Harmonic analysis of the cone flicker ERG of rabbit

Harmonic analysis was used to characterize the rabbit flicker ERG elicited by sinusoidally modulated full-field stimuli under light-adapted conditions. The frequency-response function for fundamental amplitude, derived from Fourier analysis of the ERG waveforms, exhibited two limbs, with an amplitude minimum at approximately 30Hz, and a high-frequency region peaking at around 45Hz and extending to more than 100Hz at higher adapting levels. At low frequencies (<20Hz), the fundamental response amplitude was independent of mean luminance (Weber law behavior), whereas the response amplitude at high stimulus frequencies varied nonlinearly with mean luminance. At low frequencies, intravitreal administration of L-AP4, which blocks ON-pathway activity, reduced the fundamental response amplitude and produced a phase shift. On the other hand, PDA, which reduces OFF-pathway activity, had a minimal effect on both the response amplitude and phase at low frequencies. At high frequencies, L-AP4 increased the fundamental response amplitude at low mean luminances, whereas PDA had only a small effect on amplitude and phase. Both pharmacologic agents removed the minimum in the amplitude-frequency function as well as the abrupt change in phase at stimulus frequencies near 30Hz. The results suggest that there is a nonlinear interaction between ON- and OFF-pathway activity over the entire stimulus frequency range examined in this study. These findings provide a basis for formulating protocols to evaluate the effect of pharmacologic agents and/or disease on the cone flicker ERG of rabbit.     

Characteristics of period doubling in the rat cone flicker ERG

When the eye is stimulated by a flickering light, the electroretinogram (ERG) and other electrophysiological responses in the visual pathway often exhibit period doubling. This phenomenon is manifested as an alternation in the shape of the response waveform from cycle to cycle, and also as spectral components at the half-fundamental frequency (F/2) and its odd multiples. Although period doubling has been described in humans as well as in other animals, its features in the rodent flicker ERG have not been characterized. We investigated the properties of period doubling in the rat cone flicker ERG elicited with full field, sinusoidal photic stimuli. Period doubling was observed when the temporal frequency of the stimulus was in the range of 20-30 Hz. The F/2 component of the Fourier spectrum of the ERG was more pronounced than its odd harmonics. The magnitude of the cycle-to-cycle variation in amplitude differed depending on whether measurements were based on peak-to-trough or trough-to-peak amplitudes, owing to the relative phase relationship between F/2 and F as a function of stimulus frequency. The frequency-response characteristics of period doubling varied with stimulus contrast, such that reducing the contrast shifted the peak F/2 amplitude to a lower stimulus frequency. Period doubling was evident in rat eyes in which PDA was administered intravitreally, indicating that the phenomenon can occur independently of OFF pathway activity in the rat retina. The period doubling properties we observed in the flicker ERG response of the rat cone system provide constraints on the nature of the nonlinear feedback mechanism presumed to underlie the period doubling phenomenon.     

Light adaptation, rods, and the human cone flicker ERG.

During the course of light adaptation, the amplitude and implicit time of the human cone ERG change systematically. In the present study, the effect of adapting field luminance on these ERG changes was assessed, and the hypothesis that light adaptation of the rod system is the primary determining factor was evaluated. Cone ERG responses, isolated through the use of 31.1-Hz flicker, were obtained from two visually normal subjects, initially under dark-adapted conditions and then repeatedly for 30 min following the onset of each of a series of ganzfeld adapting fields with luminances that ranged from -1.2 to 2.1 log cd/m2. The increase in flicker ERG amplitude and decrease in implicit time during light adaptation were greatest at the highest adapting field luminances. Photopically equivalent achromatic and long-wavelength adapting fields induced comparable increases in flicker ERG amplitude, while scotopically equivalent adapting fields had considerably different effects. This latter finding demonstrates that the rod system is not a major determinant of the adaptation-induced increase in cone ERG amplitude.     

