Pattern electroretinogram, blue-yellow visual evoked potentials and the risk of developing visual field defects in glaucoma suspects: a longitudinal "survival" analysis with a very long follow-up.

PURPOSE: Estimating glaucoma suspects' risk for visual field defects helps to avoid under- and over-treatment. In this retrospective, longitudinal cohort study with a very long follow-up, we studied whether pattern electroretinograms (PERG) amplitudes and blue-on-yellow visual evoked potential (BY-VEP) latencies can predict visual field defects. METHODS: Participants of the Erlangen Glaucoma Study were examined with PERG and BY-VEP between 9/1991 and 8/2001. Stimuli were created using an optical bench with Maxwellian view and consisted of vertical gratings (0,88 cpd) in a 32° field for both PERG and BY-VEP. Patients were treated according to clinical standards and performed standard automated perimetry (SAP) annually. Retrospectively, patients with normal SAP at baseline were selected. Primary endpoint was conversion to perimetric glaucoma. Predictive value was modeled using Kaplan-Meier analyses and a multivariate cox proportional hazards model with the continuous variables PERG amplitude, BY-VEP peak time and SAP square-root of loss variance (sLV) after stratification for Jonas classification of the optic discs. RESULTS: Of 412 patients (288: Jonas 0, 103: I, and 21: II; baseline age: 20-60 years), 65 converted to perimetric glaucoma during follow-up (0.5-23.3 years; median 5.5 years). Optic disc classification was a strong risk factor for conversion (log rank p < 0.0001), and patients with more advanced changes progressed earlier. In the multivariate analysis (log rank p = 0.005), only PERG amplitude remained an independent risk factor after stratification for optic disc morphology (p = 0.021), with a ~ 30% higher risk per µV amplitude decrease. CONCLUSIONS: PERG helps to estimate glaucoma suspects' risk for visual field defects.

Objective detection of visual field defects with multifrequency VEPs.

PURPOSE: To correlate multifrequency pattern reversal VEPs in quadrants (QmfrVEPs) with perimetric field losses for objective detection of visual field losses. METHODS: QmfrVEP measurements were performed using four LED-based checkerboard stimulators to stimulate the four quadrants of the visual field. QmfrVEPs were measured monocularly in 5 normal subjects and in 5 glaucoma patients who showed losses in conventional Octopus perimetry. The pattern reversal frequency varied slightly between the stimulators: (11.92, 12.00, 12.08 and 12.16 reversals/sec). The responses to the different stimuli were identified by discrete Fourier analysis. VEPs were recorded using different electrode configurations, and the recording with the highest signal-to-noise ratio (SNR) was used for further analysis. RESULTS: QmfrVEP responses from the different quadrants can be reliably measured and separated using the 0.08 reversals/sec interstimulus reversal frequency differences. The signal-to-noise ratio in the four quadrants was significantly correlated with the equivalent visual field losses obtained with perimetry (Spearman rank correlation: P < 0.001). In the five glaucoma patients, the SNR was reduced in 15 out of the 16 quadrants with a perimetric defect, in comparison to the results in quadrants of healthy subjects. This confirms the sensitivity of the procedure. CONCLUSION: QmfrVEP responses can be measured reliably. This pilot study suggests that high SNR values exclude visual field defects and that focal defects can be identified in glaucoma patients. TRIAL REGISTRATION: www. CLINICALTRIALS: gov . NCT00494923.

Chromatic Pupillometry - a New Technique for Assessing Function in Glaucoma? / Chromatische Pupillometrie ­ ein neuer Weg zur funktionellen Glaukomdiagnostik?

Chromatic pupillometry allows quantification of photoreceptor-driven (extrinsic) and melanopsin-driven (intrinsic) responses of the intrinsic-photosensitive retinal ganglion cells (ipRGCs). This small subpopulation of retinal ganglion cells is also affected by glaucoma, making chromatic pupillometry a potential diagnostic tool. Studies show reduced phasic and tonic responses in glaucoma patients. The diagnostic value in earlier studies depended on the technical details and the study design. The purpose of this article is to give an introduction into the principles of chromatic pupillometry and to discuss the potential applications in the management of glaucoma.

