Chromatic visual evoked potentials: A review of physiology, methods and clinical applications.

Objective assessment of the visual system can be performed electrophysiologically using the visual evoked potential (VEP). In many clinical circumstances, this is performed using high contrast achromatic patterns or diffuse flash stimuli. These methods are clinically valuable but they may only assess a subset of possible physiological circuitries within the visual system, particularly those involved in achromatic (luminance) processing. The use of chromatic VEPs (cVEPs) in addition to standard VEPs can inform us of the function or dysfunction of chromatic pathways. The chromatic VEP has been well studied in human health and disease. Yet, to date our knowledge of their underlying mechanisms and applications remains limited. This likely reflects a heterogeneity in the methodology, analysis and conclusions of different works, which leads to ambiguity in their clinical use. This review sought to identify the primary methodologies employed for recording cVEPs. Furthermore cVEP maturation and application in understanding the function of the chromatic system under healthy and diseased conditions are reviewed. We first briefly describe the physiology of normal colour vision, before describing the methodologies and historical developments which have led to our understanding of cVEPs. We thereafter describe the expected maturation of the cVEP, followed by reviewing their application in several disorders: congenital colour vision deficiencies, retinal disease, glaucoma, optic nerve and neurological disorders, diabetes, amblyopia and dyslexia. We finalise the review with recommendations for testing and future directions.

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.

Pre-stimulus bioelectrical activity in light-adapted ERG under blue versus white background.

To compare the baseline signal between two conditions used to generate the photopic negative response (PhNR) of the full-field electroretinogram (ERG): red flash on a blue background (RoB) and white flash on a white background (LA3). The secondary purpose is to identify how the level of pre-stimulus signal affects obtaining an unambiguous PhNR component. A retrospective chart review was conducted on four cohorts of patients undergoing routine ERG testing. In each group, LA3 was recorded the same way while RoB was generated differently using various luminances of red and blue light. The background bioelectrical activity 30 ms before the flash was extracted, and the root mean square (RMS) of the signal was calculated and compared between RoB and LA3 using Wilcoxon test. Pre-stimulus noise was significantly higher under RoB stimulation versus LA3 in all four conditions for both right and left eyes (ratio RoB/LA3 RMS 1.70 and 1.57 respectively, p < 0.033). There was also no significant difference between the RMS of either LA3 or RoB across protocols, indicating that the baseline noise across cohorts were comparable. Additionally, pre-stimulus noise was higher in signals where PhNR was not clearly identifiable as an ERG component versus signals with the presence of unambiguous PhNR component under RoB in all four groups for both eyes (p < 0.05), whereas the difference under LA3 was less pronounced. Our study suggests that LA3 produces less background bioelectrical activity, likely due to decreased facial muscle activity. As it seems that the pre-stimulus signal level affects PhNR recordability, LA3 may also produce a better-quality signal compared to RoB. Therefore, until conditions for a comparable bioelectrical activity under RoB are established, we believe that LA3 should be considered at least as a supplementary method to evaluate retinal ganglion cell function by ERG.

Single opsin driven white noise ERGs in mice.

Purpose: Electroretinograms elicited by photopigment isolating white noise stimuli (wnERGs) in mice were measured. The dependency of rod- and cone-opsin-driven wnERGs on mean luminance was studied. Methods: Temporal white noise stimuli (containing all frequencies up to 20 Hz, equal amplitudes, random phases) that modulated either rhodopsin, S-opsin or L*-opsin, using the double silent substitution technique, were used to record wnERGs in mice expressing a human L*-opsin instead of the native murine M-opsin. Responses were recorded at 4 mean luminances (MLs).Impulse response functions (IRFs) were obtained by cross-correlating the wnERG recordings with the corresponding modulation of the photopigment excitation elicited by the stimulus. So-called modulation transfer functions (MTFs) were obtained by performing a Fourier transform on the IRFs.Potentials of two repeated wnERG recordings at corresponding time points were plotted against each other. The correlation coefficient (r2repr) of the linear regression through these data was used to quantify reproducibility. Another correlation coefficient (r2ML) was used to quantify the correlations of the wnERGs obtained at different MLs with those at the highest (for cone isolating stimuli) or lowest (for rod isolating stimuli) ML. Results: IRFs showed an initial negative (a-wave like) trough N1 and a subsequent positive (b-wave like) peak P1. No oscillatory potential-like components were observed. At 0.4 and 1.0 log cd/m2 ML robust L*- and S-opsin-driven IRFs were obtained that displayed similar latencies and dependencies on ML. L*-opsin-driven IRFs were 2.5-3 times larger than S-opsin-driven IRFs. Rhodopsin-driven IRFs were observed at -0.8 and - 0.2 log cd/m2 and decreased in amplitude with increasing ML. They displayed an additional pronounced late negativity (N2), which may be a correlate of retinal ganglion cell activity.R2repr and r2ML values increased for cones with increasing ML whereas they decreased for rods. For rhodopsin-driven MTFs at low MLs and L*-opsin-driven MTFs at high MLs amplitudes decreased with increasing frequency, with much faster decreasing amplitudes for rhodopsin. A delay was calculated from MTF phases showing larger delays for rhodopsin- vs. low delays for L*-opsin-driven responses. Conclusion: Opsin-isolating wnERGs in mice show characteristics of different retinal cell types and their connected pathways.

