The aim of this work was to investigate the mechanisms of lateral interactions involved in flicker perception. Furthermore, the spatial properties of the monoptic and dichoptic components of these mechanisms were studied. We quantified the perceived flicker strength (PFS) in the center of a test stimulus, which was simultaneously modulated with a surround stimulus of variable size. The modulation depth of a separate stimulus, identical to the center test stimulus but without the surround, was determined using a two-alternative forced choice procedure. Using LCD goggles synchronized to the frame rate of a CRT screen, the center and surround of the test stimulus were presented either monoptically or dichoptically. In the monoptic condition, center-surround interactions have subcortical and cortical origins. In the dichoptic condition, center-surround interactions must have a cortical origin. The difference between the dichoptic and the monoptic data is an estimate of the contribution of the subcortical mechanisms. At each condition (surround stimulus size; monoptic or dichoptic presentation), the PFS was measured for phase differences between center and surround stimuli. The PFS changed systematically with phase difference. It also was observed that the PFS in the center stimulus changed merely be the presence of a surround stimulus independently of the center-surround phase difference. We propose that this is a phase-independent mechanism related to contrast adaptation owing to the presence of surround modulation. Our data suggest that both phase-dependent and -independent mechanisms have cortical and subcortical origins. There were no systematic differences between the spatial properties of subcortical and cortical components involved in PFS modulation.
Electroretinographic measurement instruments allow the variation of several stimulation parameters enabling to study a wide range of retinal processes. The purpose of the present study was to measure human flicker electroretinograms (ERGs) varying temporal modulation, temporal frequency and mean luminance in the photopic and higher mesopic ranges where the change from cone to rod dominance occurs.
L- and M-cone driven on- and off- ERG responses and their interactions were examined using full field stimuli with sawtooth temporal profiles. The effects of temporal frequency and contrast were studied. ERG recordings were obtained from 21 trichromatic, 1 protanopic, and 1 deuteranopic subjects. ERGs to L-cone increments and decrements resembled those to M-cone decrements and increments, respectively (i.e., of the opposite polarity). Temporal frequency and contrast had little effect on the implicit times. All response components varied linearly with contrast. When stimulated simultaneously, the responsivities of most components were larger for counterphase than for inphase modulation. The retinal processing leading to an ERG response is reversed for L- and M-cone driven responses.
Electroretinograms (ERGs) elicited by transient, square-wave L- and M-cone isolating stimuli were recorded from human trichromatic (n=19) and dichromatic (n=4) observers. The stimuli were generated on a four primary LED stimulator and were equated in terms of cone modulation (cone contrast=0.11) and retinal illuminance (12,000 trolands). L- and M-cone isolated ERGs had waveforms similar to those observed for luminance responses. However, M-cone ERGs exhibited a phase reversal in their responses to onset and offset stimuli relative to the L-cone responses. This on-off response reversal was observed in trichromats but not dichromats. Simultaneous counterphase and inphase combinations of L- and M-cone isolating stimuli generated responses that reflected chromatic and luminance processing, respectively. We conclude that L- and M-cone specific ERGs provide a measure of how photoreceptors contribute to postreceptoral mechanisms.
Cone isolating stimuli were used to assess the temporal frequency response characteristics of L- and M-cone electroretinograms (ERGs) in nine trichromatic and four dichromatic human observers. The stimuli comprised sinusoidal temporal modulations varying from 5 to 100 Hz. ERGs were recorded using corneal fiber electrodes and subjected to fast Fourier transform analysis. At low temporal frequencies (<10??Hz) the L- and M-cone ERGs had similar amplitude and exhibited minimal differences in apparent latency. At higher flicker rates (>20??Hz) L-cone ERGs had greater amplitudes and shorter apparent latencies than the M-cone responses. These differences between the L- and M-cone ERGs are consistent with their mediation by chromatic and luminance postreceptoral processing pathways at low and high temporal frequencies, respectively.