Photoreceptor-Specific Temporal Contrast Sensitivities in RP1L1-Associated Occult Macular Dystrophy.

Purpose: The purpose of this study was to compare L-, M-, S-cone-, and rod-driven temporal contrast sensitivities (tCS) in patients with RP1L1-associated autosomal-dominant occult macular dystrophy (OMD), and to investigate how photoreceptor degeneration determines which post-receptoral channels dominate perception. Methods: Photoreceptor isolating stimuli were created with the silent substitution technique. Photoreceptor-selective tCS deviations (D L-cone/M-cone/S-cone/Rod) were obtained as a function of temporal frequency with identical retinal adaptation, by subtracting tCS from age-corrected normal values. A linear-mixed effects model was used for analysis. Results: Eleven genetically confirmed patients were included (7 women, 5 men; age = 52.27 ± 14.44 years). Overall, L- and M-cone-driven sensitivity deviations (DL-cone and DM-cone) were more negative than DS-cone; DRod was normal at frequencies between 8 and 12 Hz in all subjects. Rod-driven tCS functions allowed identification of two subgroups of patients: one with band-pass properties and one with low-pass properties, suggesting dominance of different post-receptoral filters. The same filtering properties were observed in L-cone-driven tCS functions. Furthermore, the two subgroups also differed in clinical parameters (spherical equivalent, BCVA, perimetry, and ocular coherence tomography (OCT) reflectivity of the ellipsoid zone relative to the RPE). Conclusions: OMD was characterized predominantly by deterioration of L- and M-cone-cone driven function in the perifovea. Rod-driven functions were normal. Differences in the photoreceptor signals were further modified by postreceptoral filters.

Modulation of flash ERGs by dynamic backgrounds.

PURPOSE: The aim of this study was to characterize the signal processing mechanisms that lead to an ERG response and to use this characterization for obtaining more robust responses in patients who display feeble responses with standard recordings. We studied the influence of sinusoidally modulating backgrounds on flash ERGs and the relationship between the ERG components' amplitudes and the momentary Weber fraction of the flash stimulus. METHODS: ERG recordings were performed in nine healthy subjects and three RP patients. In four normal subjects, we measured the response to flashes (500 cd/m2, 1 ms duration) on a steady background (50 cd/m2) and on a sine wave (50 cd/m2 mean luminance) modulating background at 1, 5, 10, and 25 Hz temporal frequencies. The flashes were delivered at eight different phases (0-315° in a step of 45°) during the modulating background sine wave. The responses to the backgrounds were also recorded and subtracted from the responses to flash plus modulating backgrounds to obtain the flash ERGs at the different phases. The recordings in the remaining five normal subjects and the RP patients were performed with a subset of these stimuli. RESULTS: The flash ERGs were strongly modulated by the backgrounds particularly at low frequencies and were enhanced when the momentary Weber fraction was large. The amplitudes of the components could be described by the Weber fraction plus a saturating nonlinearity and a delay in the processing of background luminance. The strength of the modulation decreased with increasing peak time of the component. Furthermore the background luminance delay was positively correlated with the peak time. The effect was also present in RP patients. CONCLUSIONS: A sine wave background of about 1 Hz can be used to enhance ERG responses. Weber fraction of the flashes is an adequate quantification of stimulus for describing the amplitudes of the ERGs. The data provide basic information on how background luminance is processed in ERG generating mechanisms. The response enhancement can be used in clinical applications to obtain a more robust comparison between normal and patient data.

Selective Stimulation of the Different Photoreceptor Classes by Silent Substitution in Psychophysical and Electroretinographic Measurements. / Selektive Stimulation der verschiedenen Fotorezeptorklassen mittels Silent Substitution bei psychophysischen und elektroretinografischen Messungen.

