August 2011
Volume 52, Issue 9
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Clinical Trials  |   August 2011
Heterochromatic Flicker Electroretinograms Reflecting Luminance and Cone Opponent Activity in Glaucoma Patients
Author Affiliations & Notes
  • Mirella Telles Salgueiro Barboni
    From the Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany; and
    the Neuroscience and Behavior Center and Department of Experimental Psychology, University of São Paulo, São Paulo, Brazil.
  • Gobinda Pangeni
    From the Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany; and
  • Dora Fix Ventura
    the Neuroscience and Behavior Center and Department of Experimental Psychology, University of São Paulo, São Paulo, Brazil.
  • Folkert Horn
    From the Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany; and
  • Jan Kremers
    From the Department of Ophthalmology, University Hospital Erlangen, Erlangen, Germany; and
  • Corresponding author: Jan Kremers, Department of Ophthalmology, University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany; jan.kremers@uk-erlangen.de
Investigative Ophthalmology & Visual Science August 2011, Vol.52, 6757-6765. doi:10.1167/iovs.11-7538
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      Mirella Telles Salgueiro Barboni, Gobinda Pangeni, Dora Fix Ventura, Folkert Horn, Jan Kremers; Heterochromatic Flicker Electroretinograms Reflecting Luminance and Cone Opponent Activity in Glaucoma Patients. Invest. Ophthalmol. Vis. Sci. 2011;52(9):6757-6765. doi: 10.1167/iovs.11-7538.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: 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.

Methods.: Flicker ERGs were recorded from one randomly chosen dilated eye of 32 patients (mean age, 61 ± 11 years; 15 men, 17 women) from the Erlangen Glaucoma Registry and from 24 healthy volunteers (mean age, 43 ± 11 years; 14 men, 10 women). Red and green light-emitting diodes in a Ganzfeld stimulator were sine wave-modulated in counterphase. The responses were measured at 36 Hz, the frequency at which ERGs reflect activity of the luminance pathway, and at 12 Hz, the frequency at which ERGs reflect chromatic activity.

Results.: Response amplitudes were similar in glaucoma patients and controls. Phase differences were observed in patients with visual field defects (perimetric) compared with the control group at 36 and 12 Hz in the first harmonic and second harmonic responses. Patients without visual field defects (preperimetric) showed phase differences for the second harmonic component at 36 Hz. No age effect on response amplitudes and phases was found in any of the subject groups (controls and patients).

Conclusions.: The responses displayed phase differences but not amplitude differences in perimetric glaucoma patients at both 36 and 12 Hz, suggesting that both magnocellular and parvocellular pathways are affected. Preperimetric glaucoma patients also showed phase differences. The response phase may be sensitive to early dysfunction of the inner retina. (ClinicalTrials.gov number, NCT00494923.)