The howler monkeys (Alouatta sp.) are the only New World primates to exhibit routine trichromacy. Both males and females have three cone photopigments. However, in contrast to Old World monkeys, Alouatta has a locus control region upstream of each opsin gene on the X-chromosome and this might influence the retinal organization underlying its color vision. Post-mortem microspectrophotometry (MSP) was performed on the retinae of two male Alouatta to obtain rod and cone spectral sensitivities. The MSP data were consistent with only a single opsin being expressed in each cone and electrophysiological data were consistent with this primate expressing full trichromacy. To study the physiological organization of the retina underlying Alouatta trichromacy, we recorded from retinal ganglion cells of the same animals used for MSP measurements with a variety of achromatic and chromatic stimulus protocols. We found MC cells and PC cells in the Alouatta retina with similar properties to those previously found in the retina of other trichromatic primates. MC cells showed strong phasic responses to luminance changes and little response to chromatic pulses. PC cells showed strong tonic response to chromatic changes and small tonic response to luminance changes. Responses to other stimulus protocols (flicker photometry; changing the relative phase of red and green modulated lights; temporal modulation transfer functions) were also similar to those recorded in other trichromatic primates. MC cells also showed a pronounced frequency double response to chromatic modulation, and with luminance modulation response saturation accompanied by a phase advance between 10-20 Hz, characteristic of a contrast gain mechanism. This indicates a very similar retinal organization to Old-World monkeys. Cone-specific opsin expression in the presence of a locus control region for each opsin may call into question the hypothesis that this region exclusively controls opsin expression.
To psychophysically determine macular pigment optical density (MPOD) employing the heterochromatic modulation photometry (HMP) paradigm by estimating 460 nm absorption at central and peripheral retinal locations.
Light-dependent conductance changes of voltage-gated Cav1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the ?1F subunit of Cav1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Cav1.4 channels, but some mutations alter the channels' gating properties and, presumably, disturb Ca(2+) influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (?Ex14-17) in a longitudinal study up to eight months of age. In ?Ex14-17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca(2+) responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca(2+) response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ?Ex14-17 mutants.
The study investigated possible asymmetric dysfunction of the ON and OFF visual mechanisms in DMD (Duchenne muscular dystrophy) patients associated with specific genetic alterations. Methods: nineteen DMD patients and 7 heterozygous dmd carriers were tested, as well as 19 age-matched controls.Full-field ergs were recorded using mesopic (1 cd/m(2)) and photopic (250 cd/m(2)) sawtooth luminance modulations as stimuli: rapid increase and ramping decrease (to isolate ON responses) or rapid decrease and ramping increase (for OFF responses). In addition, a psychophysical study comprised contrast sensitivity tests using two checkerboard stimuli at either higher (ON) or lower (OFF) luminance relative to the background: 0.3 cycles per degree (cpd) presented for 33 ms (low spatial frequency, short duration) and 2 cpd presented for 1500 ms (high spatial frequency, long duration).
Monocarboxylate transporter 8 (MCT8) is a thyroid hormone (TH)-specific transporter. Mutations in the MCT8 gene are associated with Allan-Herndon-Dudley Syndrome (AHDS), consisting of severe psychomotor retardation and disturbed TH parameters. To study the functional consequences of different MCT8 mutations in detail, we combined functional analysis in different cell types with live-cell imaging of the cellular distribution of seven mutations that we identified in patients with AHDS. We used two cell models to study the mutations in vitro: 1) transiently transfected COS1 and JEG3 cells, and 2) stably transfected Flp-in 293 cells expressing a MCT8-cyan fluorescent protein construct. All seven mutants were expressed at the protein level and showed a defect in T3 and T4 transport in uptake and metabolism studies. Three mutants (G282C, P537L, and G558D) had residual uptake activity in Flp-in 293 and COS1 cells, but not in JEG3 cells. Four mutants (G221R, P321L, D453V, P537L) were expressed at the plasma membrane. The mobility in the plasma membrane of P537L was similar to WT, but the mobility of P321L was altered. The other mutants studied (insV236, G282C, G558D) were predominantly localized in the endoplasmic reticulum. In essence, loss of function by MCT8 mutations can be divided in two groups: mutations that result in partial or complete loss of transport activity (G221R, P321L, D453V, P537L) and mutations that mainly disturb protein expression and trafficking (insV236, G282C, G558D). The cell type-dependent results suggest that MCT8 mutations in AHDS patients may have tissue-specific effects on TH transport probably caused by tissue-specific expression of yet unknown MCT8-interacting proteins.