The silent substitution technique allows creating photoreceptor-selective stimuli for psychophysical and electrophysiological tests. In contrast to other techniques, the purpose of silent substitution is not to make the targeted photoreceptor type more sensitive in comparison to the other types, but to make the stimulus invisible ("silent") to the other photoreceptor types. This allows selectivity independent of the retinal state of adaptation and enables comparing photoreceptor types under identical conditions. The foundations of these techniques will be explained in this paper. Furthermore, the importance of postreceptoral processing for the perception of photoreceptor-selective stimuli is discussed here. Although this technique is currently only available in specialized vision science labs, there is an enormous potential for clinical application.

Chromatic discrimination measures in mature observers depend on the response window.

Our past anecdotal evidence prompted that a longer response window (RW) in the Trivector test (Cambridge Colour Test) improved mature observers' estimates of chromatic discrimination. Here, we systematically explored whether RW variation affects chromatic discrimination thresholds measured by the length of Protan, Deutan and Tritan vectors. We employed the Trivector test with three RWs: 3 s, 5 s, and 8 s. Data of 30 healthy normal trichromats were stratified as age groups: 'young' (20-29 years), 'middle-aged' (31-48 years), and 'mature' (57-64 years). We found that for the 'young' and 'middle-aged', the thresholds were comparable at all tested RWs. However, the RW effect was apparent for the 'mature' observers: their Protan and Tritan thresholds decreased at 8-s RW compared to 3-s RW; moreover, their Tritan threshold decreased at 5-s RW compared to 3-s RW. Elevated discrimination thresholds at shorter RWs imply that for accurate performance, older observers require longer stimulus exposure and are indicative of ageing effects manifested by an increase in critical processing duration. Acknowledging low numbers in our 'middle-aged' and 'mature' samples, we consider our study as pilot. Nonetheless, our findings encourage us to advocate a RW extension in the Trivector protocol for testing mature observers, to ensure veridical measures of their chromatic discrimination by disentangling these from other ageing effects-slowing down of both motor responses and visual processing.

SRD5A3-CDG: Twins with an intragenic tandem duplication.

Steroid 5α-reductase type 3 congenital disorder of glycosylation (SRD5A3-CDG) is a rare metabolic disease mainly characterized by psychomotor disability, visual impairment, and variable eye malformations caused by bi-allelic pathogenic variants in SRD5A3. So far, only 23 distinct mutations were described. Exome sequencing in 32-year old monozygotic male twins revealed only the heterozygous splice variant c.562+3delG in SRD5A3, but no second variant. The twins presented with psychomotor deficit and a complex eye disease including retinal dystrophy, pallor of the papilla, nystagmus, and strabismus suggestive of SRD5A3-CDG. Only when applying exome-based copy number analysis, we identified as a second compound heterozygous variant a previously not reported tandem duplication of exons 2-4 in SRD5A3. Next to the typical skeletal anomalies of SRD5A3-CDG such as kyphosis and scoliosis, extension deficits of the proximal interphalangeal (PIP) joints IV were observed. Since similar contractures were described once in a patient with SRD5A3-CDG, we suggest that this rare symptom is possibly associated with SRD5A3-CDG. Our findings further expand the mutational and clinical spectrum of SRD5A3-CDG and emphasize the importance of an intragenic copy number analysis in patients with strong clinical suspicion of SRD5A3-CDG and only one detectable sequence variant.

Blue-Yellow VEP with Projector-Stimulation in Glaucoma.