Glaucomatous degeneration of retinal ganglion cells is often induced by elevated intraocular pressure (IOP), leading to progressive visual loss. 1 Glaucoma may primarily affect larger optic nerve fibers. 2,3 In contrast, experimental glaucoma in nonhuman primates has been shown recently to lead to progressive nonselective neural impairment of visual pathways and dysfunction of both large and small retinal ganglion cells and their magnocellular (M) and parvocellular (P) geniculocortical projections. 4 6 Using a psychophysical paradigm designed to selectively detect the function of the M and P visual pathways, 7 glaucomatous visual dysfunction was indeed found in both pathways. 8 10  
Several electrophysiological 11 13 and psychophysical 14 17 studies have assessed the activity of specific visual pathways in glaucoma patients. 18 20 One noninvasive electrophysiological tool that is available to study the functional integrity of the human retina is the electroretinogram (ERG), which measures a complex electrical mass response of the retina recorded at the cornea and generated by different cell groups. 21 Many studies sought to elucidate the cellular origins of the flash ERG, 22,23 flicker ERG, 24,25 and pattern ERG. 26  
Flicker ERG responses appear to be mainly generated by postreceptoral retinal activity. 27,28 At temporal frequencies above approximately 30 Hz, the flicker ERG is dominated by the fundamental Fourier component (first harmonic), 29 although higher harmonic components that are thought to be generated in the inner retina may sometimes be present. 24 The different harmonic components may reflect the activity of distinct retinal cells. 30 At intermediate temporal frequencies (between 10 and 20 Hz), in addition to the fundamental component, there is a substantial second harmonic contribution to luminance photopic flicker ERG responses. 29 Photoreceptors appear to make a larger contribution to the first harmonic component, 31 and the second harmonic component may represent the spiking activity of the inner retina. 25  
Although postreceptoral mechanisms strongly influence flicker ERG responses, correlating the ERG response with M and P activity in the retinogeniculate pathways is difficult. Flicker ERGs measured with heterochromatic stimuli at high temporal frequencies (above approximately 20 Hz) display properties that are similar to those of the magnocellularly based psychophysical luminance channel. 32 35 Recently, work from our laboratory provided evidence that the high-frequency flicker ERG probably directly reflects M activity. Additionally, under specific conditions (stimulus frequency of approximately 12 Hz and the presence of a red-green chromatic component), the first harmonic component of flicker ERG responses reflects cone opponent pathway activity. 36,37 The results of these studies suggest that objectively investigating the responses of the M and P pathways in glaucoma patients is possible. Furthermore, electrophysiological measurements offer the ability to study not only the amplitudes but also the phases of the responses, which is not possible with psychophysical techniques. Based on these results, the present study determined how early postreceptoral retinogeniculate pathways are affected by glaucoma. 
Subjects and Methods
Subjects
Measurements were performed in one randomly chosen eye of 32 glaucoma patients (mean age, 61 ± 11 years; 15 men, 17 women) from the Erlangen Glaucoma Registry. This group of patients was divided into two subgroups, those with perimetric open-angle glaucoma (n = 20; mean age, 63 ± 11 years; 10 men, 10 women) and those with preperimetric open-angle glaucoma (n = 12; mean age, 59 ± 11 years; 5 men, 7 women) in accordance with the criteria previously established for conventional white-on-white automated perimetry, visual acuity, and IOP. 38 Briefly, the inclusion criteria were open chamber angle, increased IOP (measurements >21 mm Hg), and abnormal appearance of the optic nerve head. Additionally, all perimetric glaucoma patients displayed glaucomatous visual field defects. Mean deviations ranged from 1.2 to 23.8 (average, 9.6 ± 6.9) in the perimetric group and from −2.2 to 2.1 (average, 0.2 ± 1.4) in the preperimetric group. The control group consisted of 24 healthy volunteers (mean age, 43 ± 11 years; 14 men, 10 women). 
All glaucoma patients (and the control subjects) were phakic and had clear lenses. Any eye disorder (such as cataract) other than glaucoma was an exclusion criterion. In addition, normal color vision was established. All participants underwent anomaloscope (HMC Anomaloskop; Oculus, Wetzlar, Germany) examination before the ERG measurements. The anomalous quotient (AQ) was within the normal range (0.7–1.4), according to the manufacturer's instruction manual. Perimetric patients had AQs from 0.9 to 1.4 (average, 1.19 ± 0.14), and preperimetric patients had AQs from 0.8 to 1.2 (average, 1.02 ± 0.17). Controls had AQs from 0.7 to 1.2 (average, 0.91 ± 0.03). The experiments adhered to the tenets of the Declaration of Helsinki and were approved by the institutional ethics committee. Signed informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study. 
Visual Stimulation
The stimuli were described previously. 37,39 Briefly, a Ganzfeld stimulator (Q450 SC; Roland-Consult, Bradenburg, Germany) with six differently colored light-emitting diodes (LEDs) was used. Only the red (638 nm; bandwidth at half height, 19 nm; CIE coordinates: x, 0.6957; y, 0.2966) and green (523 nm; bandwidth at half height, 36 nm; CIE coordinates: x, 0.2016; y, 0.7371) LED arrays were used in the present experiment. The mean luminance of the red and green LED arrays was 100 cd/m2 each, resulting in a mean retinal illuminance of approximately 104 td (assuming a 8-mm pupil diameter) with a yellow mean chromaticity of 0.5813 (x) and 0.4030 (y) (CIE 1931 coordinates). 
Figure 1A shows a sketch of the luminance outputs of the red and green LED arrays. Three different stimulus conditions were used, in which the fraction of red modulation depth was varied. At R/(R+G) = 0, only the green LEDs were modulated with 100% contrast, whereas the red LEDs were not modulated and had a constant output of 100 cd/m2. At the R/(R+G) = 0.5 condition, the red and green LEDs were modulated in counterphase with 50% contrast. R/(R+G) = 1 indicates the condition in which only the red LEDs were modulated with 100% contrast; the output of the green LEDs was 100 cd/m2 and unmodulated. The measurements were repeated at two temporal frequencies, 36 Hz and 12 Hz. 
Figure 1.
 
(A) Description of the stimuli. Red and green LEDs were modulated in counterphase in a Ganzfeld stimulator with a 100 cd/m2 mean luminance of each LED. Three different stimulus conditions with different fractions of red modulation (red/[red+green]) were used: R/(R+G) = 0.0 (only the green LED is modulated, whereas the red LED is constant at 100 cd/m2), R/(R+G) = 0.5 (red and green LEDs are modulated simultaneously in counterphase), and R/(R+G) = 1 (only the red LED is modulated; the green LED is constant at 100 cd/m2). Conditions were presented at two temporal frequencies (36 and 12 Hz). (B, l eft) Resultant luminance modulation obtained by the addition of the red and green LED outputs. Time-averaged luminance output was 200 cd/m2 for all stimulus conditions. The output of the cone opponent system (right) was obtained by the subtraction of the red and green LED outputs. This subtraction resulted in identical sinusoidal cone opponent modulations for all conditions. Furthermore, cone opponent modulation had the same phase in all conditions. Reprinted with permission from Barboni MT, Ventura DF, Kremers J. Absence of ocular interaction in flicker ERG responses reflecting cone opponent and luminance signals. Doc Ophthalmol. 2010;121:69–75. © Springer.
Figure 1.
 
