the light offset response. We sought a corresponding anomalous pupillary light reflex (PLR) using a 4-primary ganzfeld as stimulator and pupillometer. Increments and decrements of white light were compared with M- and L-cone onsets and offsets using silent substitution. Luminance bias (LB) could be added to or subtracted from the cone-isolating stimuli. There was a normal PLR to L-cone increments, but the pupil constricted mainly to M-cone decrements. Changing LB produced a neutral point where on and off responses were balanced. The results reflect ERG and psychophysical studies. This observation may be linked to the antagonistic nature of the M-cone input to cone opponent mechanisms.

Panel-Based Clinical Genetic Testing in 85 Children with Inherited Retinal Disease.

PURPOSE: To assess the clinical usefulness of genetic testing in a pediatric population with inherited retinal disease (IRD). DESIGN: Single-center retrospective case series. PARTICIPANTS: Eighty-five unrelated children with a diagnosis of isolated or syndromic IRD who were referred for clinical genetic testing between January 2014 and July 2016. METHODS: Participants underwent a detailed ophthalmic examination, accompanied by electrodiagnostic testing (EDT) and dysmorphologic assessment where appropriate. Ocular and extraocular features were recorded using Human Phenotype Ontology terms. Subsequently, multigene panel testing (105 or 177 IRD-associated genes) was performed in an accredited diagnostic laboratory, followed by clinical variant interpretation. MAIN OUTCOME MEASURES: Diagnostic yield and clinical usefulness of genetic testing. RESULTS: Overall, 78.8% of patients (n = 67) received a probable molecular diagnosis; 7.5% (n = 5) of these had autosomal dominant disease, 25.4% (n = 17) had X-linked disease, and 67.2% (n = 45) had autosomal recessive disease. In a further 5.9% of patients (n = 5), a single heterozygous ABCA4 variant was identified; all these participants had a spectrum of clinical features consistent with ABCA4 retinopathy. Most participants (84.7%; n = 72) had undergone EDT and 81.9% (n = 59) of these patients received a probable molecular diagnosis. The genes most frequently mutated in the present cohort were CACNA1F and ABCA4, accounting for 14.9% (n = 10) and 11.9% (n = 8) of diagnoses respectively. Notably, in many cases, genetic testing helped to distinguish stationary from progressive IRD subtypes and to establish a precise diagnosis in a timely fashion. CONCLUSIONS: Multigene panel testing pointed to a molecular diagnosis in 84.7% of children with IRD. The diagnostic yield in the study population was significantly higher compared with that in previously reported unselected IRD cohorts. Approaches similar to the one described herein are expected to become a standard component of care in pediatric ophthalmology. We propose the introduction of genetic testing early in the diagnostic pathway in children with clinical and/or electrophysiologic findings, suggestive of IRD.

Slowed dark adaptation in older eyes; effect of location.

PURPOSE: The rate of rod sensitivity recovery following a photobleach is a basic measure of the integrity of the outer retina. Rods are selectively impaired in aging and many disorders of the retina, notably Age-Related Macular Degeneration (AMD). It is not known for certain whether the age-related deficit is a pan-retinal effect or if there are localised regions of impaired rod function. To address this important issue a dual arc stimulus was developed that samples sensitivity recovery in two retinal locations. METHODS: Arc-shaped stimuli were presented on a black CRT screen at two locations, in the inferior visual field. Following a bleach, which was localised to the stimuli, recovery of sensitivity was measured using a modified method of adjustment technique. Neutral density filters were used to extend the luminance range of the CRT. Sensitivity recovery functions were fitted by non-linear regression to a seven-parameter model. RESULTS: Pairs of sensitivity recovery functions were generated from the stimuli. The cone phases of these functions were identical. The slopes of the S2 sections of the curves were steeper for the outer stimuli for both young (p < 0.001) and older (p = 0.003) observers. The difference between the two was the same for the two groups. The α point was reached slightly earlier for the young observers and with the outer stimulus but neither of these effects reached statistical significance. The ß point occurred earlier for the outer stimuli and this effect was statistically significant only for the older group. CONCLUSIONS: The method places minimal demands on observers. The fact that rod sensitivity recovery is slowed in the older normal eye to the same extent in the two locations suggests that this deficit may be uniform across the retina. As there are localised losses in scotopic function in AMD, the technique is ideally suited to distinguishing impaired recovery dynamics due to normal ageing from those caused by disease.

A dim view of M-cone onsets.

We investigated the brightness (i.e., perceived luminance) of isolated L- and M-cone pulses to seek a perceptual correlate of our previous reports that M-on electroretinograms resemble L-off responses, implying the operation of post-receptoral opponent processing. Using triple silent substitutions, cone increments were generated in a 4-primary ganzfeld, masked by random positive or negative luminance bias. The results show that M-cone increments decrease in brightness, while L-cone increments increase. These differences became smaller as field size reduced; this was not eccentricity or area dependent. We speculate about early retinal input into brightness perception.

