The human electroretinogram (ERG) recorded at the cornea

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1 Recording Multifocal Electroretinogram On and Off Responses in Humans Mineo Kondo, Yozo Miyake, Masayuki Horiguchi, Satoshi Suzuki, and Atsuhiro Tanikawa PURPOSE. TO record the on and off responses of the multifocal photopic electroretinogram (ERG) from the human retina and to explore how each ERG component (a-, b-, and d-waves) changes at different retinal eccentricities. METHODS. Multifocal ERGs were recorded with the visual evoked response imaging system. Sixtyone densely packed stimulus elements were square wave-modulated between stimulus on and stimulus off, according to a binary m-sequence at a rate of 4.7 Hz under a constant background illumination. The ERGs were recorded with a bipolar Burian-Allen bipolar contact lens electrode from five normal subjects. Response densities (amplitude per retinal area) were calculated for five different eccentric rings. RESULTS. The response densities for all components (a-, b-, and d-waves) decreased with increasing retinal eccentricities. The ratio of the d-wave to b-wave amplitudes was minimal in the central retina and increased toward the periphery. The ratio of a-wave to b-wave amplitudes also increased toward the peripheral retina. CCLUSIS. These results demonstrate that the on and off responses of the human cone ERGs have different spatial distributions across the human retina, and they suggest a change in the photopic retinal circuitry with increasing retinal eccentricities. (Invest Ophthalmol Vis Set. 1998;39: ) The human electroretinogram (ERG) recorded at the cornea in response to a flash stimulus is the sum of the component potentials generated by retinal processing by the different groups of cells from different retinal locations. For instance, under photopic conditions that suppress rod activity, a brief flash stimulus elicits an initial negative wave (a-wave) followed by a positive component (b-wave). However, when a long-duration stimulus is used under photopic conditions, the human ERG contains an "on" response (a- and b-waves) at light onset and a separate positive wave called the "off response (d-wave) at light offset. 1 " 4 The long-duration stimulus has been reported to be suitable for dissecting the contribution of the retinal cells to the photopic ERG, which involves the cone photoreceptors and the cone on and off pathways. 5 " 8 Clinical applications of the long-duration stimulus to some retinal diseases also have been reported. 9 " 11 However, diseases can affect the retina unevenly, and little is known about how the on and off responses of the human ERG vary with retinal location. Recently, the technique of recording multifocal ERGs was developed by Sutter and colleagues. l2 " 14 This technique allows the simultaneous recording of focal ERGs from multiple retinal locations in a single recording session by using cross-correlation techniques. A recent study showed that the negative and From the Department of Ophthalmology, Nagoya University School of Medicine, Nagoya, Japan. Submitted for publication May 12, 1997; revised October 6, 1997; accepted November 4, Proprietary interest category: N. Reprint requests: Mineo Kondo, Department of Ophthalmology, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan. 574 positive components obtained in the multifocal ERG behave as do the a-waves and positive peaks of the traditional full-field ERG. 15 The purpose of the present study was to use the multifocal ERG technique to record photopic on (a- and b-waves) and off (d-waves) responses from different retinal locations in humans and to determine how each component of the ERG varies with different retinal locations. Our results showed that these components have different spatial distributions, suggesting that there are topographic variations in retinal cone circuitry across the human retina. MATERIALS AND METHODS Subjects Five healthy Japanese men 26 to 31 years of age were studied. Except for refractive errors of 1.00 to 3.00 diopters, there were no ophthalmologic or neurologic abnormalities. Informed consent was obtained after a full explanation of the procedures. All studies were conducted in accordance with the principles embodied in the Declaration of Helsinki. Stimulation The basic principles for recording the multifocal ERG have been described in detail previously by Sutter and Tran. 12 Multifocal ERGs were obtained with the visual evoked response imaging system. The stimulus array consisted of 61 densely packed hexagonal elements that were displayed on a CRT color monitor (GDM; Sony, Tokyo, Japan) and were driven at a rate of 75 frames/second. The diameter of the entire visual field was approximately 50 at a viewing distance of 27 cm (Fig. la). Investigative Ophthalmology & Visual Science, March 1998, Vol. 39, No. 3 Copyright Association for Research in Vision and Ophthalmology