L, M and L-M hybrid cone photopigments in man: deriving lambda max from flicker photometric spectral sensitivities

Using heterochromatic flicker photometry, we have measured the corneal spectral sensitivities of the X-chromosome-linked photopigments in 40 dichromats, 37 of whom have a single opsin gene in their tandem array. The photopigments encoded by their genes include: the alanine variant of the normal middle-wavelength sensitive photopigment, M(A180); the alanine and serine variants of the normal long-wavelength sensitive photopigment, L(A180) and L(S180); four different L-M hybrid or anomalous photopigments, L2M3(A180), L3M4(S180), L4M5(A180) and L4M5(S180); and two variants of the L-cone photopigment, encoded by genes with embedded M-cone exon two sequences, L(M2; A180) and L(M2; S180). The peak absorbances (lambda max) of the underlying photopigment spectra associated with each genotype were estimated by correcting the corneal spectral sensitivities back to the retinal level, after removing the effects of the macular and lens pigments and fitting a template of fixed shape to the dilute photopigment spectrum. Details of the genotype-phenotype correlations are summarized elsewhere (Sharpe, L. T., Stockman, A., J?gle, H., Knau, H., Klausen, G., Reitner, A. et al. (1998). J. Neuroscience, 18, 10053-10069). Here, we present the individual corneal spectral sensitivities for the first time as well as details and a comparison of three analyses used to estimate the lambda max values, including one in which the lens and macular pigment densities of each observer were individually measured.     

Stimulus chromatic properties affect period doubling in the human cone flicker ERG

Period doubling in the full-field cone flicker electroretinogram (ERG) refers to an alternation in waveform amplitude and/or shape from cycle to cycle, presumably owing to the operation of a nonlinear gain control mechanism. This study examined the influence of stimulus chromatic properties on the characteristics of period doubling in order to better understand the underlying mechanism. ERGs were acquired from 5 visually normal subjects in response to sinusoidally modulated flicker presented at frequencies from 25 to 100 Hz. The test stimuli and the pre-stimulus adaptation were either long wavelength (R), middle wavelength (G), or an equal combination of long and middle wavelengths (Y), all equated for photopic luminance. Fourier analysis was used to obtain the response amplitude at the stimulus frequency F and at a harmonic frequency of 3F/2, which was used as the index of period doubling. The frequency-response function for 3F/2 typically showed two peaks, occurring at approximately 33.3 and 50 Hz. However, the magnitude of period doubling within these frequency regions was dependent on the chromatic properties of both the test stimulus and the pre-stimulus adaptation. Period doubling was generally smallest when an R test was used, even though the stimuli were luminance-equated and the amplitude of F did not differ between the various conditions. The pattern of results indicates that the mechanism that generates period doubling is influenced by chromatic signals from both the test stimulus and the pre-stimulus adaptation, even though the high stimulus frequencies presumably favor the achromatic luminance system.     

Absence of ocular interaction in flicker ERG responses reflecting cone opponent and luminance signals

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.     

Nucleotide polymorphisms upstream of the X-chromosome opsin gene array tune L:M cone ratio

In support of the long-held idea that cone ratio is genetically determined by variation linked to the X-chromosome opsin gene locus, the present study identified nucleotide differences in DNA segments containing regulatory regions of the L and M opsin genes that are associated with significant differences in the relative number of L versus M cones. Specific haplotypes (combinations of genetic differences) were identified that correlated with high versus low L:M cone ratio. These findings are consistent with the biological principle that DNA sequence variations affect binding affinities for protein components of complexes that influence the relative probability that an L versus M opsin gene will be silenced during development, and in turn, produce variation in the proportion of L to M cones.     

The L:M cone ratio in males of African descent with normal color vision

Among Caucasian males with normal color vision, long-wavelength-sensitive (L) cones outnumber middle-wavelength-sensitive (M) cones by nearly three to one, on average, and the L and the M cone opsin genes are arrayed on the X-chromosome with the L opsin gene being closest to an upstream enhancer element termed the locus control region (LCR). Interaction between an opsin gene promoter and the LCR is required to mediate normal opsin gene expression, and the relative proximity of the L opsin gene promoter (4,000 base pairs for L compared to 40,000 base pairs for the M opsin gene promoter) has been proposed to endow the L gene with the advantage in competing for interaction with the LCR, thereby accounting for the nearly 3:1 ratio of L:M cones. This proximal advantage hypothesis predicts that the L:M cone ratio will be similar among populations that share the same X-chromosome opsin gene array organization. Here, we tested this hypothesis by examining a sample of males of African descent and found them to have a significantly different average L:M ratio compared to Caucasian males, even though their X-chromosome opsin gene arrays were indistinguishable from arrays in males of Caucasian descent. How these observations might be reconciled is discussed.     