(A) Description of the stimuli. Red and green LEDs were modulated in counterphase in a Ganzfeld stimulator with a 100 cd/m2 mean luminance of each LED. Three different stimulus conditions with different fractions of red modulation (red/[red+green]) were used: R/(R+G) = 0.0 (only the green LED is modulated, whereas the red LED is constant at 100 cd/m2), R/(R+G) = 0.5 (red and green LEDs are modulated simultaneously in counterphase), and R/(R+G) = 1 (only the red LED is modulated; the green LED is constant at 100 cd/m2). Conditions were presented at two temporal frequencies (36 and 12 Hz). (B, l eft) Resultant luminance modulation obtained by the addition of the red and green LED outputs. Time-averaged luminance output was 200 cd/m2 for all stimulus conditions. The output of the cone opponent system (right) was obtained by the subtraction of the red and green LED outputs. This subtraction resulted in identical sinusoidal cone opponent modulations for all conditions. Furthermore, cone opponent modulation had the same phase in all conditions. Reprinted with permission from Barboni MT, Ventura DF, Kremers J. Absence of ocular interaction in flicker ERG responses reflecting cone opponent and luminance signals. Doc Ophthalmol. 2010;121:69–75. © Springer.
Figure 1B displays the luminance (left) and red-green color opponent (right) output of the 12-Hz stimuli. When R/(R+G) = 0 and 1, the luminance modulation was large and of equal amplitude. At R/(R+G) = 0.5, no luminance was output (note that the luminance output was based on a luminance system with a spectral sensitivity according to the Vλ 40 ; different persons may display deviating spectral sensitivities so that the luminance output may differ slightly in them). The luminance modulation followed the green LEDs at R/(R+G) = 0 and red LEDs at R/(R+G) = 1, so a 180° phase luminance shift occurred between the two conditions. Not only the amplitude but also the phase of the cone opponent system's output was the same in all three conditions because the stimulus was redder in the first half of a stimulus period and greener in the second half under all conditions. The sinusoidal shape of the luminance output and cone opponent system had constant time-averaged values, indicating that the state of adaptation was the same for all stimulus conditions (assuming that the 36-Hz and 12-Hz stimulus frequencies were too high for adaptation processes). See Kremers et al. 37 for details regarding a protocol that allows the measurement of different R/(R+G) ratios and Barboni et al. 39 for details regarding the short version of the protocol that was used in the present work. The short protocol does not allow the study of individual equiluminant points, which may vary. On the other hand, one intermediate condition was considered useful to verify whether the response amplitudes and phases followed the characteristic responses previously reported, 37 for a large population of healthy subjects in the present study. In addition, this short protocol, taking approximately 5 minutes of measurement, can be easily applied in patients. 
Figure 2 shows the estimated response amplitudes (Fig. 2A, in arbitrary units) and phases (Fig. 2B, in degrees) of luminance (black) and red-green cone opponent (blue) mechanisms as a function of the stimulus condition. The S-cone input is assumed to be negligible, and the total mean luminance of 200 cd/m2 is assumed to be too high for substantial rod input. The absence of an S-cone and a rod-driven signal was shown previously by performing the measurements in a dichromatic subject. 37 Subsequently, we confirmed these data on other dichromats (unpublished data, 2010). Observe that the estimated response amplitudes and phases follow the stimulus luminance and cone opponent modulation, respectively (Fig. 1B). 
Figure 2.
 
Calculated response amplitudes (A, arbitrary units) and phases (B, degrees) of the luminance (black) and red-green cone opponent (blue) output as a function of the stimulus condition.
Figure 2.
 
Calculated response amplitudes (A, arbitrary units) and phases (B, degrees) of the luminance (black) and red-green cone opponent (blue) output as a function of the stimulus condition.
ERG Recordings
One eye was dilated with a drop of mydriaticum (0.5% tropicamide), and corneal ERG responses were measured with a DTL fiber electrode attached from the outer to inner canthus of the eye. The reference and ground skin electrodes were attached to the ipsilateral temple and forehead, respectively. The signals were amplified 100,000×, filtered between 1 and 300 Hz, and sampled at 1024 Hz using an electroretinography system (RetiPort; Roland Consult). At least 24 (36-Hz) and 48 (12-Hz) 1-second episodes were averaged to obtain a large signal-to-noise ratio at both temporal frequencies. 
Statistical Analysis
The recordings were Fourier analyzed using self-written software (MatLab; The MathWorks, Natick, MA). The amplitudes (in microvolts) and phases (in degrees) of the first (fundamental) and second harmonic components were statistically compared using analysis of variance (ANOVA) with subsequent multiple comparisons (including a Bonferroni post hoc correction for multiple testing) between controls and glaucoma patients. Pearson correlation coefficients were used to verify the age effect. Noise was quantified by the average of the response amplitudes of the 35- and 37-Hz components and the 11- and 13-Hz components for the 36- and 12-Hz stimulus conditions, respectively. Phase values were disregarded when the signal-to-noise ratio was <3. 
Results
Figure 3 shows individual recordings of one control, one perimetric, and one preperimetric patient at 36 Hz (upper recordings) and 12 Hz (lower recordings). Consistent with previous data, 37,39 the 36-Hz response amplitudes of all subjects depended strongly on the R/(R+G) values. When R/(R+G) = 0 and 1, the responses were large, whereas they were small in the condition R/(R+G) = 0.5. Close inspection shows that the responses at R/(R+G) = 0 and 1 were in counterphase with each other. The 12-Hz responses displayed relatively constant amplitudes and phases for the different stimulus conditions. However, substantial frequency doubling was observed in all subjects, especially for the condition R/(R+G) = 1. 
Figure 3.
 