Therapeutic approaches to retinal disease require a continuous monitoring of functional improvement over lesion areas that sometimes cannot be shown in full-field ERG. The aim of this study was to assess the usefulness of multifocal electroretinograms (mfERGs) under visual control using scanning laser ophthalmoscopy (SLO) for evaluation of local retinopathy in mice.
Photoreceptor cells encode light signals over a wide range of intensities with graded changes in their membrane potential. At their highly specialized ribbon synapses they transmit the signals to the postsynaptic neurons by the tonic release of glutamate, which is continuously adjusted to changes in light intensity. Such a level of performance requires adaptive mechanisms, and it is suggested that illumination-dependent changes in ribbon shape and size are one of these adaptive processes. In this study we compared structural properties of synaptic ribbons under various illumination conditions between three mouse strains: the pigmented C57BL/6 and the two albino strains Balb/c and B6(Cg)-Tyr(c-2J) /J (coisogenic to C57BL/6). In addition, electroretinograms (ERGs) recorded in the same groups were compared. In the C57BL/6 mouse a change in illumination did not result in structural alterations of the synaptic ribbon. Similarly, in the B6(Cg)-Tyr(c-2J) /J mouse only minor structural changes were detected. In contrast, the state of adaptation had a large influence on the ribbon structure of the Balb/c mouse. The ERG recordings showed only small functional differences between C57BL/6 and B6(Cg)-Tyr(c-2J) /J mice, but the retinal function of Balb/c mice was strongly compromised. We conclude that illumination-dependent changes of photoreceptor ribbon structure differ between strains and thus cannot be regarded as a general mechanism for light adaptation.
Piccolo is one of the largest cytomatrix proteins present at active zones of chemical synapses, where it is suggested to play a role in recruiting and integrating molecules relevant for both synaptic vesicle exo- and endocytosis. Here we examined the retina of a Piccolo-mutant mouse with a targeted deletion of exon 14 in the Pclo gene. Piccolo deficiency resulted in its profound loss at conventional chemical amacrine cell synapses but retinal ribbon synapses were structurally and functionally unaffected. This led to the identification of a shorter, ribbon-specific Piccolo variant, Piccolino, present in retinal photoreceptor cells, bipolar cells, as well as in inner hair cells of the inner ear. By RT-PCR analysis and the generation of a Piccolino-specific antibody we show that non-splicing of intron 5/6 leads to premature translation termination and generation of the C-terminally truncated protein specifically expressed at active zones of ribbon synapse containing cell types. With in situ proximity ligation assays we provide evidence that this truncation leads to the absence of interaction sites for Bassoon, Munc13, and presumably also ELKS/CAST, RIM2, and the L-type Ca(2) (+) channel which exist in the full-length Piccolo at active zones of conventional chemical synapses. The putative lack of interactions with proteins of the active zone suggests a function of Piccolino at ribbon synapses of sensory neurons different from Piccolos function at conventional chemical synapses.
There is evidence that multifocal visual evoked potentials (VEPs) can be used as an objective tool to detect visual field loss. The aim of this study was to correlate multifocal VEP amplitudes with standard perimetry data and retinal nerve fibre layer (RNFL) thickness.
Retinitis pigmentosa (RP) is a group of human retinal disorders, with more than 100 genes involved in retinal degeneration. Canine and murine models are useful for investigating human RP based on known, naturally occurring mutations. In Schapendoes dogs, for example, a mutation in the CCDC66 gene has been shown to cause autosomal recessively inherited, generalized progressive retinal atrophy (gPRA), the canine counterpart to RP. Here, a novel mouse model with a disrupted Ccdc66 gene was investigated to reveal the function of protein CCDC66 and the pathogenesis of this form of gPRA. Homozygous Ccdc66 mutant mice lack retinal Ccdc66 RNA and protein expression. Light and electron microscopy reveal an initial degeneration of photoreceptors already at 13 days of age, followed by a slow, progressive retinal degeneration over months. Retinal dysfunction causes reduced scotopic a-wave amplitudes, declining from 1 to 7 months of age as well as an early reduction of the photopic b-wave at 1 month, improving slightly at 7 months, as evidenced by electroretinography. In the retina of the wild-type (WT) mouse, protein CCDC66 is present at highest levels after birth, followed by a decline until adulthood, suggesting a crucial role in early development. Protein CCDC66 is expressed predominantly in the developing rod outer segments as confirmed by subcellular analyses. These findings illustrate that the lack of protein CCDC66 causes early, slow progressive rod-cone dysplasia in the novel Ccdc66 mutant mouse model, thus providing a sound foundation for the development of therapeutic strategies.