The 30-Hz flicker cone ERG for monitoring the early course of central retinal vein occlusion

PURPOSE: Investigation of the full-field ERG (electoretinography) within 24 hours after the onset of symptoms in central retinal vein occlusion, and repeated examination within the following days and weeks. METHODS: Seven patients with central retinal vein occlusion were examined with full-field ERG within less than 24 hours after the onset of their symptoms and every second to third day during the first 3 weeks. They were then followed for a period of time of six months. RESULTS: The amplitudes as well as the implicit times in the 30 Hz flicker ERG changed considerably within the first three weeks. In the three patients who developed rubeosis, the cone b-wave implicit times as well as the amplitudes were altered considerably. In the patients that did not develop rubeosis, the cone b-wave implicit times as well as the amplitudes in the 30 Hz flicker ERG were more stable. CONCLUSION: The full-field cone ERG reflects the retinal function and the ERG parameters change considerably during the first weeks after the onset of the central retinal vein occlusion. The 30 Hz flicker ERG gives important information concerning the early development of the disease. The optimal time to do a predictive ERG could be after three weeks.     

Two distinct processes are evident in rat cone flicker ERG responses at low and high temporal frequencies

The electroretinogram (ERG) provides a noninvasive, objective measure of retinal function, and is one of the most widely used diagnostic tools in the study of visual disorders. Although rodents are often used in the study of retinal disease, the properties of the flicker ERG of the rodent retina have not been fully characterized. Here, we show that the fundamental response of the rat ERG to sine-wave flicker exhibited a low-pass pattern in the frequency range from 2 to 30 Hz, whereas the second harmonic (F2) showed a more complex frequency-response relation. The F2 component represented only a small fraction of the ERG response at low temporal frequencies (below 12 Hz), but it made a substantial contribution to responses at high frequencies. The contrast-response relation was linear when tested with a low-frequency (6 Hz) stimulus, but saturated in response to a high-frequency (20 Hz) stimulus. After intravitreal injection of L-AP4, a specific blocker of the retinal ON pathway, the flicker responses elicited by either 6- or 20-Hz stimuli were greatly reduced in amplitude, whereas only a very slight enhancement was seen after the application of PDA, a drug that blocks retinal OFF-pathway activity. Based on the observed differences in the degree of nonlinearity, and contrast-response properties of the rat flicker ERG at low and high frequencies, as well as the pharmacological results, we postulate that sustained and transient ON bipolar cells generate the flicker ERG responses elicited at low and high temporal frequencies, respectively.     

Evaluating the human X-chromosome pigment gene promoter sequences as predictors of L:M cone ratio variation

Men with normal color vision vary widely in the ratio of long- (L) to middle-wavelength sensitive (M) cones. This variation provides opportunities to test models for the mechanism that produces L versus M cones during development. The L and M photopigment genes lie in a tandem array. Each gene has a promoter, and upstream of each array there is a genetic element, termed the locus control region (LCR), that is required for the expression of both L and M pigment genes. During development, for each cell that has been determined to be an L or M cone, it has been proposed that the LCR acts as a stochastic selector which chooses one gene from the array to be expressed. In this model, the L and M promoters compete for contact with the LCR in each photoreceptor. Theoretically, the promoter that, by chance, is the first to successfully form a stable and permanent complex with the LCR commits the cell to a lifetime of exclusive expression of its associated gene. Under this model, it has been suggested that nucleotide differences in the promoters influence their ability to compete in forming a complex with the LCR. Thus, normal variation in L:M cone ratio is predicted to be associated with nucleotide polymorphisms in the promoters. Here we tested this hypothesis by comparing the L and M promoter sequences for 73 males with normal color vision for whom L:M cone ratio estimates had been obtained previously. The M gene promoter sequences were found to be identical for all 73 males and the L gene promoters were identical for 71 out of the 73 males. Two males had mutations where in each case the L promoter differed by one nucleotide substitution compared to normal. Both of the males with promoter mutations had unusual cone ratios which is consistent with the growing body of evidence indicating that the relative ability of the promoters to form a complex with the LCR is a factor in determining cone ratio. However, the vast majority of cone ratio differences were not associated with any difference in the promoter sequence. To explain the high degree of cone ratio variation among normal males, the mechanism that determines whether a cone is L or M must involve genetic elements that have a high degree of genetic polymorphism in the normal population. The results presented here indicate that there are additional genetic components of the mechanism which remain to be identified and incorporated into the present hypotheses.     