Individual recordings of one control, one preperimetric patient, and one perimetric patient for the three conditions tested at 36 Hz (upper) and 12 Hz (lower). The epoch shown is 250 ms. At 36 Hz, the response amplitudes changed strongly with R/(R+G) values. Responses at R/(R+G) = 0 and R/(R+G) = 1 display larger amplitudes and are in counterphase to each other. At 12 Hz, the responses display relatively constant amplitudes and phases for the different conditions.
Figure 3.
 
Individual recordings of one control, one preperimetric patient, and one perimetric patient for the three conditions tested at 36 Hz (upper) and 12 Hz (lower). The epoch shown is 250 ms. At 36 Hz, the response amplitudes changed strongly with R/(R+G) values. Responses at R/(R+G) = 0 and R/(R+G) = 1 display larger amplitudes and are in counterphase to each other. At 12 Hz, the responses display relatively constant amplitudes and phases for the different conditions.
Figure 4 shows the averaged (± SE) response amplitudes using 36-Hz (left) and 12-Hz (right) stimuli measured in perimetric patients (circles), preperimetric patients (triangles), and controls (squares). The upper plots show the first harmonic data, and the lower plots show the second harmonic data as a function of red fraction (R/[R+G]). Figure 5 displays the corresponding response phases. A single asterisk indicates statistically significant differences (P < 0.05, Bonferroni correction for multiple tests) compared with controls, and double asterisks indicate statistically significant differences compared with the other two groups. 
Figure 4.
 
Averaged (± SE) response amplitudes at 36 Hz (left) and 12 Hz (right) in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic amplitudes; lower: second harmonic amplitudes. No significant differences were observed (36 Hz, P = 0.08; 12 Hz, P = 0.09; ANOVA followed by Bonferroni post hoc correction).
Figure 4.
 
Averaged (± SE) response amplitudes at 36 Hz (left) and 12 Hz (right) in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic amplitudes; lower: second harmonic amplitudes. No significant differences were observed (36 Hz, P = 0.08; 12 Hz, P = 0.09; ANOVA followed by Bonferroni post hoc correction).
Figure 5.
 
Averaged (± SE) response phases at 36 Hz (left) and 12 Hz (right) temporal frequencies in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic phases; lower: second harmonic phases. *Statistically significant difference compared with controls only. **Statistically significant difference compared with the other two groups. A significant phase change was observed in perimetric patients compared with the control group for the first harmonic response in condition R/(R+G) = 1 at 36 Hz (P = 0.0004). The second harmonic response phases at 36 Hz in conditions R/(R+G) = 0 and 1 are different between perimetric patients and controls (P = 0.03 and P = 0.001, respectively) and also between preperimetric patients and controls (P = 0.04 and P = 0.03, respectively). At 12 Hz, the first harmonic response phases were higher for controls (P = 0.0001) and preperimetric patients (P = 0.01) than for perimetric patients, and the second harmonic response phases were lower for controls (P = 0.0008) and preperimetric patients (P = 0.01) than for perimetric patients.
Figure 5.
 