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.

Retinal dystrophins and the retinopathy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by X-linked inherited or de novo DMD gene mutations predominantly affecting males who develop early-onset muscle degeneration, severely affecting their quality of life and leading to reduced life expectancy. DMD patients may also develop proliferative retinopathy, cataract, ERG abnormalities, altered contrast sensitivity, color vision losses, and elevated flash detection thresholds during dark adaptation. Depending on the position of the genetic alteration in the large DMD gene, it is associated with a lack of the full-length dystrophin protein possibly with an additional loss of one or several other dystrophins, which are normally transcribed from internal promoters in retina and crystalline lens. During the last decades, the properties of the dystrophins have been characterized in patients with different genetic alterations and in genetic mouse models of DMD. The complex expression pattern of the dystrophins in photoreceptors, Müller glial cells and astrocytes, likely influences synaptic transmission, ionic balance and vascular integrity of the retina. However, the specific function of each retinal dystrophin remains largely unknown. This review describes the current knowledge on dystrophin expression, the putative molecular, structural, and physiological properties of retinal dystrophins, and the main clinical implications associated with the loss of dystrophins in DMD patients and mouse models. Current data and working hypotheses warrant future research on retinal dystrophins to increase our understanding of dystrophin function in the central nervous system in general and to unveil new retinal mechanisms and therapeutic avenues for retinal diseases.

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.

Luminance white noise electroretinograms (wnERGs) in mice.

Purpose: To record and analyse electroretinograms (ERGs) to luminance stimuli with white noise temporal profiles in mice. White noise stimuli are expected to keep the retina in a physiologically more natural state than, e.g., flashes. The influence of mean luminance (ML) was studied. Methods: Electroretinograms to luminance temporal white noise (TWN) modulation (wnERGs) were measured. The white noise stimuli contained all frequencies up to 20 Hz with equal amplitudes and random phases. Responses were recorded at 7 MLs between -0.7 and 1.2 log cd/m2. Impulse response functions (IRFs) were calculated by cross correlating the averaged white noise electroretinogram (wnERG) responses with the stimulus. Amplitudes and latencies of the initial trough and subsequent peak in the IRFs were measured at each ML. Fourier transforms of the IRFs resulted in modulation transfer functions (MTFs). wnERGs were averaged across different animals. They were measured twice and the responses at identical instances in the 1st and 2nd recordings were plotted against each other. The correlation coefficient (r 2 repr) of the linear regression quantified the reproducibility. The results of the first and second measurement were further averaged. To study the underlying ERG mechanisms, the ERG potentials at the different MLs were plotted against those at the lowest and highest ML. The correlation coefficients (r 2 ML) were used to quantify their similarities. Results: The amplitudes of the initial (a-wave-like) trough of the IRFs increased with increasing ML. The following positive (b-wave-like) peak showed a minimum at -0.4 log cd/m2 above which there was a positive correlation between amplitude and ML. Their latencies decreased monotonously with increasing ML. In none of the IRFs, oscillatory potential (OP)-like components were observed. r 2 repr values were minimal at a ML of -0.1 log cd/m2, where the MTFs changed from low-pass to band-pass. r 2 ML values increased and decreased with increasing ML when correlated with responses obtained at the highest or the lowest ML, respectively. Conclusion: White noise electroretinograms can be reliably recorded in mice with luminance stimuli. IRFs resemble flash ERGs superficially, but they offer a novel procedure to study retinal physiology. New components can be described in the IRFs. The wnERGs are either rod- or cone-driven with little overlap.

Electroretinographic responses to luminance and cone-isolating white noise stimuli in macaques.