The percept of a time-varying light depends on the temporal properties of light within the surrounding area. The locus of the neural mechanism mediating this lateral interaction is controversial; neural mechanisms have been posited at the LGN (Kremers et al., 2004) or cortical level (DAntona & Shevell, 2007). To determine the neural locus, changes in perceived temporal variation were compared with ipsilateral versus contralateral surrounding context. In both cases, a temporally varying central field was viewed within a temporally varying surround; relative phase between center and surround was varied. Perceived modulation depth in the central field depended strongly on the relative phase between center and surround, in both the ipsilateral and contralateral conditions. The results revealed lateral interactions arising from both a weak monocular (plausibly LGN) and a stronger central (cortical) mechanism. The monocular contribution was similar over the range of temporal frequencies tested (approx. 3-12Hz), while the central component showed low-pass temporal-frequency selectivity.
The electroretinographic response to L- and M-cone isolating stimuli was measured at different luminance levels to study the effect of retinal illuminance on amplitude and phase, and how this may influence estimates of L:M ratios in the retina. It was found that the amplitude of L- and M-cone driven responses increases differently with increasing retinal illuminance: L-cone responses increase more quickly than those of M-cones. The L:M ratio does not change strongly with retinal illuminance. The phase of both L- and M-cone driven responses advances with increasing retinal illuminance. There is considerable interindividual variability in the phase difference between the two, but generally M-cone driven responses are phase advanced.
Autocrine, paracrine, and juxtacrine are recognized modes of action for mammalian EGFR ligands including EGF, TGF-? (TGF?), amphiregulin (AREG), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, epiregulin, and epigen. We identify a new mode of EGFR ligand signaling via exosomes. Human breast and colorectal cancer cells release exosomes containing full-length, signaling-competent EGFR ligands. Exosomes isolated from MDCK cells expressing individual full-length EGFR ligands displayed differential activities; AREG exosomes increased invasiveness of recipient breast cancer cells 4-fold over TGF? or HB-EGF exosomes and 5-fold over equivalent amounts of recombinant AREG. Exosomal AREG displayed significantly greater membrane stability than TGF? or HB-EGF. An average of 24 AREG molecules are packaged within an individual exosome, and AREG exosomes are rapidly internalized by recipient cells. Whether the composition and behavior of exosomes differ between nontransformed and transformed cells is unknown. Exosomes from DLD-1 colon cancer cells with a mutant KRAS allele exhibited both higher AREG levels and greater invasive potential than exosomes from isogenically matched, nontransformed cells in which mutant KRAS was eliminated by homologous recombination. We speculate that EGFR ligand signaling via exosomes might contribute to diverse cancer phenomena such as field effect and priming of the metastatic niche.
The aim of this study was to determine the influence of temporal frequency of temporal contrast adaptation on contrast sensitivity in healthy subjects. Temporal contrast sensitivities (TCS) were measured monocularly in seven healthy subjects with a modified ERG full-field bowl stimulator at eight different test temporal frequencies (9, 15, 20, 25, 31, 37, 44, 51 Hz) using a two-alternative-forced-choice strategy. Before each presentation of the test stimulus, a 100% contrast adapting flicker stimulus was presented (frequencies: 9, 15, 20, 25, 31, 37, 44, 51, 100 Hz). At each adapting frequency, a complete set of TCSs was measured. All temporal contrast sensitivities decreased with increasing temporal frequencies. Adaptation led to a general temporal contrast sensitivity decrease. Largest adaptation effects were seen at an adaptation frequency of 25 Hz. Reduction of contrast sensitivity was significantly larger at 25 Hz adaptation than at 9 Hz adaptation (t-test of paired samples, Bonferroni corrected). The results of this study showed a general TCS decrease with the largest effect at an adaptation frequency of 25 Hz. This finding indicates that the contrast adaptation probably occurred in the magnocellular-pathway. In future clinical studies adaptation effects could be investigated in patients with reduced temporal contrast sensitivity.
Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the ?-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.
