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(Investigative Ophthalmology and Visual Science. 2002;43:2500-2507.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Post-Photoreceptoral Activity Dominates Primate Photopic 32-Hz ERG for Sine-, Square-, and Pulsed Stimuli

Mineo Kondo1,2 and Paul A. Sieving1,3

1 From the Department of Ophthalmology and Visual Sciences, W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan; the 2 Department of Ophthalmology; Nagoya University School of Medicine, Nagoya, Japan; and the 3 National Eye Institute, National Institutes of Health, Bethesda, Maryland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate the relative contributions of photoreceptors and postphotoreceptoral activity to the primate 32-Hz flicker electroretinogram (ERG) elicited by sine-wave, square-wave, and pulse stimuli.

METHODS. Flicker 32-Hz ERGs were evoked from four adult rhesus (Macaca mulatta) monkeys using sine-wave, square-wave, and 4-ms pulse trains and xenon photostrobe flicker stimuli. All stimuli had time-averaged luminance of 2.11 log cd/m2 and were presented on a 1.63-log cd/m2 white background. Intravitreal injections of DL-2-amino-4-phosphonobutyric acid (APB) and cis-2,3-piperidinedicarboxylic acid (PDA) were given to block activity of ON- and OFF-bipolar cells, respectively.

RESULTS. Flicker harmonic analysis showed that the fundamental frequency component provided nearly 75% of the sine-wave and square-wave ERGs versus 63% for 4-ms pulse stimuli and only 49% for strobe flicker. Strobe-flicker responses contained the greatest contribution from higher harmonic components. Removing the ON component with APB increased the fundamental component’s amplitudes by more than 30% with sine-wave and square-wave ERGs but had a lesser effect on responses to 4-ms pulses and strobe flicker. When cone responses were isolated by synaptic blockade with APB+PDA, the fundamental component’s amplitude was reduced to less than 20% of control for all four stimuli. Postsynaptic ON and OFF components were characterized by amplitude and phase vectors, and sine-wave and square-wave stimuli gave a large phase difference (138°) between ON and OFF components, which resulted in greater response self-cancellation than with the 4-ms pulse train (121° phase difference) or for strobe flicker (118°).

CONCLUSIONS. The major decrease in flicker responses after photoreceptor synaptic blockade implicates a major contribution from postphotoreceptoral activity to the fundamental flicker component, regardless of the stimulus type. Sine-wave and square-wave stimuli produced larger phase differences between ON- and OFF-pathway components, thereby causing more complete self-cancellation of postphotoreceptoral contributions and revealing slightly greater relative contribution directly from cone photoreceptors with these stimuli than with pulsed stimuli. The direct cone contribution was always small, however, and the clinical point is that 32-Hz flicker ERG amplitudes do not provide an unambiguous assessment of direct cone photoreceptor contribution with any of these stimuli.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Electroretinography (ERG) is a valuable technique for studying normal retinal function in vivo and for diagnosing retinal disease in human patients. However, the exact retinal cells and circuitry that contribute to particular ERG responses are highly dependent on the specific stimulus conditions. Under photopic light-adapted conditions, which saturate the rod photoreceptors, the ERG is driven primarily by cones and the cone system. Cone activity can be isolated further by eliciting the ERG with stimuli that are flickering more rapidly than the rod flicker fusion frequency—that is, with stimuli in general higher than 20 Hz.

In the clinical setting, the fast-flicker ERG is commonly elicited by stroboscopic flashes near 30 Hz, although sine-wave or square-wave stimuli are used in some situations.1 2 3 4 5 6 However, it is not known whether sine-wave, square-wave, and pulsed flicker stimuli yield similar results. Nor is it known whether the contributions from the cone photoreceptors and postphotoreceptoral retinal activity are comparable for these different stimuli.

We have studied the origins of fast-flicker ERGs in primates by isolating retinal components with glutamate analogues.7 8 9 We found that the corneal ERG elicited by fast flicker was virtually eliminated by the combination of DL-2-amino-4-phosphonobutyric acid (APB)10 and cis-2,3-piperidinedicarboxylic acid (PDA),11 which respectively block transmission from photoreceptors to ON- and OFF-bipolar cells, (as well as affecting signaling to cells more proximal in the ON pathway, in the case of APB, and signaling to horizontal cells and cells more proximal in both the ON and OFF pathways in the case of PDA).12 From these results we concluded that cells postsynaptic to the cones play a major role in generating fast-flicker ERG responses.