Averaged (± SE) response phases at 36 Hz (left) and 12 Hz (right) temporal frequencies in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic phases; lower: second harmonic phases. *Statistically significant difference compared with controls only. **Statistically significant difference compared with the other two groups. A significant phase change was observed in perimetric patients compared with the control group for the first harmonic response in condition R/(R+G) = 1 at 36 Hz (P = 0.0004). The second harmonic response phases at 36 Hz in conditions R/(R+G) = 0 and 1 are different between perimetric patients and controls (P = 0.03 and P = 0.001, respectively) and also between preperimetric patients and controls (P = 0.04 and P = 0.03, respectively). At 12 Hz, the first harmonic response phases were higher for controls (P = 0.0001) and preperimetric patients (P = 0.01) than for perimetric patients, and the second harmonic response phases were lower for controls (P = 0.0008) and preperimetric patients (P = 0.01) than for perimetric patients.
Consistent with previous data, 37 the first harmonic responses at 36 and 12 Hz followed the luminance and cone opponent output of the stimuli, respectively (Fig. 2, upper graph). Accordingly, the three groups displayed a minimal first harmonic response amplitude in condition R/(R+G) = 0.5 at 36 Hz and did not vary with stimulus condition at 12 Hz (Fig. 4, upper graphs). These data support the possibility of assessing different visual mechanisms using heterochromatically modulating stimuli, as previously proposed. 37 At 36 Hz, the first harmonic response amplitudes were similar for both groups of glaucoma patients and the controls at all stimulus conditions. At 12 Hz, the first harmonic amplitude was larger for perimetric patients in condition R/(R+G) = 0, but the difference was not significant (P = 0.09). 
The second harmonic response amplitudes at 36 Hz were larger in perimetric patients in condition R/(R+G) = 1 than in controls, but the difference was also not significant (P = 0.08). The second harmonic amplitudes were similar for the three groups at 12 Hz for all conditions. The second harmonic component at 12 Hz showed a similar dependency on R/(R+G) as the first harmonic component at 36 Hz; the responses were small for R/(R+G) = 0.5 and large for R/(R+G) = 0 and 1, with the response larger for the red fraction of R/(R+G) = 1 than R/(R+G) = 0 values (Fig. 4, lower graphs). This might indicate a residual input from a luminance mechanism at 12 Hz. 
Phase data are shown in Figure 5. At 36 Hz, a 180° difference was observed between the first harmonic phases in conditions R/(R+G) = 0 and R/(R+G) = 1 for the three groups. In contrast, at 12 Hz, the first harmonic response phases were relatively constant for patients and controls (Fig. 5, upper graphs; note the different scales of the y-axes for the 36-Hz and 12-Hz data). This is consistent with the proposal that luminance and cone opponent mechanisms determine the responses at 36 and 12 Hz, respectively 37 (see also Fig. 2, lower graph). 
Phase differences between patients and controls were found at 36 and 12 Hz (Fig. 5). The first-harmonic response phases at 36 Hz were higher for the controls, indicating a response delay in the patients compared with controls, but only in condition R/(R+G) = 1 was the difference between perimetric patients and controls significant (P = 0.0004). The second harmonic response phases at 36 Hz in conditions R/(R+G) = 0 and 1 were different between perimetric patients and controls (P = 0.03 and P = 0.001, respectively) and also between preperimetric patients and controls (P = 0.04 and P = 0.03, respectively). 
The estimated time delay of the 36-Hz first harmonic responses was 2 ms for the perimetric patients compared with the controls (there was no significant difference between perimetric and preperimetric glaucoma patients). For the 36-Hz second harmonic responses, the estimated time delays between glaucoma patients (both perimetric and preperimetric) and controls were approximately 1.8 and 3 ms for the R/(R+G) = 0 and R/(R+G) = 1 conditions, respectively (assuming that the delay is less than one period). Although these time delays are small, the phases can be measured very reliably so that very small delay changes can be detected. 
At 12 Hz, the first and second harmonic response phases were different between perimetric patients and the other two groups in condition R/(R+G) = 0. The first harmonic response phases were higher for the controls (P = 0.0001) and preperimetric patients (P = 0.01) than for the perimetric patients. The second harmonic response phases were lower for the controls (P = 0.0008) and preperimetric patients (P = 0.01) than for the perimetric patients. 
The estimated time delay for the perimetric patients was approximately 10 and 5.5 ms compared with the preperimetric patients and the controls at 12 Hz in the first and second harmonic components respectively. 
To verify the relationship between visual field losses and ERG response phases in glaucoma patients, response phases were plotted as a function of mean deviation values in perimetric (circles) and preperimetric (triangles) glaucoma (Fig. 6) patients. The parameters used for this comparison were the first and the second harmonic response phases for condition R/(R+G) = 0, in which phases are significantly different between perimetric and preperimetric patients. There were significant correlations between mean deviation values and response phases for both first (P = 0.01 and r 2 = 0.25) and second (P = 0.04 and r 2 = 0.15) harmonic response phases. 
Figure 6.
 
First harmonic (left) and second harmonic (right) response phases at 12 Hz [condition R/(R+G) = 0] as a function of the visual field index: mean deviation. C ircles: perimetric patients; triangles: preperimetric patients. The parameters used for this comparison were significantly different between perimetric and preperimetric patients. There were significant correlations between mean deviation values and response phases for both first (P = 0.01 and r 2 = 0.25) and second (P = 0.04 and r 2 = 0.15) harmonic response phases.
Figure 6.
 