Macular pigment and the edge hypothesis of flicker photometry

Heterochromatic flicker photometry is commonly used to measure macular pigment optical density (MPOD) in the human retina. It has been proposed, and accepted by many, that the MPOD so measured represents the value at a retinal location corresponding to the edge of the flickering, circular stimulus. We have investigated this proposal by using a series of annular stimuli to determine the MPOD distribution in the central 1.5 degrees of the retina for both eyes of 10 subjects. The MPOD obtained using a 1.5 degrees circular stimulus matched the MPOD distribution at a retinal eccentricity that was always less than the stimulus radius, and averaged, for the 10 subjects, 51% of the stimulus radius. Similar results were obtained using a 1 degrees stimulus. Thus the edge hypothesis is inconsistent with our data. We suggest that involuntary eye movements may be responsible for an apparent edge effect.     

Analysis of nonlinearities in the flicker ERG.

The electroretinogram (ERG) response to steadily flickering fields shows significant nonlinear components. These nonlinear components have been used to investigate the ERG. We find the following. (1) The fundamental retinal response can be recorded at frequencies greater than 100 Hz and the second and higher harmonic responses have measurable response frequencies as high as 200 Hz. Both the fundamental response component and the second harmonic response component have multiple local maxima as a function of frequency. (2) Measurement of the response of the retina to the sum of two sine waves indicates that there is an early low-pass temporal filter in the retina. This early filter has a cutoff frequency between 40 and 50 Hz. (3) The high frequency nonlinearity is not a compressive nonlinearity.     

The influence of suppression on the flicker ERG

Summary The evoked occipital responses on flicker stimulation of the attentively reading eye have a significantly higher amplitude than the same responses on flicker stimulation of the suppressed eye.An effect of attention in the flicker ERG is not sufficiently demonstrated. If anything could be said about the influence of attention on the ERG it is the reverse of that on the occipital responses, i.e. attention lowers the amplitude of the ERG.

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Zusammenfassung Die Amplituden der Reizantworten des occipitalen Cortex auf Flimmerlicht-reizung des lesenden Auges sind höher als die auf Flimmerlichtreizung des supprimierten Auges.Ein Einfluss der Aufmerksamkeit war im ERG nicht deutlich erkennbar. Man möchte eher sagen, dass ein Einfluss in gegenseitiger Richtung zu finden war. Das heisst: die Aufmerksamkeit verkleinert die Amplituden des ERG.

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Résumé Les réponses évoquées du cortex occipital à la stimulation lumineuse intermittente (S.L.I.) de l'oeil attentivement lisant sont d'amplitude plus haut que les mêmes réponses au S.L.I. de l'oeil supprimé.Dans l'ERG au S.L.I. un effet de l'attention n'était pas clairement démontrable. Il y avait quelquefois une indication d'une influence de l'attention sur l'ERG en sens opposé. C'est à dire l'attention diminuait l'amplitude des réponses électro-rétinographiques.

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Eye Clinic, Rotterdam Medical School.     

Measurement of macular pigment: Raman spectroscopy versus heterochromatic flicker photometry

PURPOSE: There are several techniques for measuring macular pigment (MP) in vivo, of which Raman spectroscopy (RS) is a recently developed objective METHOD: This study reports the reproducibility, test-retest variability, and validity of RS MP readings, by comparing them with heterochromatic flicker photometry (HFP). METHODS: MP was measured with HFP and RS in 120 healthy subjects, and the latter technique was also used on two separate occasions in a sample of 20 subjects to investigate the intersessional variability of readings. Intrasessional reproducibility of RS MP measurements was also calculated. In addition, serum concentrations of lutein (L) and zeaxanthin (Z) were measured and correlated with both RS and HFP MP readings. RESULTS: Mean (+/-SD) MP in the right eye was 0.279 +/- 0.145 and 0.319 +/- 0.155 with RS and HFP, respectively. The differences between corresponding MP readings taken on RS and HFP lay within the Bland-Altman 95% limits of agreement for the two instruments in 93.6% and 94.4% of cases in the right and left eyes, respectively. Intrasessional reproducibility of RS readings, expressed as the coefficient of variation, was 8.42% +/- 7.12%. Ninety-five percent of MP readings taken with RS on two separate occasions lay within the 95% limits of agreement for the two sessions. A positive, but insignificant, relationship was observed between RS and HFP MP readings and serum concentrations of L and Z (RS, P = 0.356; HFP, P = 0.540). CONCLUSIONS: RS, an objective method of measuring MP levels in vivo, exhibits acceptable reproducibility and test-retest variability. The results demonstrated good correlation between RS and HFP measurements of MP, thus authenticating RS against a validated psychophysical technique of measuring MP. However, investigators should use only one of these instruments for the duration of any given study because of differences in the scientific rationale, and the factors that influence RS and HFP measurements of MP.     