BACKGROUND AND AIM: In the past, increased latencies of the blue-on-yellow pattern visually evoked potentials (BY-VEP), which predominantly originate in the koniocellular pathway, have proven to be a sensitive biomarker for early glaucoma. However, a complex experimental setup based on an optical bench was necessary to obtain these measurements because computer screens lack sufficient temporal, spatial, spectral, and luminance resolution. Here, we evaluated the diagnostic value of a novel setup based on a commercially available video projector. METHODS: BY-VEPs were recorded in 126 participants (42 healthy control participants, 12 patients with ocular hypertension, 17 with "preperimetric" glaucoma, and 55 with perimetric glaucoma). Stimuli were created with a video projector (DLP technology) by rear projection of a blue checkerboard pattern (460 nm) for 200 ms (onset) superimposed on a bright yellow background (574 nm), followed by an offset interval where only the background was active. Thus, predominantly S-cones were stimulated while L- and M-cone responses were suppressed by light adaptation. Times of stimulus onset to VEP onset-trough (N-peak time) and offset-peak (P-peak time) were analyzed after age-correction based on linear regression in the normal participants. RESULTS: The resulting BY-VEPs were quite similar to those obtained in the past with the optical bench: pattern-onset generated a negative deflection of the VEP, whereas the offset-response was dominated by a positive component. N-peak times were significantly increased in glaucoma patients (preperimetric 136.1 ± 10 ms, p < 0.05; perimetric 153.1 ± 17.8 ms, p < 0.001) compared with normal participants (123.6 ± 7.7 ms). Furthermore, they were significantly correlated with disease severity as determined by visual field losses retinal nerve fiber thinning (Spearman R = -0.7, p < 0.001). CONCLUSIONS: Video projectors can be used to create optical stimuli with high temporal and spatial resolution, thus potentially enabling sophisticated electrophysiological measurements in clinical practice. BY-VEPs based on such a projector had a high diagnostic value for detection of early glaucoma. Registration of study Registration site: www. CLINICALTRIALS: gov Trial registration number: NCT00494923.

Perifoveal Cone- and Rod-Mediated Temporal Contrast Sensitivities in Stargardt Disease/Fundus Flavimaculatus.

Purpose: The purpose of this study was to compare L-cone-driven, S-cone-driven, and rod-driven temporal contrast sensitivities (tCSs) in patients with Stargardt disease 1/fundus flavimaculatus (STGD1/FF). Methods: Fourteen patients (eight male, six female; mean age, 43.21 ± 13.18 years) with genetically confirmed STGD1/FF participated in this study. A dedicated light-emitting diode stimulator was used to measure perifoveal tCSs in an annular test field (1°-6° of visual eccentricity) at temporal frequencies between 1 and 20 Hz. Photoreceptor classes were isolated with the triple silent substitution technique. To compare functional damage among photoreceptor classes, sensitivity deviations (decibels) were calculated based on age-related normal values and then averaged across those frequencies where perception is mediated by the same post-receptoral pathway (L-cone red-green opponent pathway: 1, 2, 4 Hz; luminance pathway: 12, 16, 20 Hz; S-cone pathway: 1, 2, 4 Hz; fast rod pathway: 8, 10, 12 Hz). Sensitivity deviations were compared with infrared scanning laser ophthalmoscopy (IR-SLO) and standard automated perimetry (SAP). Results: Photoreceptor-driven tCSs were generally lower in patients with STGD1/FF than in normal subjects but were without systematic differences among photoreceptors. Although sensitivity deviations were significantly correlated between each other, only luminance-driven L-cone sensitivity deviations were significantly correlated with the IR-SLO area of hyporeflectance (AoH) and SAP central mean deviation within 6° eccentricity (MD6deg). Conclusions: No systematic differences between photoreceptor classes were detected; however, our data suggest that temporal contrasts detected by the luminance pathway were closely correlated with other clinical parameters (AoH and MD6deg) and might be most useful as functional biomarkers in clinical trials.

Responses of Postreceptoral Pathways Elicited by L- and M-Cone Isolating ON- and OFF-Electroretinograms in Glaucoma Patients.