First harmonic (left) and second harmonic (right) response phases at 12 Hz [condition R/(R+G) = 0] as a function of the visual field index: mean deviation. C ircles: perimetric patients; triangles: preperimetric patients. The parameters used for this comparison were significantly different between perimetric and preperimetric patients. There were significant correlations between mean deviation values and response phases for both first (P = 0.01 and r 2 = 0.25) and second (P = 0.04 and r 2 = 0.15) harmonic response phases.
The average age of the control group (43 ± 11 years) in the present study was 18 years younger than the average age of the glaucoma patients (61 ± 11 years). To verify whether the phase differences between patients and controls were not attributable to differences in age, response phases were plotted as a function of age. Figures 7 and 8 show individual first (upper graphs) and second (lower graphs) harmonic response phases at 36 Hz (left) and 12 Hz (right) as a function of age for the controls and patients, respectively. No correlation was found between age and response phases in either controls (P > 0.4) or patients (P > 0.2). 
Figure 7.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in controls as a function of age. No correlation was found between age and response phases (P > 0.6, Pearson correlation).
Figure 7.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in controls as a function of age. No correlation was found between age and response phases (P > 0.6, Pearson correlation).
Figure 8.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in the glaucoma group (including perimetric and preperimetric patients) as a function of age. No correlation was found between age and response phases (P > 0.2, Pearson correlation).
Figure 8.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in the glaucoma group (including perimetric and preperimetric patients) as a function of age. No correlation was found between age and response phases (P > 0.2, Pearson correlation).
Although no amplitude differences were found between subject groups, the same analysis was performed for the response amplitudes (data not shown), and no correlation was found in controls (P > 0.2) or patients (P > 0.7). 
Discussion
Phase differences were evident in glaucoma patients at 36 Hz (for the perimetric group in the first harmonic and for both glaucoma groups in the second harmonic) and 12 Hz (only for the perimetric group in both the first and second harmonics). No amplitude differences were observed. In addition, the visual field loss was correlated with the response phases in conditions in which perimetric and preperimetric patients displayed different response phases. 
The first harmonic components of responses to heterochromatic modulation were previously proposed to reflect the activity of the luminance mechanism at high temporal frequencies and the red-green cone opponent mechanism at intermediate temporal frequencies (12 Hz). 36,37 At 12 Hz, the flicker ERG that uses L- and M-cone–isolating stimuli reflects postreceptoral mechanisms that belong to the cone opponent channel because L- and M-cone–driven signals are in counterphase with each other and have approximately equal amplitudes, indicating cone opponent response properties. Less inter-individual variability is also observed at 12 Hz, and it is less influenced by chromatic adaptation. At higher temporal frequencies (approximately 30 Hz and higher), the flicker ERG for L- and M-cone–isolating stimuli may be mediated by the luminance mechanism because the modulation is beyond the fusion frequency of chromatic flicker. The physiological implication is that the flicker ERG that uses L- and M-cone–isolating stimuli is assumed to be determined by M pathway activity at higher temporal frequencies and may reflect P pathway activity at 12 Hz. 36,37,41 43 The present data show that it is possible to assess the activity of luminance and cone opponent mechanisms using a fast protocol that allows testing patients. In addition, we suggest that the second harmonic components may be also a valuable index for investigating retinal abnormalities in glaucoma. 
Previous studies 24,25,27,29 31,44 provided a better understanding of the contribution of different components of flicker ERG responses. However, assigning a specific response component to a group of cells was not possible. The present study showed that the second harmonic response amplitude was relatively small at 36 Hz. Nevertheless, closer inspection of the second harmonic components at 36 Hz showed that they and the first harmonic component similarly depend on the stimulus condition (Figs. 4, 5, left graphs). This indicates that the ERG responses are not purely sinusoidal. However, intrusion from another pathway is not apparent. In contrast, at 12 Hz, the first and second harmonic response components differently depended on the red fraction of the red-green counterphase-modulated stimulus (Figs. 4, 5, right graphs), indicating that they may reflect the activity of distinct pathways. At this temporal frequency, the second harmonic component and the 36-Hz first harmonic responses similarly depended on the red fraction (Figs. 4, 5, upper left graphs). The present data, therefore, suggest that the activity of the luminance mechanism may determine the second harmonic component of heterochromatic-modulated flicker ERGs at 12 Hz. 
A previous study on L- and M-cone–driven ERGs showed that the response phases in full-field ERGs may be a more sensitive indicator than response amplitudes of the differences between control subjects and glaucoma patients (Link et al. IOVS 2007;48:ARVO E-Abstract 527). Compared with controls, glaucoma patients with visual field defects had different response phases in the ERG signals driven by the P pathway. Consistent with these findings, our data suggest that response phases may be a more sensitive indicator of glaucomatous changes than amplitudes. However, the study by Link et al. (IOVS 2007;48:ARVO E-Abstract 527) and the present work have four main differences: in their study, the patients were only perimetric glaucoma subjects, a CRT monitor was used as a stimulator, the stimuli were L- and M-cone isolating stimuli rather than red-green modulations, and, finally, their stimuli silenced the rods. The present study used stimuli that in principle also stimulated rods and S-cones. By comparing the responses with those in dichromats, we showed that neither played a role in the responses. 37 Subsequent measurements with dichromats (unpublished data, 2010) confirmed this conclusion. Additionally, in the present study, an LED Ganzfeld stimulator with narrow band emission spectra was used. As a result, larger cone contrasts, higher mean luminances, and therefore larger responses were feasible than were possible with a CRT monitor. The larger responses enabled a more reliable phase analysis. 
The present results in glaucoma patients with visual field losses (perimetric group) showed phase changes at both temporal frequencies, 36 and 12 Hz, which may represent luminance and cone opponent mechanisms, respectively. The changes probably occurred on a postreceptoral level; if the changes were in the cone-driven responses, we would have expected to find the same differences in the response phases at 12 and 36 Hz, which was not the case. 
The data presented here are consistent with previous psychophysical 8,9 and electrophysiological 13,45 data that showed nonselective dysfunction of retinogeniculate visual pathways in glaucoma patients. The glaucoma patients without visual field defects (preperimetric group) showed phase changes for the second harmonic responses at 36 Hz. Spiking activity in the inner retina has been shown to contribute to the second harmonic responses of photopic flicker ERGs, 25 suggesting that these phases may be correlated with spiking activity. This is possibly consistent with data that showed early changes in the pattern ERG in glaucoma patients. 46,47 The response phases of flicker ERGs have been previously shown to be useful for detecting retinal abnormalities. 48 50 The amplitude and phase responses may represent two distinct aspects of neural activity. 
In contrast with previous data, 51 54 we did not find an age effect either on response amplitudes or on phases. However, age-related effects are mainly observed with short-wavelength stimuli, possibly through age-related changes in preretinal absorption, which may primarily affect S-cone–driven ERGs. 55 In the present study, we used stimuli in which short wavelengths are absent. This may explain the absence of a clear age-related effect. Additionally, ERG changes caused by lens senescence would affect the response amplitude but not necessarily the response phase. Thus, the method described here may be less sensitive to age-related effects. 
Conclusion
The present data showed that ERG responses to heterochromatic stimuli might have altered phases but no amplitude changes in perimetric glaucoma patients. Glaucoma patients without visual field defects (preperimetric) show phase changes at 36 Hz in the second harmonic component, possibly representing early dysfunction. Moreover, the correlation between response phases and visual field index indicates that the method may be important for monitoring glaucoma progression and possible therapeutic effects in the future. Heterochromatic flicker ERG responses provide a noninvasive functional assessment of the luminance and cone opponent retinal mechanisms without sensitivity to the effects of age. We therefore conclude that the ERG method presented here may become a valuable diagnostic tool. 
Footnotes
 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2010.
Footnotes
 Supported by German Research Council Grant KR1317/9-1. MTSB received an FAPESP fellowship for graduate students (07/55125-1). DFV is a CNPq Research Fellow. JK is Fellow in the Excellence Program of the Hertie Foundation.
Footnotes
 Disclosure: M.T.S. Barboni, None; G. Pangeni, None; D.F. Ventura, None; F. Horn, None; J. Kremers, None
The authors thank Sylvia Rühl, Anja Erhardt, Astrid Kraus, and Edith Monczak for their collaboration with the measurements. 
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Figure 1.
 