To investigate the role of enhanced antigen presentation in dendritic cell (DC)-based immunotherapy. Here, we describe the development of a cell-penetrating mucin 1 (MUC1) antigen and its immunotherapeutic potential against tumors. After animal groups received two immunizations of MUC1-MPA(11)P-pulsed DCs, we observed a marked tumor regression compared with the mice treated with DCs alone or DCs pulsed with MUC1 peptide. We confirmed the migration and homing of DCs in the popliteal lymph node using magnetic resonance imaging during the study. In summary, enhanced antigen uptake using an MPA(11)P delivery molecule improves cell therapy.
Recent studies suggest a diagnostic value of the photopic negative response (PhNR) with a long-duration stimulus. The aim of this study was to record the on and off responses of the photopic fullfield electroretinogram (ERG) in normal subjects and glaucoma patients. We focused on different waves of the responses after onset and offset of the long-duration stimulus ERG. Photopic fullfield ERGs were recorded in response to a white bright LED flash on a white 20 cd/m(2) background. Stimulus luminances were 40, 60 and 80 cd/m(2). Responses were averaged using a flash duration of 240 ms and an offset period of 500 ms. We examined 19 healthy subjects, 27 patients with glaucomatous optic disc atrophy and 7 ocular hypertensive patients. The amplitudes and implicit times of the on and off responses of the human ERG depended on flash luminance. Comparing patients with glaucoma and healthy subjects for the 60 cd/m² flash, there was a significant change in the PhNRs (at onset: P < 0.01, at offset: P < 0.001) of the d-wave and of the i-wave at offset (P < 0.01). No significant difference was found for peak times of the fullfield ERG and for a- and b-wave amplitudes. PhNR amplitudes were significantly correlated with mean thickness of retinal nerve fibre layer as measured with OCT. In comparison with the normal photopic long-flash ERG, glaucoma patients showed changes in the PhNR amplitude following stimulus onset and in waves following stimulus offset.
A transfecting agent-coated hybrid imaging nanoprobe (HINP) composed of visible and near-infrared (NIR) light emitting quantum dots (QDs) tethered to superparamagnetic iron oxide (SPIO) nanoparticles was developed. The surface modification of QDs and SPIO particles and incorporation of dual QDs within the SPIO were characterized by dynamic light scattering (DLS), quartz crystal microbalance (QCM) analysis and atomic force microscopy (AFM). The optical contrasting properties of HINP were characterized by absorption and photoluminescence spectroscopy and fluorescence imaging. Multicolor HINP was used in imaging the migration of dendritic cells (DCs) by optical, two-photon and magnetic resonance imaging techniques.
To measure heterochromatic flicker electroretinograms (ERGs) at high (36 Hz) and intermediate (12 Hz) temporal frequencies to evaluate luminance and cone opponent responses, respectively, in glaucoma eyes with (perimetric) and without (preperimetric) visual field defects.
Full-field electroretinograms were recorded from five normal human subjects using white light (mean luminance: 250 cd/m2) sine wave stimuli at different frequencies and contrasts. In agreement with previous studies, we found that the amplitude of the fundamental component displayed a dip at about 12 Hz, coinciding with a maximum in the second harmonic component, indicating frequency doubling of the responses. By including measurements at different contrasts, we were able to recognize two (sine-like and transient) response components. We found that the waveform of the transient response was relatively frequency independent. An algorithm to separate the two components was developed. The interaction between these two components can explain the frequency-doubled responses around 12 Hz. The sine-like component is more linear and prominent in the low-frequency region, whereas the transient seems to be more nonlinear and prominent in the high-frequency region.
To examine the interplay between tumor cells and the microenvironment during early breast cancer metastasis, we developed a technique for ex vivo imaging of murine tissue explants using two-photon microscopy. Cancer cells in the liver and the lung were compared by imaging both organs at specific time points after the injection of the same polyomavirus middle T-initiated murine mammary tumor cell line. Extravasation was greatly reduced in the lung compared with the liver, with 56% of tumor cells in the liver having extravasated by 24 hours, compared with only 22% of tumor cells in the lung that have extravasated. In the liver, imaged cells continually transitioned from an intravascular location to an extravascular site, whereas in the lung, extravasation rates slowed after 6 hours. Within the liver microenvironment, the average size of the imaged micrometastatic lesions increased 4-fold between days 5 and 12. Histologic analysis of these lesions determined that by day 12, the micrometastases were heterogeneous, consisting of both tumor cells and von Willebrand factor-positive endothelial cells. Further analysis with intravenously administered lectin indicated that vessels within the micrometastatic tumor foci were patent by day 12. These data present the use of two-photon microscopy to directly compare extravasation times in metastatic sites using the same tumor cell line and highlight the differences in early events and metastatic patterns between two important secondary sites of breast cancer progression with implications for future therapy.
Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles. Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion. This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.
To identify age-dependent regulated aqueous humor (AH) factors in DBA2/J (D2J) mice and to correlate them with optic nerve degeneration and intraocular pressure (IOP) by population and individual analysis.
The aim of the study was to investigate whether there is an ocular interaction in the flicker ERG responses reflecting luminance and cone opponency in normal human subjects. Flicker ERGs were recorded from one dilated eye of 10 healthy volunteers. Each subject was tested twice: once with and once without occluding the opposite eye. Red and green LEDs were modulated in counterphase in a Ganzfeld stimulator. ERG responses were recorded for different ratios of the modulation in the red and green LEDs and at 12 and 36 Hz. The amplitudes and phases of the fundamental components were compared between the conditions with and without occlusion. The 12-Hz flicker ERGs reflected activity of the cone opponent channel, whereas the 36-Hz data reflected luminance activity. There were no significant differences between the conditions with and without occluding the opposite eye for any of the stimulus protocols. Ocular interaction is absent in flicker ERGs reflecting cone opponent and luminance activity.
The purpose of the present study was to investigate whether L- and M-cone driven responses can be influenced by concomitant modulation in the rods or the S-cones. In addition, it was studied whether a change in the state of adaptation in L- or M-cones can have a different influence on ERG data when simultaneously the mean number of photoisomerizations in either rods or S-cones is altered. It was found that rods and/or S-cones cannot be neglected when measuring L- or M-cone driven ERGs.
The aim of this study was to determine up to which extent the specific characteristics of cathode ray tube (CRT) and liquid crystal display (LCD) monitors influence the retinal biosignal when used as stimulators in ocular electrophysiology. In a conventional CRT monitor, each pixel lights up only for a duration of a few milliseconds during each frame. In contrast, liquid crystal displays are quasi-static, i.e. each pixel has a constant luminance during the whole length of the frame, but lights up only with a certain delay after the trigger. These different display characteristics may affect the mfERG signal. The temporal and spatial luminance distributions of a CRT and an LCD monitor were measured in white flashes. The total amount of emitted light was calculated by integration of the intensity versus time curves. By means of an mfERG recording system (RETIsystem, Roland Consult, Brandenburg, Germany) first-order kernel (FOK) mfERG signals were computed and then analysed using customized MATLAB (TheMathWorks, Natick, MA, USA) software. With the two stimulator monitors, differences in the mfERG signal were observed. The latencies of mfERG responses recorded with the LCD monitor were significantly increased by 7.1 ms for N1 and 9.5 ms for P1 compared to the CRT. Due to a higher luminance, the N1 amplitude was significantly higher by approx. 2 dB in measurements with the LCD monitor while no significant difference could be detected with regard to the more contrast sensitive P1 amplitude. When using LCD monitors as stimulators the increase in latencies and differences in the luminance versus time profile must be taken into account. Prior to clinical application, the establishment of guidelines for the use of LCD monitors is recommended.
We found that photoconversion is fairly common among orange and red fluorescent proteins, as in a screen of 12 proteins, 8 exhibited photoconversion. Specifically, three red fluorescent proteins could be switched to a green state, and two orange variants could be photoconverted to a far-red state. The orange proteins are ideal for dual-probe highlighter applications, and they exhibited the most red-shifted excitation of all fluorescent proteins described to date.