In reviewing our results from two previous studies, we noted that after the activity of the ON pathway was blocked by APB, the peak-to-peak amplitude of the 30-Hz strobe-flicker ERGs decreased by nearly 40%,8 whereas 32-Hz flicker ERG responses to sine-wave stimuli actually increased by approximately 30%.9 This disparity suggested that the relative contributions from retinal ON and OFF components to fast-flicker ERGs are different for different stimuli.

We have now explored this further and have performed a direct comparison of the flicker ERG responses elicited by sine-wave, square-wave, and brief-pulse stimuli, before and after application of glutamate analogues. We matched equal time-constant luminosity and background illumination of the stimuli and used the same stimulus frequency of 32 Hz for all conditions. Responses were analyzed by vector modeling9 to investigate how photoreceptoral and postreceptoral ON and OFF components contributed to the fundamental component of each type of flicker response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Preparation
Experiments were conducted in accordance with National Institutes of Health guidelines on animal use and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Four adult rhesus (Macaca mulatta) monkeys were studied. The animals were sedated with intramuscular ketamine hydrochloride (7 mg/kg; 5–10 mg/kg per hour maintenance dose) and xylazine (0.6 mg/kg). Supplemental oxygen was provided by nasal cannula during the entire anesthesia period. Respiration and heart rate were monitored, and hydration was maintained with continuous, slow, subcutaneous infusion of lactated Ringer’s solution. Corneal anesthesia (topical proparacaine 0.5%) and full pupil dilation (topical phenylephrine HCl 2.5% and atropine 1%) were used.

Drug Application
The drugs and intravitreal injection technique have been described in detail.13 The drugs (Sigma Chemical Co., St. Louis, MO) were injected into the vitreous with a 30-gauge needle inserted through the pars plana, approximately 5 to 6 mm posterior to the limbus—a route that is atraumatic to the eye. The drugs were dissolved in sterile saline and injected in amounts of 0.05 to 0.07 mL of 40 mM APB or 200 mM PDA. Drug effects were monitored by ERG recordings to track the photopic b-wave after APB and the d-wave after PDA. Study recordings were begun after full drug effects were seen—normally, approximately 60 minutes to 90 minutes after drug injections. The studies were completed within 5 hours. Only one eye was studied at a time, except for terminal recordings.

Visual Stimulation
ERG responses were elicited by 32-Hz sine-wave, square-wave, and brief-pulse stimuli. Intensities were adjusted to the same time-averaged luminance for all stimuli (Fig. 1) . Light adaptation effects on the flicker ERG14 15 16 were minimized by maintaining a continuous white background of 1.63 log cd/m2 that was uninterrupted throughout the lengthy recording sessions and by accumulating data beginning only after 10 minutes of stimulus presentation. This background is equivalent (using human physiological optics calculations) to approximately 3.3 log scotopic troland, which effectively suppresses rod activity.



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  		Figure 1. Schematic diagram of the four types of 32-Hz flicker stimuli. Sine-wave, square-wave, and 4-ms pulse stimuli were elicited by red LEDs (623 nm peak wavelength; 8 nm half-width). Strobe flicker stimulus was produced by xenon lamp (I-16 setting) and Ganzfeld dome. All stimuli were adjusted to a time-averaged luminosity of 2.11 log cd/m2, and all were presented on a constant white background of 1.63 log cd/m2.

 
The luminance of the xenon photostrobe (PS-22; Grass Instruments, Quincy, MA) in the Ganzfeld stimulus bowl was measured with an integrating radiometer (40X-Spotmeter; United Detector Technology, Hawthorne, CA). The 32-Hz xenon flash train had a time-averaged measured luminosity of 2.11 log cd/m2 (maximum intensity I-16 setting, 0.60 log cd/s · m2 per flash). This stimulus was presented on the constant white background of 1.63 log cd/m2.