(A) Description of the stimuli. Red and green LEDs were modulated in counterphase in a Ganzfeld stimulator with a 100 cd/m2 mean luminance of each LED. Three different stimulus conditions with different fractions of red modulation (red/[red+green]) were used: R/(R+G) = 0.0 (only the green LED is modulated, whereas the red LED is constant at 100 cd/m2), R/(R+G) = 0.5 (red and green LEDs are modulated simultaneously in counterphase), and R/(R+G) = 1 (only the red LED is modulated; the green LED is constant at 100 cd/m2). Conditions were presented at two temporal frequencies (36 and 12 Hz). (B, l eft) Resultant luminance modulation obtained by the addition of the red and green LED outputs. Time-averaged luminance output was 200 cd/m2 for all stimulus conditions. The output of the cone opponent system (right) was obtained by the subtraction of the red and green LED outputs. This subtraction resulted in identical sinusoidal cone opponent modulations for all conditions. Furthermore, cone opponent modulation had the same phase in all conditions. Reprinted with permission from Barboni MT, Ventura DF, Kremers J. Absence of ocular interaction in flicker ERG responses reflecting cone opponent and luminance signals. Doc Ophthalmol. 2010;121:69–75. © Springer.
Figure 1.
 
(A) Description of the stimuli. Red and green LEDs were modulated in counterphase in a Ganzfeld stimulator with a 100 cd/m2 mean luminance of each LED. Three different stimulus conditions with different fractions of red modulation (red/[red+green]) were used: R/(R+G) = 0.0 (only the green LED is modulated, whereas the red LED is constant at 100 cd/m2), R/(R+G) = 0.5 (red and green LEDs are modulated simultaneously in counterphase), and R/(R+G) = 1 (only the red LED is modulated; the green LED is constant at 100 cd/m2). Conditions were presented at two temporal frequencies (36 and 12 Hz). (B, l eft) Resultant luminance modulation obtained by the addition of the red and green LED outputs. Time-averaged luminance output was 200 cd/m2 for all stimulus conditions. The output of the cone opponent system (right) was obtained by the subtraction of the red and green LED outputs. This subtraction resulted in identical sinusoidal cone opponent modulations for all conditions. Furthermore, cone opponent modulation had the same phase in all conditions. Reprinted with permission from Barboni MT, Ventura DF, Kremers J. Absence of ocular interaction in flicker ERG responses reflecting cone opponent and luminance signals. Doc Ophthalmol. 2010;121:69–75. © Springer.
Figure 2.
 
Calculated response amplitudes (A, arbitrary units) and phases (B, degrees) of the luminance (black) and red-green cone opponent (blue) output as a function of the stimulus condition.
Figure 2.
 
Calculated response amplitudes (A, arbitrary units) and phases (B, degrees) of the luminance (black) and red-green cone opponent (blue) output as a function of the stimulus condition.
Figure 3.
 