Visual adaptation and the cone flicker electroretinogram

This study examined the hypothesis that changes in the response properties of the human cone ERG during light adaptation represent the recovery of cone system responsiveness toward a dark-adapted value after an initial decrease in responsiveness at adapting field onset. The electroretinographic (ERG) responses to 31.1 Hz flicker were obtained under both dark-adapted and light-adapted conditions for stimulus luminances ranging from -1.42(-)+0.82 log cd sec/m2. At low stimulus luminances, flicker ERG amplitudes were larger under dark-adapted than under light-adapted conditions, consistent with the hypothesis. However, at high stimulus luminances, flicker ERG amplitudes obtained under light-adapted conditions were approximately double those recorded from the dark-adapted eye. Therefore, the increase in cone ERG amplitude that occurs during light adaptation at high stimulus luminances does not represent a return toward a dark-adapted level but instead entails a substantial enhancement above the dark-adapted value, by a mechanism that is presently unidentified.     

Development of the cone ERG in infants

PURPOSE: To assess cone photoreceptor and cone-mediated postreceptoral retinal function in infants. METHODS: ERG responses to a 1.8-log unit range of long-wavelength flashes on a white, rod-saturating background were recorded in 4-week-old (n = 22) and 10-week-old (n = 28) infants and control adults and children, 8 to 40 years of age (n = 13). A model of the activation of cone phototransduction was fit to the a-waves. Sensitivity (S(CONE)) and saturated-response amplitude (R(CONE)) were calculated. The amplitude and implicit time of the b-wave were examined as a function of stimulus intensity. The cone photoresponse parameters were compared to the rod photoresponse parameters (S(ROD) and R(ROD)) in the same subjects. RESULTS: S(CONE) and R(CONE) in infants were significantly smaller than in the mature control subjects. The mean S(CONE) was 64% and 68%, and the mean R(CONE) was 63% and 72% in 4- and 10-week-olds, respectively. The mean rod photoresponse parameters were considerably less mature, as the mean S(ROD) was 35% and 46%, and the mean R(ROD) was 39% and 43% of mature values at 4 and 10 weeks. The b-wave stimulus-response functions in the 4- and 10-week-old infants did not show the photopic hill that was characteristic of the children's and adults' photopic b-waves. CONCLUSIONS: Peripheral cone function is relatively more mature than rod function in young infants. The lack of a photopic hill is hypothesized to result from immaturity in the relative contributions of ON and OFF bipolar cell responses.     

Characteristics of late negative ERG responses elicited by sawtooth flicker

Background

This study aimed to determine whether the properties of the late negative responses (LNRs) of the electroretinogram (ERG) elicited by sawtooth flicker are consistent with the characteristics of the photopic negative response generated by a light pulse (PhNR_pulse).

Methods

ERG recordings were obtained from 10 visually normal individuals and from 6 patients with optic atrophy (OA) in response to 8-Hz rapid-on and rapid-off sawtooth flicker and to brief (4 ms) light pulses. All stimuli were either long wavelength (R), middle wavelength (G), or a combination of equal luminances of long and middle wavelengths (Y) presented on a short-wavelength, rod-saturating adapting field. Amplitudes of LNRs were obtained in response to rapid-on (LNR_on) and rapid-off (LNR_off) sawtooth flicker and were also derived from the sum of the ERG waveforms to the two sawtooth phases (LNR_add).

Results

For the control subjects, PhNR_pulse amplitude varied with stimulus wavelength, being largest in response to a long-wavelength pulse, as expected. However, the amplitudes of LNR_on, LNR_off, and LNR_add were not significantly different for R, Y, and G sawtooth flicker. Despite the absence of a chromatic effect, LNR_off and LNR_add amplitudes were significantly smaller in the OA patients than in the controls, similar to the results for the PhNR_pulse, implying an inner retinal origin for the LNR_off and LNR_add. However, LNR_on amplitudes did not differ significantly between the OA patients and controls, although there was a significant correlation between the LNR_on and PhNR_pulse for R stimuli.

Conclusion

We conclude that LNR_off and LNR_add but not LNR_on can be useful measures to assess the integrity of the inner retina that can complement the PhNR_pulse.