The DBA/2J (D2J) is a genetic mouse model for glaucomatous neurodegeneration because the animals develop anatomical and functional retinal deficits that partially can be correlated with elevated intraocular pressure (IOP). The IOP starts to increase at an age of about 6 months as a result of morphological changes within the anterior eye segment, e.g., pigment dispersion and iris synechiae. The purpose of the present study was to investigate how ERG responses change in individuals at different ages in D2J mice and to compare these changes with normal aging effects in pigmented C57/B6 (B6) mice. IOP was measured in awake, non-sedated D2J and B6 mice with a rebound tonometer. At ages between 2-3 and 10 months, scotopic flash ERGs were measured five times with about 2 months intervals. In addition, light adapted flicker ERGs were recorded. Our data show that the D2J shows lower flicker ERG responses than the B6 mice already at an age of 2-3 months. Dark adapted flash ERG responses are not decreased at this age. In both mouse strains the ERG responses decrease as a function of age, but there is a stronger decrease in the D2J mice. The data of flicker ERGs suggest the presence of early functional deficits in the D2J retina that possibly have a post-receptoral origin. The scotopic flash ERG reveals a functional deficit that occurs at a later stage and that possibly is IOP dependent. But, the deficits appear at an age at which the IOP is still lower than in the B6 mouse, indicating that other factors play an additional role.
The parallel processing of information forms an important organisational principle of the primate visual system. Here we describe experiments which use a novel chromatic–achromatic temporal compound stimulus to simultaneously identify colour and luminance specific signals in the human electroretinogram (ERG). Luminance and chromatic components are separated in the stimulus; the luminance modulation has twice the temporal frequency of the chromatic modulation. ERGs were recorded from four trichromatic and two dichromatic subjects (1 deuteranope and 1 protanope). At isoluminance, the fundamental (first harmonic) response was elicited by the chromatic component in the stimulus. The trichromatic ERGs possessed low-pass temporal tuning characteristics, reflecting the activity of parvocellular post-receptoral mechanisms. There was very little first harmonic response in the dichromats ERGs. The second harmonic response was elicited by the luminance modulation in the compound stimulus and showed, in all subjects, band-pass temporal tuning characteristic of magnocellular activity. Thus it is possible to concurrently elicit ERG responses from the human retina which reflect processing in both chromatic and luminance pathways. As well as providing a clear demonstration of the parallel nature of chromatic and luminance processing in the human retina, the differences that exist between ERGs from trichromatic and dichromatic subjects point to the existence of interactions between afferent post-receptoral pathways that are in operation from the earliest stages of visual processing.
The aim of this study is to measure the on- and off-responses and their response asymmetries elicited by sawtooth stimuli in normal subjects and glaucoma patients. Furthermore, the correlation between the ERGs and other functional and structural parameters are investigated. Full-field stimuli were produced using a Ganzfeld bowl with Light Emitting Diodes (LEDs) as light sources. On- and off-response ERGs were recorded from 17 healthy subjects, 12 pre-perimetric and 15 perimetric glaucoma patients using 4-Hz luminance rapid-on and rapid-off sawtooth stimuli (white light; mean luminance 55 cd/m(2)) at 100% contrast. The on- and off-responses were added to study response asymmetries. In addition, flash ERGs were elicited by red stimuli (200 cd/m(2)) on a blue background (10 cd/m(2)). The mean deviations (MD) of the visual field defects were obtained by standard automated perimetry. The retinal nerve fibre layer thickness (RNFLT) was measured with Spectral Domain Optical Coherence Tomography (SOCT). We studied the correlation between ERG response amplitudes, visual field mean deviation (MDs) and RNFLT values. The on-responses showed an initial negative (N-on) followed by a positive (P-on), a late positive (LP-on) and a late negative responses (LN-on). The off-responses showed an initial positive (P-off) a late positive (LP-off) and a late negative response (LN-off). The addition of on- and off-responses revealed an initial positive (P-add) and a late negative response (LN-add). The on-response components (N-on, P-on and LN-on) in the glaucoma patients were relatively similar to those of the control subjects. However, the LP-on was significantly elevated (p = 0.03) in perimetric patients. The LP-off was significantly elevated (p < 0.001), and the amplitude of LN-off was significantly reduced in perimetric patients (p = 0.02). The LN-add amplitude was significantly reduced (p < 0.001) and delayed (p = 0.03) in perimetric patients. The amplitudes of the LN-off and LN-add ERG components were significantly correlated with the PhNR in the flash ERG (LN-off: p = 0.01; LN-add: p < 0.001) and with RNFLT (LN-off: p = 0.006; LN-add: p = 0.001). On- and off-response ERGs and their response asymmetries, elicited by sawtooth stimuli, are altered in the glaucoma patients. The late components are affected. Changes in the late negative components are correlated with structural and other functional changes.
