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.

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.

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.

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.

Genetic disruption of bassoon in two mutant mouse lines causes divergent retinal phenotypes.

Bassoon (BSN) is a presynaptic cytomatrix protein ubiquitously present at chemical synapses of the central nervous system, where it regulates synaptic vesicle replenishment and organizes voltage-gated Ca2+ channels. In sensory photoreceptor synapses, BSN additionally plays a decisive role in anchoring the synaptic ribbon, a presynaptic organelle and functional extension of the active zone, to the presynaptic membrane. In this study, we functionally and structurally analyzed two mutant mouse lines with a genetic disruption of Bsn-Bsngt and Bsnko -using electrophysiology and high-resolution microscopy. In both Bsn mutant mouse lines, full-length BSN was abolished, and photoreceptor synaptic function was similarly impaired, yet synapse structure was more severely affected in Bsngt/gt than in Bsnko/ko photoreceptors. The synaptic defects in Bsngt/gt retina coincide with remodeling of the outer retina-rod bipolar and horizontal cell sprouting, formation of ectopic ribbon synaptic sites-and death of cone photoreceptors, processes that did not occur in Bsnko/ko retina. An analysis of Bsngt/ko hybrid mice revealed that the divergent retinal phenotypes of Bsngt/gt and Bsnko/ko mice can be attributed to the expression of the Bsngt allele, which triggers cone photoreceptor death and neurite sprouting in the outer retina. These findings shed new light on the existing Bsn mutant mouse models and might help to understand mechanisms that drive photoreceptor death.

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.

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.

Altered visual processing in the mdx52 mouse model of Duchenne muscular dystrophy.

The mdx52 mouse model of Duchenne muscular dystrophy (DMD) is lacking exon 52 of the DMD gene that is located in a hotspot mutation region causing cognitive deficits and retinal anomalies in DMD patients. This deletion leads to the loss of the dystrophin proteins, Dp427, Dp260 and Dp140, while Dp71 is preserved. The flash electroretinogram (ERG) in mdx52 mice was previously characterized by delayed dark-adapted b-waves. A detailed description of functional ERG changes and visual performances in mdx52 mice is, however, lacking. Here an extensive full-field ERG repertoire was applied in mdx52 mice and WT littermates to analyze retinal physiology in scotopic, mesopic and photopic conditions in response to flash, sawtooth and/or sinusoidal stimuli. Behavioral contrast sensitivity was assessed using quantitative optomotor response (OMR) to sinusoidally modulated luminance gratings at 100% or 50% contrast. The mdx52 mice exhibited reduced amplitudes and delayed implicit times in dark-adapted ERG flash responses, particularly in their b-wave and oscillatory potentials, and diminished amplitudes of light-adapted flash ERGs. ERG responses to sawtooth stimuli were also diminished and delayed for both mesopic and photopic conditions in mdx52 mice and the first harmonic amplitudes to photopic sine-wave stimuli were smaller at all temporal frequencies. OMR indices were comparable between genotypes at 100% contrast but significantly reduced in mdx52 mice at 50% contrast. The complex ERG alterations and disturbed contrast vision in mdx52 mice include features observed in DMD patients and suggest altered photoreceptor-to-bipolar cell transmission possibly affecting contrast sensitivity. The mdx52 mouse is a relevant model to appraise the roles of retinal dystrophins and for preclinical studies related to DMD.

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.

The spatial distribution of ERGs reflecting luminance and L-/M-cone-opponent signals.