Sine-wave and square-wave flicker stimuli were produced by a 5-cm-diameter densely packed array of 102 red LEDs (623 nm peak wavelength; 8 nm half-width) that was positioned 10 cm from the eye to illuminate a ping-pong ball hemisphere (40 mm diameter) that was placed immediately in front of the eye to give wide-field stimulation.9 LED modulation was controlled by a digital function generator (Model 39; Wavetek, San Diego, CA), which was fed into a linear power amplifier. The sine-wave and square-wave stimuli had minimum and maximum intensities of 1.35 and 2.37 log cd/m2, giving the same mean luminance of 2.11 log cd/m2 that was used for both of the xenon photostrobe 32-Hz flicker stimuli, and, as with these other stimuli, the sine-wave and square-wave stimuli were presented on the constant white background of 1.63 log cd/m2, giving a net modulation depth of 62%.

Because the strobe flicker ERG was elicited by a "white" flash, whereas the sine-wave and square-wave flicker ERGs were elicited with the "red" LED stimulus, we also compared results with 4-ms brief-pulse, 32-Hz flicker stimuli evoked with the 623-nm red LED array. We used 4-ms red pulses, because this was the shortest duration capable of giving a mean luminance of 2.11 log cd/m2, equal to the other stimuli (Fig. 1) . This stimulus also was presented on the white background of 1.63 log cd/m2.

Recording and Analysis
After an initial 10 minutes of light adaptation by the white background of 1.63 log cd/m2, steady state flicker ERGs were recorded using a Burian-Allen bipolar corneal contact lens electrode (Hansen Ophthalmic Development Laboratories, Iowa City, IA). A ground electrode was placed on the ipsilateral ear. Responses were amplified with a 3-dB band pass of 0.1 to 1000 Hz and digitized at a 5102-Hz rate. Twenty to 50 response epochs of 800 ms each were averaged into the single traces in Figure 2 .



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  		Figure 2. Three left columns: Response waveforms of 32-Hz flicker ERGs from three different animals elicited by sine-wave, square-wave, 4-ms pulse, and strobe flicker stimuli. Right column: averaged response from the three animals, derived by summing the three different responses and dividing by three. The dotted lines beneath the responses show the stimuli monitored by a photodiode.

 
The amplitude and phase of the fundamental components were analyzed with Fourier coefficients of the response.17 18 The phase lag was specified as positive values and was drawn on polar plots counterclockwise from the positive x-axis, as in our earlier studies.8 9 18


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
32-Hz Flicker Control ERGs
The waveforms of the 32-Hz flicker ERGs elicited by the sine-wave, square-wave, 4-ms pulse train, and strobe-flicker stimuli are shown in Figure 2 . Response amplitudes were smallest with sine-wave and square-wave, intermediate with 4-ms pulse, and largest with strobe-flicker stimuli, with mean peak-to-peak amplitude ratios of 1.0, 1.3, 2.4, and 4.3 (measured from trough to peak, and given relative to the sine-wave response). Thus, even under constant luminosity, the amplitudes of the 32-Hz flicker ERG differ considerably for the four different stimuli. The waveforms also appeared quite different for the four types of stimuli and were more sinusoidal with sine-wave and square-wave stimuli than for the brief-pulse stimuli.

Harmonic analysis8 18 was used to determine the extent to which the fundamental and higher harmonic components were represented in the ERG responses elicited by the four types of stimuli (Fig. 3) . The response spectra contained peaks at the stimulus frequency (fundamental component, 1F) and higher harmonics (2F–6F) with all four stimuli. The majority of the response was contained in the fundamental component with sine-wave (73.2% ± 8.1%, n = 4 animals) and for square-wave (74.8% ± 5.5%, n = 4) stimulation, but to a lesser extent for the 4-ms pulse train (63.0% ± 6.9%, n = 4) and for xenon strobe flicker (49.7% ± 3.1%, n = 4). The second harmonic component was present to a greater degree in responses to the 4-ms pulse train and to the strobe flicker than in those to sine-wave and square-wave stimuli. The similarity of response harmonic content in response to sine-wave and square-wave stimulation (and the difference of both compared with response to the 4-ms pulse train) is interesting, because square-wave stimuli are harmonically complex compared to the single, fundamental component of a sine-wave stimulus, indicating that the retinal ERG generators are not responding as Fourier devices. No period-doubling subharmonic component was evident, even when we analyzed only a single response epoch to a single train of flashes to avoid possible masking of subharmonics that might occur by asynchronous averaging with respect to the subharmonic period (e.g., the need to average precisely across every second flicker stimulus period to observe the one-half harmonic).