2 Multifocal On and Off Responses TOYS, March 1998, Vol. 39, No a. Stimulus array U 120cd/m2 _. 20cd/m2 20 b. Sequences of TV frames and the - and OFF-responses b-wave d-wave 213 ms B FIGURE 1. (a) Stimulus array of 6l hexagonal elements. The diameter of the entire visual field was approximately 50. The luminance of on and off was 120 and 20 cd/m2, respectively. The luminance of the periphery of the television screen was 20 cd/m2. (b) Principle of the present method. Each hexagon was modulated between stimulus A (eight consecutive white frames after eight consecutive dark frames) and stimulus B (16 consecutive dark frames), according to a binary m-sequence. Each focal electroretinogram (ERG) was calculated as the difference between the mean response to stimulus A and the mean response to stimulus B. The right-hand portion of the figure shows a typical ERG waveform recorded in this study. On (aand b-waves) and off (d-wave) responses can be seen. A small red fixation spot was placed at the center of the stimulus matrix. To record the multifocal on and off responses, each hexagonal element was modulated between stimulus A (eight consecutive white frames after eight consecutive dark frames) and stimulus B (16 consecutive dark frames) according to a binary m-sequence, as shown in Figure lb. The m-sequence stimulation rate was, therefore, 4.7 frames/second, and the base interval was msec. Focal responses were calculated as the difference between the mean response to stimulus A and the mean response to stimulus B. The luminance of the white frame was candela (cd)/m2 (the maximum intensity of the television screen). To minimize both the rod activity and the scattered light, a luminance of 20 cd/m2 was used for both the dark frames and the periphery of the TV monitor (Fig. la). Recording and Data Analysis The subject's pupils were fully dilated with a combination of 0.5% tropicamide and 0.5% phenylephrine hydrochloride, and they were 9 mm or more in diameter at the beginning and end of each recording session. A Burian-Allen bipolar contact lens electrode was used for the ERG recording. A ground electrode was attached to the ipsilateral earlobe. The opposite eye was occluded. After each subject's vision had been corrected optically to the best acuity, the subject was asked to look at the fixation spot. The signals were amplified by 100 K and were filtered between 3 and 300 Hz (RPS-107; Grass, West Warwick, Rl). The data-sampling rate was 1200 Hz, and an artifact rejection technique was used.12 The length of the m-sequence used for

3 576 Kondo et al. IOVS, March 1998, Vol. 39, No. 3 Number of the stimulus frames 1 frame 2 frames 50 cd/m* 20 cd/m* V\^~ A- 5pV L FIGURE 2. (a) Effect of the stimulus duration. Spatially summed responses at various stimulus durations in a normal subject. The stimulus and background intensity were 120 and 20 cd/m 2, respectively. The m-sequence stimulation rate was 4.7 times/second, (b) Effect of the stimulus intensity. Spatially summed responses at various stimulus intensities in a normal subject. The background intensity was 20 cd/m 2. The stimulus duration was 106 msec (eight consecutive white frames), (c) Effect of the background intensity, (c) Effect of the background intensity. Spatially summed responses at various background intensities in a normal subject. The stimulus intensity was 120 cd/m 2. The stimulus duration was 106 msec. the present study was 2'' 1. Thus, the total recording took 8 minutes and was divided into 16 segments. The local responses were extracted by use of the multi-input technique described by Sutter et al. l2 The array of local responses was plotted in the same manner as the visual field. The amplitudes of the a-, b-, and d-waves were measured from the baseline to the negative trough, from the negative trough to the top of the positive peak, and from the plateau to the top of positive peak, respectively, as shown in Figure lb. Statistical analysis was performed with Spearman's rank correlation coefficient (r^. RESULTS Effect of Stimulus Duration, Stimulus Intensity, and Background Intensity Initially, we studied the basic characteristics of the on and off responses obtained in the present system as a function of stimulus duration, stimulus intensity, and background intensity in normal subjects. Figure 2a shows the spatially summed responses for various stimulus durations. With a shortening of the stimulus duration, the amplitude of the d-wave became smaller. With the shortest duration stimulus, one white frame, the b- and d-waves merged; the b-wave was largest. Figure 2b shows the spatially summed responses at various stimulus intensities. With increasing stimulus intensity, the amplitude of all components (a-, b-, and d-waves) increased, but the overall waveform shape did not change much. Figure 2c shows the spatially summed responses at various background intensities. The amplitudes of the a-, b-, and d-waves did not change significantly at background intensities of 5 to 20 cd/m 2, but they decreased at the highest background intensity of 40 cd/ m 2. From these results, we selected as the standard stimulus conditions an intensity of 120 cd/m 2 with a duration of 106 msec, and we presented the stimulus on a 20-cd/m 2 background. These stimulus parameters were well suited to elicit on and off responses under light-adapted conditions. An Example of 61 Multifocal On and Off Responses Figure 3 shows an example of 61 multifocal on and off responses recorded from the left eye of a normal subject under the stimulus conditions mentioned. Each local response was noisier than that obtained with conventional fast-flicker multifocal stimulation, presumably as a result of the short length of the m-sequence used in the present study. However, each component of the focal photopic on (a- and b-waves) and off (d-wave) responses was identifiable. The response amplitude was weak in the area corresponding to the blind spot (arrow). The residual response can be explained by the fact that the size of the stimulus hexagon at this point was larger than that of the optic disc as described in previous reports. 12 " 14 On and Off Responses at Different Retinal Eccentricities We next studied how the photopic on and off responses changed with the distance from the fovea. For this purpose, multifocal on and off responses were recorded from five normal subjects under the standard recording and stimulus conditions mentioned above. Figure 4 shows representative focal responses averaged from five annuli of increasing eccentricity. The scale of the response density (amplitude per retinal area) was constant in the left row of Figure 4 (response density scale). It is clear that the response density of each component decreased with increasing eccentricity. The scales were varied in the right row of Figure 4 to obtain an approximately equal size for all five responses. It was also noted that the d-wave became larger and the a-wave became deeper with increasing eccentricity when compared with the b-wave. Figure 5 shows the mean ± SEM of the response density (amplitude per retinal area) at different eccentricities for one session withfivenormal subjects. With increasing eccentricity, the response densities of all components decreased significantly (a-wave, r s = 0.77, P < 0.01; b-wave, r s = -0.83, P < 0.01; and d-wave, r s = 0.67, P < 0.05). To study the waveform change at different eccentricities, the mean ± SEM of the ratio of the d-wave to b-wave amplitudes (d/b ratio) and the ratio of the a-wave to