Purpose: To compare the electroretinographical (ERG) responses elicited by L- and M-cone isolating ON- and OFF-sawtooth stimuli in normal subjects and glaucoma patients. Methods: Twenty-one normal subjects and 44 primary open-angle glaucoma patients participated in the study. L- and M-cone isolating (18% cone contrast; 284 cd/m2) rapid ON- and rapid OFF-sawtooth (4 Hz) stimuli with two stimulus sizes (full-field (FF) and central 70° diameter) were generated using the triple silent substitution technique. ON- and OFF-response asymmetries were studied by adding the two (to obtain L-add and M-add responses). The initial positive (P) and subsequent late negative (LN) components of the L-add and M-add ERGs were compared between the subject groups and correlated with retinal nerve fiber layer thickness (RNFLT) and pattern ERG responses. Results: The responses to L-ON and to M-OFF stimuli and vice versa resembled each other particularly with 70° stimuli. The PL-add amplitudes were not significantly different between the normal subjects and glaucoma patients, whereas the LNL-add amplitude was significantly (P < 0.01) smaller in the glaucoma patients. Both PM-add and LNM-add were not significantly different between the subject groups. The PERG amplitude with 0.8° check sizes and the 0.8°/16° amplitude ratio (PERG ratio) were significantly (P < 0.05) different between the subject groups. The 70° LNL-add amplitude and the 0.8° PERG amplitude were significantly correlated with RNFLT. Conclusions: The ERGs to 70° cone isolating sawtooth stimuli reflect cone opponency. The cone opponent ERG responses were not significantly different between glaucoma patients and normal subjects. Luminance driven L-add responses were significantly different, indicating that central luminance signals are mainly affected in glaucoma.

Summation of Temporal L-Cone- and M-Cone-Contrast in the Magno- and Parvocellular Retino-Geniculate Systems in Glaucoma.

Purpose: The purpose of this study was to characterize summation of temporal L- and M-cone contrasts in the parvo- (P-) and magnocellular (M-) pathways in glaucoma and the relationship between the respective temporal contrast sensitivities (tCS) and clinical parameters. Methods: Perifoveal tCS to isolated or combined L- and M-cone contrasts (with different contrast ratios, and therefore different luminance and chromatic components) were measured at different temporal frequencies (at 1 or 2 Hz and at 20 Hz) using triple silent substitution in 73 subjects (13 healthy, 25 with glaucoma, and 35 with perimetric glaucoma). A vector summation model was used to analyze whether perception was driven by the P-pathway, the M-pathway, or both. Using this model, L- and M-cone input strengths (AL, AM) and phase differences between L- and M-cone inputs were estimated. Results: Perception was always mediated by the P-pathway at low frequencies, as indicated by a median phase angle of 179.84 degrees (cone opponency) and a median AL/AM ratio of 1.04 (balanced L- and M-cone input strengths). In contrast, perception was exclusively mediated by the M-pathway at higher frequencies (input strength not balanced: AL/AM = 2.94, median phase angles = 130.17 degrees). Differences in phase were not significant between diagnosis groups (Kruskal-Wallis = 0.092 for P- and 0.35 for M-pathway). We found differences between groups only for the M-pathway (L-cone tCS deviations at 20 Hz were significantly lower in the patients with glaucoma P = 0.014, with a strong tendency in M-cones P = 0.049). L-cone driven tCS deviations at 20 Hz were linearly correlated with perimetric mean defect (MD) and quadratically correlated with retinal nerve fiber layer (RNFL) thickness. Conclusions: Unaltered phase angles between L- and M-cone inputs in glaucoma indicated intact temporal processing. Only in the M-pathway, contrast sensitivity deviations were closely related to diagnosis group, MD, and RNFL thickness, indicating M-pathway involvement.

The influence of temporal frequency and stimulus size on the relative contribution of luminance and L-/M-cone opponent mechanisms in heterochromatic flicker ERGs.