Spatial properties of L- and M-cone driven incremental (On-) and decremental (Off-) electroretinograms: evidence for the involvement of multiple post-receptoral mechanisms.

An overview of electroretinogram response components to incremental and decremental steps in L- and M-cone excitation was obtained in human observers, while varying the spatial properties of the stimulus. Responses to L- and M-cone stimuli of opposite polarity resembled each other, particularly within the central 35° of the retina. All amplitudes grew as stimulus size increased; however, earlier and later components of the On- and Off-responses showed differing degrees of dependency on stimulus size. Thus, they may reflect different proportions of responses originating in parvocellular (less stimulus size-dependent) and magnocellular (more stimulus size-dependent) streams.

Development and testing of an automated computer tablet-based method for self-testing of high and low contrast near visual acuity in ophthalmic patients.

PURPOSE: Many eye diseases require on-going assessment for optimal management, creating an ever-increasing burden on patients and hospitals that could potentially be reduced through home vision monitoring. However, there is limited evidence for the utility of current applications and devices for this. To address this, we present a new automated, computer tablet-based method for self-testing near visual acuity (VA) for both high and low contrast targets. We report on its reliability and agreement with gold standard measures. METHODS: The Mobile Assessment of Vision by intERactIve Computer (MAVERIC) system consists of a calibrated computer tablet housed in a bespoke viewing chamber. Purpose-built software automatically elicits touch-screen responses from subjects to measure their near VA for either low or high contrast acuity. Near high contrast acuity was measured using both the MAVERIC system and a near Landolt C chart in one eye for 81 patients and low contrast acuity using the MAVERIC system and a 25 % contrast near EDTRS chart in one eye of a separate 95 patients. The MAVERIC near acuity was also retested after 20 min to evaluate repeatability. RESULTS: Repeatability of both high and low contrast MAVERIC acuity measures, and their agreement with the chart tests, was assessed using the Bland-Altman comparison method. One hundred and seventy-three patients (96 %) completed the self- testing MAVERIC system without formal assistance. The resulting MAVERIC vision demonstrated good repeatability and good agreement with the gold-standard near chart measures. CONCLUSIONS: This study demonstrates the potential utility of the MAVERIC system for patients with ophthalmic disease to self-test their high and low contrast VA. The technique has a high degree of reliability and agreement with gold standard chart based measurements.

Temporal characteristics of L- and M-cone isolating steady-state electroretinograms.

Cone isolating stimuli were used to assess the temporal frequency response characteristics of L- and M-cone electroretinograms (ERGs) in nine trichromatic and four dichromatic human observers. The stimuli comprised sinusoidal temporal modulations varying from 5 to 100 Hz. ERGs were recorded using corneal fiber electrodes and subjected to fast Fourier transform analysis. At low temporal frequencies (<10 Hz) the L- and M-cone ERGs had similar amplitude and exhibited minimal differences in apparent latency. At higher flicker rates (>20 Hz) L-cone ERGs had greater amplitudes and shorter apparent latencies than the M-cone responses. These differences between the L- and M-cone ERGs are consistent with their mediation by chromatic and luminance postreceptoral processing pathways at low and high temporal frequencies, respectively.

Incremental and decremental L- and M-cone driven ERG responses: II. Sawtooth stimulation.

L- and M-cone driven on- and off- ERG responses and their interactions were examined using full field stimuli with sawtooth temporal profiles. The effects of temporal frequency and contrast were studied. ERG recordings were obtained from 21 trichromatic, 1 protanopic, and 1 deuteranopic subjects. ERGs to L-cone increments and decrements resembled those to M-cone decrements and increments, respectively (i.e., of the opposite polarity). Temporal frequency and contrast had little effect on the implicit times. All response components varied linearly with contrast. When stimulated simultaneously, the responsivities of most components were larger for counterphase than for inphase modulation. The retinal processing leading to an ERG response is reversed for L- and M-cone driven responses.

Incremental and decremental L- and M-cone-driven ERG responses: I. Square-wave pulse stimulation.

Electroretinograms (ERGs) elicited by transient, square-wave L- and M-cone isolating stimuli were recorded from human trichromatic (n=19) and dichromatic (n=4) observers. The stimuli were generated on a four primary LED stimulator and were equated in terms of cone modulation (cone contrast=0.11) and retinal illuminance (12,000 trolands). L- and M-cone isolated ERGs had waveforms similar to those observed for luminance responses. However, M-cone ERGs exhibited a phase reversal in their responses to onset and offset stimuli relative to the L-cone responses. This on-off response reversal was observed in trichromats but not dichromats. Simultaneous counterphase and inphase combinations of L- and M-cone isolating stimuli generated responses that reflected chromatic and luminance processing, respectively. We conclude that L- and M-cone specific ERGs provide a measure of how photoreceptors contribute to postreceptoral mechanisms.