PURPOSE: To study the spatial retinal distribution of electroretinographic (ERG) responses that reflect signals in the L-/M-cone-opponent and luminance post-receptoral pathways. METHODS: ERG recordings to heterochromatic stimuli (sinusoidal counter-phase modulation of red and green LED light sources) were performed, while varying fractions of red and green modulation. Two temporal frequencies of the stimuli were employed: 12 Hz to record ERGs that reflect L-/M-cone-opponent signal and 36 Hz for recording ERG signals sensitive to stimulus luminance. Stimuli were about 20° in diameter and projected on various retinal locations: the fovea and four eccentricities (10°, 19°, 28° and 35°), each presented nasally, temporally, inferiorly and superiorly from the fovea. RESULTS: The 36 Hz stimuli elicited responses that strongly varied with red fraction and were minimal at iso-luminance. Moreover, response phases changed abruptly at the minimum by 180°. In contrast, the responses to the 12 Hz stimuli had amplitudes and phases that changed more gradually with red fraction. The 36 Hz response amplitudes were maximal close to the fovea and sharply decreased with increasing distance from the fovea. The responses to 12 Hz stimuli were more broadly distributed across the retina. CONCLUSIONS: In the present study, it was found that retinal eccentricity and direction from the fovea have distinct effects on ERGs reflecting different post-receptoral mechanisms. The results are in accord with previous findings that ERGs to 12 Hz stimuli are predominantly determined by the red-green chromatic content of the stimuli, thus reflecting activation in the L-/M-cone-opponent pathway, while responses to 36 Hz stimuli manifest post-receptoral luminance-dependent activation. We found that the response in the cone-opponent pathway is broadly comparable across the retina; in comparison, response amplitude of the luminance pathway strongly depends on retinal stimulus position.

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.

Relationship between stimulus size and different components of the electroretinogram (ERG) elicited by flashed stimuli.

PURPOSE: To investigate how light stimulus conditions of varying spatial sizes affect components of the flash and long-flash electroretinogram (ERG) in normal subjects. METHOD: Three stimulus conditions were generated by a Ganzfeld stimulator: a white flash on white background (WoW), a red flash on a blue background (RoB) and an L+M-cone isolating on-off (long flash) stimulus (Cone Iso). ERGs were recorded from six subjects (5 M, 1 F) with DTL electrodes to full-field (FF), 70°, 60°, 50°, 40°, 30° and 20° diameter circular stimuli. Amplitudes and peak times for a-, b-, d- and i-wave, and PhNR were examined. PhNR amplitudes were estimated in two different ways: from baseline (fB) and from preceding b-wave peak (fP). RESULTS: With decreasing stimulus size, amplitudes for all ERG waveform components attenuated and peak times increased, although the effect varied across different components. An exponential fit described the relationship between amplitudes and size of stimulated retinal area well for most components and conditions (R2= 0.75-0.99), except for PhNR(fB) (R2= - 0.16-0.88). For peak times, an exponential decay function also fitted the data well (R2= 0.81-0.97), except in a few cases where the exponential constant was too small and a linear regression function was applied instead (a-wave Cone Iso, b- and i-wave WoW). The exponential constants for RoB amplitudes (b-wave, PhNR(fB), PhNR(fP)) were larger compared to their counterparts under WoW (p < 0.05), while there was no difference between the constants for a-wave amplitudes and peak times and for PhNR peak times. The exponential constants of amplitudes vs. area under WoW and Cone Iso were remarkably similar, while under RoB PhNR(fB) showed larger constants compared to either a- or b-wave (p < 0.05). CONCLUSION: ERG components change in a predictable way with stimulus size and spectral characteristics of the stimulus under these conditions. This predictability could allow a modified version of these sets of stimuli to be tested for clinical applicability.

Pseudorandom full-field electroretinograms reflect different light adaptation mechanisms.