Electroretinograms (ERGs) are mass potentials with a retinal origin that can be measured non-invasively. They can provide information about the physiology of the retina. Often, ERGs are measured to flashes that are highly unnatural stimuli. To obtain more information about the physiology of the retina, we measured ERGs with temporal white noise (TWN) stimuli that are more natural and keep the retina in a normal range of operation. The stimuli can be combined with the silent substitution stimulation technique with which the responses of single photoreceptor types can be isolated. We characterized electroretinogram (ERG) responses driven by luminance activity or by the L- or the M-cones. The ERGs were measured from five anesthetized macaques (two females) to luminance, to L-cone isolating and to M-cone isolating stimuli in which luminance or cone excitation were modulated with a TWN profile. The responses from different recordings were correlated with each other to study reproducibility and inter-individual variability. Impulse response functions (IRFs) were derived by cross-correlating the response with the stimulus. Modulation transfer functions (MTFs) were the IRFs in the frequency domain. The responses to luminance and L-cone isolating stimuli showed the largest reproducibility. The M-cone driven responses showed the smallest inter-individual variability. The IRFs and MTFs showed early (high frequency) components that were dominated by L-cone driven signals. A late component was equally driven by L- and M-cone activity. The IRFs showed characteristic similarities and differences relative to flash ERGs. The responses to TWN stimuli can be used to characterize the involvement of retinal cells and pathways to the ERG response. It can also be used to identify linear and non-linear processes.

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.

The Association Between Acquired Color Deficiency and PET Imaging of Neurodegeneration in Mild Cognitive Impairment and Alzheimer Disease.

Purpose: To evaluate color vision changes and retinal processing of chromatic and luminance pathways in subjects with Alzheimer disease (AD) and mild cognitive impairment (MCI) compared with a matched control group and whether such changes are associated with impaired brain glucose metabolism and ß-amyloid deposition in the brain. Methods: We evaluated 13 patients with AD (72.4 ± 7.7 years), 23 patients with MCI (72.5 ± 5.5 years), and 18 controls of comparable age (P = 0.44) using Cambridge color test and the heterochromatic flicker ERG (HF-ERG). The Cambridge color test was performed using the trivector protocol to estimate the protan, deutan and tritan color confusion axes. HF-ERG responses were measured at a frequency of 12 Hz, which ERGs reflect chromatic activity, and at 36 Hz, reflecting luminance pathway. A study subsample was performed using neuropsychological assessments and positron emission tomography. Results: Patients with AD presented higher mean values indicating poorer color discrimination for protan (P = 0.04) and deutan (P = 0.001) axes compared with the controls. Along the tritan axis, both patients with AD and patients with MCI showed decreased color vision (P = 0.001 and P = 0.001) compared with controls. The analyses from the HF-ERG protocol revealed no differences between the groups (P = 0.31 and P = 0.41). Diffuse color vision loss was found in individuals with signs of neurodegeneration (protan P = 0.002, deutan P = 0.003 and tritan P = 0.01), but not in individuals with signs of ß-amyloid deposition only (protan P = 0.39, deutan P = 0.48, tritan P = 0.63), regardless of their clinical classification. Conclusions: Here, patients with AD and patients with MCI present acquired color vision deficiency that may be linked with impaired brain metabolism.

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.

Cell Types and Synapses Expressing the SNARE Complex Regulating Proteins Complexin 1 and Complexin 2 in Mammalian Retina.

Complexins (Cplxs) 1 to 4 are components of the presynaptic compartment of chemical synapses where they regulate important steps in synaptic vesicle exocytosis. In the retina, all four Cplxs are present, and while we know a lot about Cplxs 3 and 4, little is known about Cplxs 1 and 2. Here, we performed in situ hybridization experiments and bioinformatics and exploited Cplx 1 and Cplx 2 single-knockout mice combined with immunocytochemistry and light microscopy to characterize in detail the cell type and synapse-specific distribution of Cplx 1 and Cplx 2. We found that Cplx 2 and not Cplx 1 is the main isoform expressed in normal and displaced amacrine cells and ganglion cells in mouse retinae and that amacrine cells seem to operate with a single Cplx isoform at their conventional chemical synapses. Surprising was the finding that retinal function, determined with electroretinographic recordings, was altered in Cplx 1 but not Cplx 2 single-knockout mice. In summary, the results provide an important basis for future studies on the function of Cplxs 1 and 2 in the processing of visual signals in the mammalian retina.

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.