The association between dark adaptation and macular pigment optical density in healthy subjects.

PURPOSE: To investigate whether macular pigment optical density (MPOD) is related to dark adaptation in healthy subjects. METHODS: Dark adaptation was measured after a minimum 30 % pigment bleach in 33 subjects (aged 15-68), using a white 1° stimulus presented 11° below fixation on a cathode ray tube monitor. The luminance range of the monitor was extended using neutral density filters. A heterochromatic flicker photometry based instrument (MPS 9000) was used to measure MPOD. RESULTS: The average MPOD for the whole group was 0.37 ± 0.21 optical density units. Subjects with lighter irides had on average 40 % lower MPOD compared to those with darker irides (0.3 ± 0.20 vs 0.5 ± 0.19). Group mean MPOD was weakly associated with second (r = 0.32, p = 0.07) and third rod-mediated recovery rates (r = 0.31, p = 0.08) and with the rod threshold (r = -0.24, p = 0.18) 30 min after the onset of bleach. MPOD was unrelated to cone time constant (r = -0.02, p = 0.91), cone threshold (r = -0.01, p = 0.96), rod-cone break (r = 0.13, p = 0.45) or the rod-rod break (r = 0.11, p = 0.52). The second rod-mediated recovery rate (S2) for the lower 10th percentile of MPOD (n = 4) was 0.18 log cd.m(-2).min(-1) and 0.24 log cd.m(-2).min(-1) for the upper 10th percentile (n = 4). The two groups were significantly different (t = -2.67, p = 0.037). CONCLUSIONS: We report a statistically significant difference between subjects falling in the 10th percentile extremes of MPOD and rod-mediated but not cone-mediated sensitivity recovery. Further investigation into the relationship between MPOD and rod function is warranted, particularly extending the work to encompass those with low MPOD and poor night vision.

Assessment of age changes and repeatability for computer-based rod dark adaptation.

PURPOSE: To characterize the rate of rod-mediated sensitivity decline with age using a PC-driven cathode ray tube (CRT) monitor. To provide data regarding the repeatability of the technique. METHODS: Dark adaptation was monitored for 30 min following a minimum 30 % pigment bleach, using a white 1° stimulus (modulated at 1 Hz), presented 11° below fixation on a CRT monitor. Thirty-three subjects with no ocular pathology and normal fundus photographs were divided into two groups: older (≥45, n = 16) and younger (<45, n = 17). RESULTS: Rod recovery was assessed using component S2 of dark adaptation. S2 was significantly slower in the older (0.19 ± 0.03 log cd.m(-2).min(-1)) compared with the younger group (0.23 ± 0.03 log cd.m(-2).min(-1), t = -4.05, p < 0.0003), despite no difference in visual acuity and fundus appearance. Faster rates of S2 recovery were correlated with lower threshold at 30 min (T30) (r = -0.49). Correlation coefficients between first and second measurements for S2 and T30 were 0.49 (p < 0.009) and 0.84 (p < 0.0001) respectively. The coefficient of repeatability was 0.07 log cd.m(-2).min(-1) for S2 and 0.35 log cd.m(-2) for T30. The coefficients of variation for S2 and T30 were 15 % and 10 % respectively. CONCLUSIONS: Dark adaptation is slowed in normal ageing. CRT-based dark adaptometry is easily implemented and highly repeatable. The technique described in this article would be useful for documenting visual changes in future clinical trials assessing retinal health in the older eye with and without ocular pathology.

Retinal gene therapy in RPE-65 gene mediated inherited retinal dystrophy.

BACKGROUND: Voretigene neparvovec (VN) is a gene therapeutic agent for treatment of retinal dystrophies caused by bi-allelic RPE65 mutations. We illustrate, both the benefits and pitfalls associated with ocular gene therapy in the same patient. METHODS: Two eyes of one patient with bi-allelic RPE65 mutations have been treated with VN. The clinical examinations included visual acuity (VA, in normal and low luminance), colour vision, contrast sensitivity, International Society for Clinical Electrophysiology of Vision (ISCEV) standard retinal electrophysiology and dark-adapted full-field stimulus threshold (FST), Goldmann VF analysis and imaging studies, including optical coherence tomography (OCT) and autofluorescence. These were performed at baseline, 2-weeks, 3 and 6-months, 1 and 2-years follow-up. RESULTS: The first eye showed improvement in rod photoreceptor function with increased peripheral and low luminance vision (baseline VA: 0.9 logMAR and 2-years post-operative VA: 0.7 logMAR). The second eye, whilst showing increased light sensitivity, suffered a drop in central vision (at 2-weeks) with loss of foveal photoreceptors as shown by the loss of ellipsoid zone on OCT scan (baseline VA: 0.6, 2-year post-operative VA: 1.2). FST improvements were maintained in both eyes indicating a sustained efficacy of VN with little waning of its effect. CONCLUSIONS: We present a previously unreported adverse complication of subretinal VN therapy in bi-allelic RPE65, indicating a probable immune response in treatment of the second eye, resulting in loss of foveal photoreceptors. This case-series highlights the potential and pitfalls of retinal gene therapy in the same patient. The immune responses of the body to a 'foreign vector', remains a challenge.