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  		Figure 3. The power spectra of 32-Hz flicker ERGs elicited with sine-wave, square-wave, 4-ms pulse, and strobe flicker stimuli. Right: power spectra determined from the waveforms averaged from three animals (from the rightmost column of Fig. 2 ). Left: amplitude mean ± SD for each harmonic component.

 
Effect of APB and PDA
APB affected the amplitude to a quite different extent with sine-wave and square-wave stimuli compared with the response amplitudes elicited by the 4-ms pulses and strobe-flicker stimuli (Fig. 4) . Compared with the no-drug control, peak-to-peak amplitude after APB actually increased with both sine-wave (134.4% ± 19.5%, n = 3) and square-wave (140.2% ± 18.9%, n = 3) stimuli, whereas amplitude after APB was nearly unchanged from control with the 4-ms pulse train (92.4% ± 11.7%, n = 3) and was decreased with the strobe flicker (66.1% ± 22.7%, n = 3). This indicates that blocking postsynaptic ON pathway activity affected the flicker response in different ways with the different types of flicker stimuli and that the sine-wave stimulus elicited markedly different responses than the strobe flicker.



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  		Figure 4. Photopic 32-Hz flicker ERGs to sine-wave, square-wave, 4m-pulse, and strobe flicker stimuli before and after glutamate analogues. Three left columns: ERGs before drugs (black), after APB alone (blue), and after APB+PDA (red) in three monkeys. Right column: ERGs before drugs (black), after PDA alone (blue), and APB+PDA (red) in one monkey. Note that the initial voltage points have no absolute value for steady state flicker responses under AC-coupled recording conditions. These traces may be shifted vertically relative to each other, and the only information that should be extracted from these steady state flicker responses is peak-to-peak amplitude and the overall waveform shape.

 
We were interested in learning how PDA (which affects OFF-pathway transmission, plus having effects on horizontal cells and third-order cells in both pathways) would change the flicker amplitude for the four types of stimuli. For one monkey we applied PDA first, followed by APB (right-most column of Fig. 4 ). The amplitude after PDA alone became larger with sine-wave and square-wave stimuli, whereas it did not change much with the 4-ms pulse flicker, and it became smaller with strobe-flicker stimuli. The amplitude change after PDA followed the same pattern as after APB alone (Fig. 4 , left columns) except that the timing of flicker ERGs after PDA alone clearly became faster than the control, whereas it was delayed after APB alone (described further below). These timing changes were more prominent with sine-wave and square-wave stimuli.

The cumulative application of both APB and PDA served to isolate cone photoreceptor activity, and this gave similar amplitudes for the four types of flicker ERGs. With all four stimulus types, the amplitudes were reduced to less than 20% of the control. This indicated that the photoreceptor contribution to the fast-flicker ERG is relatively small, regardless of whether sine-wave, square-wave, or brief-pulse stimuli are used.

We next explored which harmonic components were altered by the combination of APB+PDA, which isolated direct cone contribution. Figure 5 shows the mean amplitude (±SD) of each harmonic component of the four types of stimuli before and after APB+PDA, up to the fourth harmonic. With sine-wave and square-wave flicker, APB increased the fundamental amplitude by 51.2% ± 20.9% (n = 3) and 37.6% ± 10.7%, (n = 3) of the control, respectively. The contributions from the second and higher harmonics remained small after APB, indicating that the OFF pathway does not, of itself, produce much harmonic content in the flicker ERG. The response fundamental amplitude for both 4-ms pulse and strobe-flicker stimuli (Fig. 5 , lower two panels) changed very little after APB. However, APB reduced the second harmonic amplitude for 4-ms pulses and strobe flicker to approximately one-half of predrug control, and the third and fourth harmonic amplitudes for strobe-flicker were also attenuated by APB by more than half. Thus, the reduction in the peak-to-peak amplitude after APB for strobe-flicker stimuli was due mostly to the amplitude reduction of the second and higher harmonics. Further, because the majority of this response arises postsynaptic to the cones, the inner retina is responding in a nonlinear way to the harmonically complex impulse stimulus train.



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  		Figure 5. The amplitude of the fundamental (1F) to fourth (4F) harmonic components before drugs (control), after APB, and after APB+PDA. The mean ± SD is shown for three animals.