PURPOSE: To investigate the magnitude and time course of pseudorandom ffERG during light adaptation. METHODS: Ten healthy subjects (26 ± 10.1 years) underwent 20 min of dark adaptation, and then the ffERG was evoked by pseudorandom flash sequences (4 ms per flash, 3 cd.s/m2) driven by m-sequences (210-1 stimulus steps) using Veris Science software and a Ganzfeld dome over a constant field of light adaptation (30 cd/m2). The base period of the m-sequence was 50 ms. Each stimulation sequence lasting 40 s was repeated at 0, 5, 10, 15 and 20 min of light adaptation. Relative amplitude and latency (corrected by values found at 0 min) of the three components (N1, P1, and N2) of first-order (K1) and first slice of the second-order (K2.1) kernel at 5 time points were evaluated. An exponential model was fitted to the mean amplitude and latency data as a function of the light adaptation duration to estimate the time course (τ) of the light adaptation for each component. Repeated one-way ANOVA followed by Tukey post-test was applied to the amplitude and latency data, considering significant values of p < 0.05. RESULTS: Regarding the K1 ffERG, N1 K1, P1 K1, and N2 K1 presented an amplitude increase as a function of the light adaptation (N1 K1 τ value = 2.66 min ± 4.2; P1 K1 τ value = 2.69 min ± 2.10; and N2 K1 τ value = 3.49 min ± 2.96). P1 K1 and N2 K1 implicit time changed as a function of the light adaptation duration (P1 K1 τ value = 3.61 min ± 5.2; N2 K1 τ value = 3.25 min ± 4.8). N1 K1 had small implicit time changes during the light adaptation. All the K2,1 components also had nonsignificant changes in amplitude and implicit time during the light adaptation. CONCLUSIONS: Pseudorandom ffERGs showed different mechanisms of adaptation to retinal light. Our results suggest that K1 ffERG is generated by retinal mechanisms with intermediate- to long-term light adaptation, while K2.1 ffERG is generated by retinal mechanism with fast light adaptation course.

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.

A Multiple Piccolino-RIBEYE Interaction Supports Plate-Shaped Synaptic Ribbons in Retinal Neurons.

Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.

The photopic negative response of the Light-adapted 3.0 ERG in clinical settings.

PURPOSE: To analyze the effects of different methods of measurement on the photopic negative response (PhNR), recorded as part of a standard ISCEV Photopic 3.0 ERG responses from patients with a variety of clinical diagnoses. METHODS: ERGs were recorded from both eyes of 97 patients (187 eyes) as part of a standard clinical assessment. The average age was 56.4 ± 15.7 years, the gender balance was 35 M, 62F, and only recordable responses of acceptable quality were included. PhNR was measured at an identifiable trough before (PhNR1) and after the i-wave (PhNR2), and the amplitudes and peak times were compared with a-, b- and i-wave corresponding parameters. PhNR components were measured: from baseline and from b-wave peak. Correlation between PhNR troughs and with ERG parameters were tested for right eyes. The possibility to predict and substitute PhNR2 amplitude from PhNR1 amplitude was also tested. RESULTS: PhNR1 was recordable in 97.3% of eyes and PhNR2 in 85.6%. An identifiable PhNR2 peak was found to occur before 65 ms at ~ 50% of the records, while in ~ 38% of the cases was within 65-75 ms in ~ 12%-after 75 ms. The correlation between the PhNR1 and PhNR2 peaks was quite strong (with coefficients 0.81-0.98, depending on method of measurement, and slopes close to 1). The average difference between predicted and measured PhNR2 was reasonably small in absolute (< 2 µV) and relative (< 2%) terms. The correlations between PhNR amplitudes and other ERG component amplitudes showed different ranges of correlation coefficients depending on the method of measurement: for the a-wave amplitudes the range of coefficients was 0.48-0.73, while for the b-wave amplitudes it was 0.30-0.95 and 0.39-0.65 for i-wave. CONCLUSION: The strong correlation between the two PhNR troughs could allow using PhNR1 when PhNR2 is poorly defined due to artifacts. Different methods of PhNR measurement lead to different correlations with other ERG parameters, and this needs to be considered when analyzing and comparing PhNR data between studies.

Heterogeneous Presynaptic Distribution of Munc13 Isoforms at Retinal Synapses and Identification of an Unconventional Bipolar Cell Type with Dual Expression of Munc13 Isoforms: A Study Using Munc13-EXFP Knock-in Mice.