Individual recordings of one control, one preperimetric patient, and one perimetric patient for the three conditions tested at 36 Hz (upper) and 12 Hz (lower). The epoch shown is 250 ms. At 36 Hz, the response amplitudes changed strongly with R/(R+G) values. Responses at R/(R+G) = 0 and R/(R+G) = 1 display larger amplitudes and are in counterphase to each other. At 12 Hz, the responses display relatively constant amplitudes and phases for the different conditions.
Figure 3.
 
Individual recordings of one control, one preperimetric patient, and one perimetric patient for the three conditions tested at 36 Hz (upper) and 12 Hz (lower). The epoch shown is 250 ms. At 36 Hz, the response amplitudes changed strongly with R/(R+G) values. Responses at R/(R+G) = 0 and R/(R+G) = 1 display larger amplitudes and are in counterphase to each other. At 12 Hz, the responses display relatively constant amplitudes and phases for the different conditions.
Figure 4.
 
Averaged (± SE) response amplitudes at 36 Hz (left) and 12 Hz (right) in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic amplitudes; lower: second harmonic amplitudes. No significant differences were observed (36 Hz, P = 0.08; 12 Hz, P = 0.09; ANOVA followed by Bonferroni post hoc correction).
Figure 4.
 
Averaged (± SE) response amplitudes at 36 Hz (left) and 12 Hz (right) in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic amplitudes; lower: second harmonic amplitudes. No significant differences were observed (36 Hz, P = 0.08; 12 Hz, P = 0.09; ANOVA followed by Bonferroni post hoc correction).
Figure 5.
 
Averaged (± SE) response phases at 36 Hz (left) and 12 Hz (right) temporal frequencies in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic phases; lower: second harmonic phases. *Statistically significant difference compared with controls only. **Statistically significant difference compared with the other two groups. A significant phase change was observed in perimetric patients compared with the control group for the first harmonic response in condition R/(R+G) = 1 at 36 Hz (P = 0.0004). The second harmonic response phases at 36 Hz in conditions R/(R+G) = 0 and 1 are different between perimetric patients and controls (P = 0.03 and P = 0.001, respectively) and also between preperimetric patients and controls (P = 0.04 and P = 0.03, respectively). At 12 Hz, the first harmonic response phases were higher for controls (P = 0.0001) and preperimetric patients (P = 0.01) than for perimetric patients, and the second harmonic response phases were lower for controls (P = 0.0008) and preperimetric patients (P = 0.01) than for perimetric patients.
Figure 5.
 
Averaged (± SE) response phases at 36 Hz (left) and 12 Hz (right) temporal frequencies in perimetric patients (closed circles), preperimetric patients (closed triangles), and controls (open squares). U pper: first harmonic phases; lower: second harmonic phases. *Statistically significant difference compared with controls only. **Statistically significant difference compared with the other two groups. A significant phase change was observed in perimetric patients compared with the control group for the first harmonic response in condition R/(R+G) = 1 at 36 Hz (P = 0.0004). The second harmonic response phases at 36 Hz in conditions R/(R+G) = 0 and 1 are different between perimetric patients and controls (P = 0.03 and P = 0.001, respectively) and also between preperimetric patients and controls (P = 0.04 and P = 0.03, respectively). At 12 Hz, the first harmonic response phases were higher for controls (P = 0.0001) and preperimetric patients (P = 0.01) than for perimetric patients, and the second harmonic response phases were lower for controls (P = 0.0008) and preperimetric patients (P = 0.01) than for perimetric patients.
Figure 6.
 
First harmonic (left) and second harmonic (right) response phases at 12 Hz [condition R/(R+G) = 0] as a function of the visual field index: mean deviation. C ircles: perimetric patients; triangles: preperimetric patients. The parameters used for this comparison were significantly different between perimetric and preperimetric patients. There were significant correlations between mean deviation values and response phases for both first (P = 0.01 and r 2 = 0.25) and second (P = 0.04 and r 2 = 0.15) harmonic response phases.
Figure 6.
 
First harmonic (left) and second harmonic (right) response phases at 12 Hz [condition R/(R+G) = 0] as a function of the visual field index: mean deviation. C ircles: perimetric patients; triangles: preperimetric patients. The parameters used for this comparison were significantly different between perimetric and preperimetric patients. There were significant correlations between mean deviation values and response phases for both first (P = 0.01 and r 2 = 0.25) and second (P = 0.04 and r 2 = 0.15) harmonic response phases.
Figure 7.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in controls as a function of age. No correlation was found between age and response phases (P > 0.6, Pearson correlation).
Figure 7.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in controls as a function of age. No correlation was found between age and response phases (P > 0.6, Pearson correlation).
Figure 8.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in the glaucoma group (including perimetric and preperimetric patients) as a function of age. No correlation was found between age and response phases (P > 0.2, Pearson correlation).
Figure 8.
 
Individual first (upper) and second (lower) harmonic response phases at 36 Hz (left) and 12 Hz (right) in the glaucoma group (including perimetric and preperimetric patients) as a function of age. No correlation was found between age and response phases (P > 0.2, Pearson correlation).
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