Simultaneous chromatic and luminance human electroretinogram responses.

The parallel processing of information forms an important organisational principle of the primate visual system. Here we describe experiments which use a novel chromatic­achromatic temporal compound stimulus to simultaneously identify colour and luminance specific signals in the human electroretinogram (ERG). Luminance and chromatic components are separated in the stimulus; the luminance modulation has twice the temporal frequency of the chromatic modulation. ERGs were recorded from four trichromatic and two dichromatic subjects (1 deuteranope and 1 protanope). At isoluminance, the fundamental (first harmonic) response was elicited by the chromatic component in the stimulus. The trichromatic ERGs possessed low-pass temporal tuning characteristics, reflecting the activity of parvocellular post-receptoral mechanisms. There was very little first harmonic response in the dichromats' ERGs. The second harmonic response was elicited by the luminance modulation in the compound stimulus and showed, in all subjects, band-pass temporal tuning characteristic of magnocellular activity. Thus it is possible to concurrently elicit ERG responses from the human retina which reflect processing in both chromatic and luminance pathways. As well as providing a clear demonstration of the parallel nature of chromatic and luminance processing in the human retina, the differences that exist between ERGs from trichromatic and dichromatic subjects point to the existence of interactions between afferent post-receptoral pathways that are in operation from the earliest stages of visual processing.

The relationship between peripherally matched invariant hues and unique hues: a cone-contrast approach.

A characteristic shift in hue and saturation occurs when colored targets are viewed peripherally compared with centrally. Four hues, one in each of the red, blue, green, and yellow regions of color space, remain unchanged when presented in the peripheral field. Apart from green, these peripherally invariant hues correspond almost exactly in color space with the unique hues. We explore this puzzling observation using asymmetric color-matching and color-naming experiments and computing cone contrasts for peripheral and central stimuli. We find that the difference between cone contrasts for the peripheral and central stimuli reaches a maximum at the chromatic axis corresponding to peripherally invariant green. We speculate that the effect is linked to a weakened signal from M-cones and probably associated with a reduced number of M-cones in peripheral retina.

Real-world stimuli show perceived hue shifts in the peripheral visual field.

Certain hues undergo shifts in their appearance when they are viewed by the peripheral retina. This has often been shown on a 3-primary color CRT monitor. To investigate the possible role of metamerism, we replicated our peripheral color matching experiments using Munsell paper stimuli viewed under real and simulated daylight (using a 3-primary projection system). Using stimuli of constant value and chroma (7/4), observers adjusted the hue of a 3 deg target presented 18 deg nasally, until it matched a 1 deg target presented 1 deg nasally. The magnitude and pattern of measured hue shifts were similar to those measured using CRT stimuli. We conclude that the perceived hue shifts that have previously been reported in the peripheral retina are independent of the nature of the stimulus and of the illuminant.

Optimization of large field tritan stimuli using concentric isoluminant annuli.

Large, nominally isoluminant chromatic gratings containing a short-wavelength component are prone to luminance contrast intrusions due to retinal inhomogeneity, especially as a result of the uneven distribution of macular pigment. Isoluminance is usually determined for a relatively small, central area, but a significantly larger stimulus cannot be isoluminant across the whole field, largely due to macular pigment absorption of short-wavelength light. This confounds attempts to maintain high selectivity, particularly in suprathreshold electrophysiological and brain-imaging studies that require large stimulus fields. Here we introduce the concept of a panisoluminant grating (PIG), which comprises a series of concentric annular regions, each adjusted to location-specific isoluminance for the observer. Gratings were modulated along subject-specific tritanopic confusion lines and the selectivity of responses to the PIG was tested according to both psychophysical and electrophysiological criteria. The psychophysically-determined temporal tuning function obtained using the PIG showed lower sensitivity and lower resolution than with a conventional tritan grating of equal diameter (18°). Chromatic onset visual evoked potentials (VEPs) to the PIG were dominated by a chromatic-specific negative wave and reduced achromatic response components that were prominent in VEPs to the conventional grating. These data demonstrate that a large tritan PIG is capable of eliciting selective responses of the S-cone-driven pathway at threshold and at suprathreshold levels. The PIG stimulus may prove beneficial in investigations that require large fields such as electrophysiological and brain imaging studies of chromatic processing.