PURPOSE: To study the effect of stimulus size and temporal frequency on the relative contribution of luminance and L-/M-cone opponent signals in the ERG. METHODS: In four healthy, color normal subjects, ERG responses to heterochromatic stimuli with sinusoidal, counter-phase modulation of red and green LEDs were measured. By inverse variation of red and green contrasts, we varied luminance contrast while keeping L-/M-cone opponent chromatic contrast constant. The first harmonic components in the full field ERGs are independent of stimulus contrast at 12 Hz, while responses to 36 Hz stimuli vary, reaching a minimum close to isoluminance. It was assumed that ERG responses reflect L-/M-cone opponency at 12 Hz and luminance at 36 Hz. In this study, we modeled the influence of temporal frequency on the relative contribution of these mechanisms at intermediate frequencies, measured the influence of stimulus size on model parameters, and analyzed the second harmonic component at 12 Hz. RESULTS: The responses at all frequencies and stimulus sizes could be described by a linear vector addition of luminance and L-/M-cone opponent reflecting ERGs. The contribution of the luminance mechanism increased with increasing temporal frequency and with increasing stimulus size, whereas the gain of the L-/M-cone opponent mechanism was independent of stimulus size and was larger at lower temporal frequencies. Thus, the luminance mechanism dominated at lower temporal frequencies with large stimuli. At 12 Hz, the second harmonic component reflected the luminance mechanism. CONCLUSIONS: The ERGs to heterochromatic stimuli can be fully described in terms of linear combinations of responses in the (magnocellular) luminance and the (parvocellular) L-/M-opponent retino-geniculate pathways. The non-invasive study of these pathways in human subjects may have implications for basic research and for clinical research.

[New techniques for quantification of color vision in disorders of cone function : Cambridge color test and photoreceptor-specific temporal contrast sensitivity in patients with heterozygous RP1L1 and RPGR mutations]. / Neue Techniken zur Quantifizierung des Farbsinns bei Störungen der Zapfenfunktion : Cambridge-Color-Test und photorezeptorspezifische zeitliche Kontrastempfindlichkeit bei Patient/Innen mit heterozygoten RP1L1- und RPGR-Mutationen.

BACKGROUND: Inherited retinal diseases with cone dysfunction can be accompanied by severe visual loss and a marked loss of color vision despite relatively normal fundus appearance. Autosomal dominant occult macular dystrophy (RP1L1 gene) and X­chromosomal retinitis pigmentosa (RPGR gene, including heterozygous female carriers) are important examples. New examination techniques enable quantification of the extent of color vision disturbances. METHODS: After a thorough clinical examination, color discrimination and cone function were quantified. The Cambridge color test is a computer-based test that generates pseudo-isochromatic plates with Landolt C figures for quantifying color discrimination along several axes in color space. Examination of photorecepor-specific temporal contrast sensitivity is performed by subtle cyclic modulation of the spectral composition of a light stimulus. Molecular diagnostics were carried out by next generation sequencing (NGS)-based targeted gene panel analysis and Sanger sequencing. RESULTS: Markedly reduced color discrimination as well as reduced photoreceptor-specific temporal contrast sensitivity could be demonstrated in two patients with occult macular dystrophy and two heterozygous female carriers of RPGR mutations. CONCLUSION: The demonstration of dyschromatopsia is very helpful in the diagnosis of inherited retinal diseases, in addition to modern imaging techniques, such as optical coherence tomography (OCT) and fundus fluorescence. New functional techniques enable quantification of color vision disturbances and could be useful as outcome parameters in clinical trials of new gene and stem cell-based therapies.

Photoreceptor-Specific Loss of Perifoveal Temporal Contrast Sensitivity in Retinitis Pigmentosa.

Purpose: Inherited retinal diseases affect the L-, M-, S-cones and rods in distinct ways, which calls for new methods that enable quantification of photoreceptor-specific functions. We tested the feasibility of using the silent substitution paradigm to estimate photoreceptor-driven temporal contrast sensitivity (tCS) functions in patients with retinitis pigmentosa. Methods: The silent substitution paradigm is based on substitution of lights of different spectral composition; this offers considerable advantage over other stimulation techniques. We used a four-primary LED stimulator to create perifoveal annular stimuli (2° inner, 12° outer diameters) and used a triple silent substitution to probe photoreceptor-selective tCS. Measurements were performed in a heterogeneous cohort of 15 patients with retinitis pigmentosa and related to those in a control group of nine color-normal healthy observers. Age differences between groups were addressed with a model of age-related normal contrast sensitivity derived from measurements in 20 healthy observers aged between 23 and 83 years. Results: The age-related loss of tCS amounted to 0.1 dB/year in healthy subjects across all photoreceptor subtypes. In patients, tCS was decreased for every photoreceptor subtype; however, S-cone- and rod-driven sensitivities were most strongly affected. Postreceptoral mechanisms were not affected. Conclusions: This feasibility study provides evidence that the silent substitution technique enables the estimation of photoreceptor-selective tCS functions and can serve as an accurate biomarker of photoreceptor-specific contrast sensitivity loss in patients with retinitis pigmentosa. Translational Relevance: We aim to develop tests of visual function for clinical trials of novel therapies for inherited retinal diseases from methods that can currently be used only in vision research labs.