 
Vector Modeling Analysis of the Fundamental Component
The question then arose as to why the fundamental amplitude increased after APB with sine-wave and square-wave stimuli but did not change substantially for brief-flash flicker. We suspected that some interplay of the ON- and OFF-pathway activity was involved, and we used vector-modeling analysis to probe the mechanism for this phenomenon.9 Figure 6 shows the constituent phase and amplitude vectors for the fundamental frequency components of 32-Hz flicker ERG with the four types of stimuli. The photoreceptor component was obtained from the residual ERGs that remain after applying APB+PDA. The APB-sensitive ON component was derived by subtracting the post-APB records from the control, and the PDA-sensitive OFF component was obtained by subtracting the post-APB+PDA ERGs from the post-APB ERGs. The postsynaptic component was obtained by subtracting the APB+PDA ERGs records from the control ERG. The colored dotted bars at the top of the vectors indicate the standard deviations of the amplitude and phase averaged from three animals.



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  		Figure 6. The constituent amplitude and phase vectors for the fundamental frequency component of the 32-Hz flicker ERGs for conditions of sine-wave, square-wave, 4-ms pulse, and strobe flicker stimulation. Black arrow: control ERG; red arrow: APB-sensitive (ON) component; blue arrows: PDA-sensitive (OFF) component; cyan arrow: postsynaptic (ON + OFF) component. Phase-delay data are plotted counterclockwise as positive values. All amplitudes are shown on an absolute scale with 10-µV calibration bars for each condition.

 
The vector modeling demonstrated that the fundamental harmonic of the 32-Hz flicker ERG was composed of roughly similar factors with all the stimuli. The amplitudes of the APB-sensitive ON component (Fig. 6 , red) and the PDA-sensitive OFF component (Fig. 6 , blue) were approximately equal, but the phase differences of the vectors were 118° to 139°. Consequently, they added together to produce a large postsynaptic vector (cyan). The contribution of the photoreceptor component (Fig. 6 , green) was relatively small and was less than 20% of the postsynaptic component for each of the four types of flicker. Thus, the fundamental component of the 32-Hz flicker was dominated almost entirely by postsynaptic activity regardless of stimulus type.

We next determined whether there were any differences in the relative magnitude or phase of the ON and OFF components for the four types of flicker stimuli. The amplitude ratio of the ON to OFF component tended to be slightly smaller with sine-wave (0.83 ± 0.10, n = 3) and square-wave (0.83 ± 0.16, n = 3) stimuli than for 4-ms pulse (0.98 ± 0.13, n = 3) and xenon flash (0.97 ± 0.24, n = 3), but the differences were not statistically significant when averaged across animals.

One striking difference was found in the phase difference between the ON and OFF components for these stimuli. The relative phase difference between the ON and OFF components was closer to 180° (i.e., the opposite phase) with sine-wave (138.3° ± 2.3°, n = 3) and square-wave (138.3° ± 2.3°, n = 3; note that the values are actually identical) than for 4-ms pulse (120.6° ± 5.5°, n = 3) and xenon strobe-flicker (117.6° ± 2.1°, n = 3) stimuli. These differences for pulse stimuli relative to sine-wave and square-wave were statistically significant (P < 0.05). Consequently, with sine-wave and square-wave stimuli, the phase difference between the two large postsynaptic ON and OFF components was approximately 20° greater than for brief-flash flicker, and the resultant net (ON + OFF) response was smaller than either ON or OFF alone. This helps to explain why removing either the ON or OFF component caused the amplitude to increase with sine-wave and square-wave flickers. In contrast, for brief-pulse stimuli, the net (ON + OFF) response was nearly comparable to either the ON or OFF component alone, because the phase differences were farther from 180°.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Contribution of ON and OFF Components to Sine-Wave, Square-Wave, and Brief-Pulse Flicker ERGs
By blocking the postphotoreceptor activity with the combination of APB plus PDA, we found that fast-flicker ERG responses are dominated by activity originating proximal to the cone photoreceptors regardless of whether the flicker stimulus was a sine-wave, square wave, or brief pulse. Application of APB+PDA reduced the peak-to-peak flicker amplitudes by 80% from control amplitudes with all four stimuli, indicating that the contribution from the photoreceptors was relatively small. The vector-modeling analysis showed that the fundamental component of the 32-Hz flicker ERG was dominated by postsynaptic ON and OFF components in all four types of flicker ERGs. In this respect, the flicker ERGs elicited by sine-wave, square-wave, or brief-flash stimuli were similar with all stimulus types. The clear and considerable decrease in responses after synaptic blockade implicates a major representation of postphotoreceptoral processes in the fundamental component of the flicker response. Even though the alteration by APB+PDA could arise through postphotoreceptoral feedback onto the cone pedicles, this leaves the clinical point the same, that 32-Hz flicker ERG amplitudes do not provide an unambiguous, accurate assessment of cone activity exclusively with any of the four types of stimuli.