Munc13 isoforms are constituents of the presynaptic compartment of chemical synapses, where they govern important steps in preparing synaptic vesicles for exocytosis. The role of Munc13-1, -2 and -3 is well documented in brain neurons, but less is known about their function and distribution among the neurons of the retina and their conventional and ribbon-type chemical synapses. Here, we examined the retinae of Munc13-1-, -2-, and -3-EXFP knock-in (KI) mice with a combination of immunocytochemistry, physiology, and electron microscopy. We show that knock-in of Munc13-EXFP fusion proteins did not affect overall retinal anatomy or synapse structure, but slightly affected synaptic transmission. By labeling Munc13-EXFP KI retinae with specific antibodies against Munc13-1, -2 and -3, we found that unlike in the brain, most retinal synapses seem to operate with a single Munc13 isoform. A surprising exception to this rule was type 6 ON bipolar cells, which expressed two Munc13 isoforms in their synaptic terminals, ubMunc13-2 and Munc13-3. The results of this study provide an important basis for future studies on the contribution of Munc13 isoforms in visual signal processing in the mammalian retina.

In vivo electroretinographic differentiation of rod, short-wavelength and long/medium-wavelength cone responses in dogs using silent substitution stimuli.

The canine species has dichromatic color vision comprising short-wavelength (S-) and long/medium (L/M-) wavelength-sensitive cones with peak spectral sensitivity of 429-435 nm and 555 nm respectively. Although differentiation of rod- and cone-mediated responses by electroretinogram (ERG) in dogs is commonly performed, and standards have been developed based on standards for human observers, methods to differentiate S- and L/M-cone responses in dogs have not been described. We developed flicker protocols derived from previously published rod and cone spectral sensitivities. We used a double silent substitution paradigm to isolate responses from each of the 3 photoreceptor subclasses. ERG responses were measured to sine-wave modulation of photoreceptor excitation at different temporal frequencies (between 4 and 56 Hz) and mean luminance (between 3.25 and 130 cd/m2) on 6 different normal dogs (3 adult female, and 3 adult male beagles) and one female beagle dog with suspected hereditary congenital stationary night blindness (CSNB). Peak rod driven response amplitudes were achieved with low frequency (4 Hz, maximal range 4-12 Hz) and low mean luminance (3.25 cd/m2). In contrast, peak L/M-cone driven response amplitudes were achieved with high frequency (32 Hz, maximal range 28-44 Hz) and high mean luminance (32.5-130 cd/m2). Maximal S-cone driven responses were obtained with low frequency stimuli (4 Hz, maximal range 4-12 Hz) and 32.5-130 cd/m2 mean luminance. The dog with CSNB had reduced rod- and S-cone-driven responses, but normal/supernormal L/M cone-driven responses. We have developed methods to differentiate rod, S- and L/M-cone function in dogs using silent substitution methods. The influence of temporal frequency and mean luminance on the ERGs originating in each photoreceptor type can now be studied independently. Dogs and humans have similar L/M cone responses, whereas mice have significantly different L/M responses. This work will facilitate a greater understanding of canine retinal electrophysiology and will complement the study of canine models of human hereditary photoreceptor disorders.

Pathway-specific light adaptation in human electroretinograms.

The cellular origins of slow ERG changes during light adaptation following a dark-adapted state are still unclear. To study light adaptation, six healthy, normal trichromats were dark-adapted for 30 min prior to full-field ERG recordings to sinusoidal stimuli that isolate responses of the L- or M-cones or that stimulate luminance and chromatic mechanisms at 12 or 36 Hz. Recordings were performed for 16 min with 2-min intervals after onset of a constant background. Generally, the responses were sine-wave-like, and the first harmonic (fundamental) component dominated the Fourier spectrum except for the 12-Hz luminance stimulus in which two components, a sine-wave-like component and a transient component, determined the response profiles, leading to large second harmonic components. The amplitude of the first harmonic component (F) increased as a function of the light-adaptation time except for the 12-Hz luminance stimulus at which the F component decreased as a function of the light-adaptation period. The phase of the first harmonic component changed only slightly (less than 30°) during the light-adaptation period for all stimuli conditions. The L/M ratio in luminance reflecting ERGs decreased with increasing adaptation time. Our present data suggest that the light-adaptation process mainly reflects changes in the luminance pathway. The responses to 12-Hz luminance stimuli are determined by two different luminance driven pathways with different adaptation characteristics.