Novel truncating mutation in CACNA1F in a young male patient diagnosed with optic atrophy.

BACKGROUND: Low vision in children can be accompanied by pallor of the optic disc with little or no characteristic morphologic changes of the retina. A variety of diseases can be the underlying cause, including hereditary optic atrophy, Leber's congenital amaurosis (LCA), achromatopsia, and calcium channel, voltage-dependent, L-type, alpha-1F subunit gene (CACNA1F)-associated retinopathy (most widely known as incomplete congenital stationary night blindness: iCSNB). Differentiation at early age is desirable due to large differences in prognosis, but may be difficult because phenotypes overlap and electrophysiological testing is challenging in young patients. We present the case of a 6-year-old boy with unexplained low vision and pallor of the optic disc who originally had been diagnosed with hereditary optic atrophy in the absence of recordable full-field electroretinography (ERG) due to poor patient cooperation. MATERIALS AND METHODS: Standard Sanger sequencing excluded mutations in the OPA1 gene (autosomal-dominant optic atrophy). To identify the underlying genetic cause, whole-exome sequencing was performed on patient's DNA. Recording of the full-field ERG was successfully performed 6 months later. RESULTS: We identified a novel truncating mutation in CACNA1F gene (NM_001256789: c.3895C > T in exon 33) which led to the correct diagnosis of CACNA1F-associated retinopathy in the young boy. ERG recordings showed a negative scotopic mixed response with preserved oscillatory potentials and a flicker ERG with reduced amplitude and biphasic waveform, compatible with a CACNA1F-asssociated phenotype. CONCLUSIONS: We show that genetic testing may help to differentiate between optic atrophy, LCA, and CACNA1F-associated retinopathy at a much earlier age, in absence of electrophysiological examination and by widely overlapping phenotypes.

Photoreceptor-specific light adaptation of critical flicker frequency in trichromat and dichromat observers.

The silent substitution paradigm offers possibilities to investigate and compare the temporal properties of mechanisms driven by single photoreceptor types, including the critical flicker frequency (CFF), in which the state of adaptation can be kept as invariant. We have (1) measured CFFs using triple silent substitutions to isolate L-, M-, and S-cone as well as rod-driven pathways under identical mean luminances and chromaticities; (2) repeated the CFF measurements at different mean luminances in order to validate the Ferry-Porter law (stating that the relationship between CFF and the log retinal illuminance-log I-is linear); and (3) compared these CFF versus log I functions for L-, M-, S-cone-, and rod-isolating stimuli for five trichromats and four X-linked dichromats (two protanopes, two deuteranopes). We show that the effects of luminance on the CFFs with silent substitution are comparable to those measured previously with chromatic stimuli. We found that M-cone-driven CFFs are smaller in trichromats than in protanopes. Furthermore, the slopes of the M-cone-driven CFF versus log I functions are smaller in trichromats. Possibly, the lacking L-cones are replaced by M-cones in these two protanopes and the CFF depends on cone density. Furthermore, we found that in trichromats, the slopes of the CFF-log I functions are smaller for M-cone- than for L-cone-isolating stimuli. This contradicts the current interpretation of the CFF-log I functions for chromatic stimuli, which states that CFF is mediated by the most strongly modulated photoreceptor type. Thus, the larger slopes that were previously found with medium-wavelength chromatic stimuli compared with long-wavelength chromatic stimuli seem to be the result of an addition of signals from different photoreceptors and do not necessarily result from M-cones being inherently faster.

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.