In contrast to the cone component as isolated by synaptic blockade with the combination of APB+PDA, the actions of either APB or PDA alone on the ERGs were quite different for the four different stimuli. Either APB or PDA alone increased the response amplitudes to sine-wave and to square-wave flicker stimuli, whereas these drugs affected the flicker responses minimally to either of the brief-pulse stimuli. These results pertained, even when only the fundamental component was considered, as APB increased the fundamental amplitude by more than 30% with sine-wave and square-wave stimuli, but it did not change the amplitude with brief-pulse flicker. The vector-modeling analysis indicated that this difference was caused mainly by a differences in the relative lag of the ON and OFF phases. The ON and OFF components were approximately 20° more out of phase with sine-wave and square-wave stimuli than with brief-pulse flicker, as shown in Figure 6 . Thus, under normal conditions, our analysis indicated that the fundamental ON and OFF components counteract each other to a greater extent with sine-wave and square-wave stimuli than with brief-pulse stimuli. These data indicate that, under some conditions, the results of flicker ERGs depend on the specific type of flicker stimulus that is used, even when the mean luminance is the same. This may be even more of a factor in retinal diseases in which either the ON or OFF component may be selectively affected.19 20

The exact reason why the ON and OFF components are more out of phase with sine-wave and square-wave stimuli than with brief-pulse flicker is not clear. However, brief-pulse stimuli elicit the ON and OFF events nearly simultaneously, whereas the sine- and square-wave stimuli have a time difference between the "ON" and "OFF" portions of the stimulus cycle.

Concerning the origins of flicker response activity, after a number of years of similar experiments, it is our belief that primate flicker responses involve bipolar cell activity primarily, but we acknowledge that this is a bias on our part that the data do not absolutely resolve, because APB and PDA have effects elsewhere in the retina besides simply in the ON- and OFF-bipolar cells. Hence, our bias remains our conclusion based on what is understood about the nature of the representation of the "outer" (i.e., photoreceptor), "middle" (i.e., bipolar), and "innermost" (i.e., amacrine and ganglion cell) layers to the classic full-field ERG. The essential clinical point remains the same, that 32-Hz flicker ERG amplitudes do not reflect exclusively direct cone activity with any of these four types of stimuli.

Comparison with Flicker ERG in the Complete Type of Congenital Stationary Night Blindness
In this study, we compared the photoreceptor and the ON- and OFF-contributions with the brief-pulse 32-Hz flicker ERG fundamental component and with the sine-wave9 and square-wave flickers. Because stroboscopic flashes are widely used in the clinic, vector-modeling analysis may be useful for interpreting the changes resulting from retinal disease. For instance, Kim et al.8 analyzed the fundamental component of 30-Hz strobe flicker ERGs in patients with complete congenital stationary night blindness (cCSNB), who are thought to have a defect in signal transmission from photoreceptors to the postreceptoral ON-pathway.5 21 22 cCSNB showed a delay of the fundamental phase without a significant amplitude reduction. Similarly, these monkey studies showed that removal of the ON component by APB caused a substantial phase delay without any notable amplitude reduction for stroboscopic stimulation (Fig. 6) . This suggests that vector analysis may be useful for interpreting flicker ERG changes in presumed postsynaptic retinal diseases. One difference between the patients with cCSNB and the monkeys studied was that the delay in the monkey responses after APB was two times larger (58°) than in the patients with cCSNB (28°).8 This difference may be partly due to differences between species. Alternatively, it suggests that the pathologic course of cCSNB may be more complex than the relatively pure pharmacologic blockage of the ON component by APB. Recent genetic analysis of X-linked cCSNB identified mutations in NYX gene that encode the nyctalopin protein, and suggested that mutant nyctalopin may impair proper development of the ON pathway circuit.23 24