Electroretinographic responses to cone and rod isolating stimuli and to simultaneous L- and M-cone modulation were measured at different temporal frequencies between 2 and 60 Hz and at two mean luminances using a four primary stimulator. The responses driven by each photoreceptor type had distinct characteristics. The responses to stimuli containing L- and/or M-cone stimulation indicated the presence of two underlying mechanisms that were active in distinct frequency regions. Between 2 and 12 Hz, the responses displayed properties that were reminiscent of the L-M-cone opponent system. At higher temporal frequencies, the electroretinograms were more determined by the luminance content in the stimuli.
We analyzed mesopic rod and S-cone interactions in terms of their contributions to the blue-yellow opponent pathway. Stimuli were generated using a four-primary colorimeter. Mixed rod and S-cone modulation thresholds (constant L-, M-cone excitation) were measured as a function of their phase difference. Modulation amplitude was equated using threshold units and contrast ratios. This study identified three interaction types: (1) a linear and antagonistic rod:S-cone interaction, (2) probability summation, and (3) a previously unidentified mutual nonlinear reinforcement. Linear rod:S-cone interactions occur within the blue-yellow opponent pathway. Probability summation involves signaling by different postreceptoral pathways. The origin of the nonlinear reinforcement is possibly at the photoreceptors.
Flash electroretinogram responses were measured in normal subjects to different chromatic combinations of flashes and backgrounds. The amplitudes of the flash response components were measured at different flash strengths and could be described by a generalized Naka-Rushton function. The measurements were repeated at different background luminances to study adaptation effects. It was found that when flash strength and background luminance were expressed in photometric terms (cd s/m² and cd/m², respectively), then the responses were very similar for all chromatic combinations with the exception of the condition in which blue (peak wavelength 458 nm) was flashed upon an orange (peak wavelength 591 nm) background. We propose that in this condition, a second (possibly S-cone or rod-driven) mechanism intrudes. The negative response after the b-wave (here called "photopic negative response" or PhNR for all conditions) is thought to reflect ganglion cell activity and was also largest at this condition. Responses were measured to the 458 nm flash on 591 nm background and the reversed combination in a population of 39 normal subjects and 49 glaucoma patients. It was found that the PhNR amplitude was affected by glaucoma in all conditions. Other component parameters, reflecting responses and adaptation dynamics, were not altered. The best stimulus condition among the conditions used to separate the PhNR amplitude of normals and patients was a 1 cd s/m² 458 nm flash on a 10 cd/m² 591 nm background.
Purpose: To compare the results of Flicker Defined Form (FDF)-perimetry with standard automated perimetry (SAP) and retinal nerve-fiber-layer (RNFL) thickness measurements using spectral domain OCT. Methods: 64 healthy subjects, 45 ocular hypertensive patients, and 97 early open-angle glaucoma (OAG) patients participated in this study. Definition of glaucoma was exclusively based on glaucomatous optic disc appearance. All subjects underwent FDF-perimetry, SAP and peripapillar measurements of the RNFL thickness. FDF-perimetry and SAP were performed at identical test locations (G1 protocol). Exclusion criteria were: subjects younger than 34 years, SAP-MD > 5 dB, eye diseases other than glaucoma, or non-reliable FDF-measurements. The correlations between the perimetric data on the one hand and RNFL thicknesses on the other hand were statistically analyzed. Results: The age corrected sensitivity values and the local results from the controls were used to determine FDF mean-defect (FDF-MD). FDF-perimetry and SAP showed high concordance in this cohort of experienced patients (MD-values: R=-0.69, P <0.001). 38 of in total 42 OAG-patients with abnormal SAP-MD also displayed abnormal FDF-MD. However, FDF-MD was abnormal in 28 out of 55 OAG-patients with normal SAP-MD. FDF-MD was significantly (R=-0.61, P <0.001) correlated with RNFL-thickness with a (non-significantly) larger correlation coefficient than conventional SAP-MD (R=-0.48, P <0.001). Conclusion: FDF-perimetry is able to uncover functional changes concurrent with the changes in RNFL thickness. FDF-perimetry may be an efficient functional test to detect early glaucomatous nerve atrophy.
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