Similarity of Sine-Wave and Square-Wave Flicker ERGs
We noted that the fast-flicker ERG responses to sine-wave and square-wave stimuli were similar. First, the fundamental component dominated the 32-Hz flicker responses of both (>70%), with only a small contribution from the second and higher harmonic components. Second, the action of APB or PDA alone was similar for both types of ERGs. Third, vector-modeling analysis showed that the relative magnitude and phase difference between the ON and OFF components were very similar with these two types of flicker responses. And fourth, when we analyzed the amplitude and phase of the fundamental components with the sine-wave and square-wave flicker ERGs across stimulus frequencies of 4 to 64 Hz for the control, after APB alone, and after APB+PDA in one monkey, the frequency–amplitude and frequency–phase functions were both qualitatively similar with these two stimuli (Fig. 7) . These data suggest that the retinal origin of the fundamental component of the sine-wave flicker ERG is probably the same as that of the square-wave flicker and that a square-wave stimulus may provide an appropriate alternative to the more difficult task of generating pure sine-wave flicker stimuli. From this, it appears that the ERG response fundamental component to a sine-wave stimulus is not unique in reflecting activity of specific retinal elements (e.g., cone photoreceptors directly) in a manner different from other stimuli, as had been suggested in earlier studies.1 2 25 26



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  		Figure 7. Plots of amplitudes and phase lags of the fundamental component of the flicker ERG response to sine-wave and square-wave stimuli before and after application of glutamate analogues. (•) Control condition without drugs; ({circ}) after intravitreous injection of APB; and (X), after intravitreous injection of APB+PDA. The sine-wave and square-wave stimulus trains were produced by 623-nm red LEDs with 2.66 log cd/m2 mean luminance and 80% modulation depth.

 
Second and Higher Harmonic Components
One question yet to be resolved is which specific retinal cells or circuits contribute to the second and higher harmonic components. This is important, because the brief-pulse, fast-flicker ERG, which is widely used in diagnostic clinics, contains substantial contribution from these higher harmonics, as is shown in Figure 2 . Current-source density analyses27 have indicated that the second harmonic component of the 8-Hz sine-wave ERG has multiple origins, including the inner retina, and some clinical studies2 28 29 have shown that the second harmonic component contains contributions from the proximal retina. In the present study, the second and higher harmonic components with brief-pulse flickers were reduced by more than half after APB, and these harmonics were also greatly attenuated after additional application of PDA, indicating that there must be large contributions from postsynaptic ON and OFF components, as we also found to be the case for the fundamental component. However, we could not further characterize the second harmonic component with vector-modeling analysis, because there were considerable variations of both amplitude and phase among the three monkeys we tested. An interesting observation was that the second and higher harmonic amplitudes with strobe flashes never increased after either APB or PDA alone but always decreased—a more than 50% reduction after APB alone for each of the three monkeys and a more than 60% reduction after PDA alone in the one monkey tested.

From these results, it is reasonable to conclude that the majority of second and higher harmonic components requires signaling from both the ON and OF -pathways through some form of interaction. One possibility is that neuronal circuits interconnecting the ON and OFF pathways may play a role in generating second and higher harmonic components. Alternatively, the ON and OFF pathways’ contributions to the flicker ERG may have different dynamics, so that development of the higher harmonics lies in the formation of the ERG signal, rather than interconnecting neural circuitry. Clearly, further studies are warranted to determine the retinal origins of the second- and higher harmonic components.


   Acknowledgements
 
The authors thank Ronald A. Bush and Naheed W. Khan for advice and comments and Eric B. Arnold and Jeffery A. Jamison for assistance with software and hardware.


   Footnotes
 
Supported by National Eye Institute Grants R01-EY06094 and Vision Core Grant EY07003, The Foundation Fighting Blindness, Research to Prevent Blindness, and an Alcon Research Institute Award.

Submitted for publication October 18, 2001; revised February 6, 2002; accepted February 15, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Paul A. Sieving, National Eye Institute, Bldg. 31, Room 6A03, 31 Center Drive, MSC 2510, Bethesda, MD 20892-2510; paulsieving{at}nei.nih.gov.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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