4 Multifocal On and Off Responses JOVS, March 1998, Vol. 39, No ms 3. Multifocal photopic on and off responses recorded from the left eye of a representative normal subject. Recording time was 8 minutes. A low-amplitude response was seen at the blind spot (arrow). FIGURE b-wave amplitudes (a/b ratio) for one session from five normal subjects are plotted in Figure 6. The d/b ratio was minimal in the central retina and increased significantly (rs = 0.92, P < 0.01) with increasing eccentricities. The a/b ratio also increased significantly (rs 0.74, P < 0.01) with increasing eccentricities. To support our finding that the d/b and a/b ratios change with increasing eccentricities, ERGs were recorded with increasing stimulus intensities ( cd/m2) and at various background intensities (5-40 cd/m2) in one subject. For stim- ulus intensities and background intensities, the d/b and a/b ratios were consistently lowest in the central retina and increased toward the peripheral retina (Fig. 7). DISCUSSI It has been shown already that it is possible to record the a-waves, the b-waves, and the oscillatory potentials of the ERG from a number of retinal areas with the multifocal ERG tech- Response density scale Normalized scale 50 n 200 ms 200 ms 4. Averaged electroretinogram waveforms from five eccentric annuli. Scale of the response density (amplitude per retinal area) was constant in the left row. The scales were varied in the right row to obtain an approximately equal size of b-waves for all five responses. Note that the waveforms vary with retinal eccentricity. FIGUHE

5 578 Kondo et al. IOVS, March 1998, Vol. 39, No. 3 o> 60-50H CO CD CO i «30 H a-wave 20i b-wave d-wave FIGURE 5. Mean of the response density (amplitude per retinal area) of each electroretinogram component (a-, b-, and d- waves) as a function of retinal eccentricity. Error bars represent 1 SEM (n = 5). possible to obtain multifocal off responses (d-wave) and on responses (a- and b-waves), which is an important observation about retinal function and the unique characteristics of cone vision in humans. For at least three reasons, ERGs obtained in the present study are the same as those found in traditional full-field photopic ERGs with long-duration stimuli. First, we used filler frames to slow down (base interval, 2133 msec) the effective stimulus rate of the mutifocal ERG, which minimized the nonlinear overlapping effects of succeeding stimuli. Second, each component obtained in the present study behaved as did those of the previously reported traditional photopic ERG, that is, with the shortening of the stimulus durations the amplitude of the d-wave became smaller and the b- and d-waves merged for the shortest flash. 616 " 18 Third, it has been shown recently by Hood and colleagues 15 that the components of the multifocal ERG are the same as those underlying the full-field ERG. We found that the amplitude per degree squared of all photopic ERG components (a-, b-, and d-waves) was maximal at the fovea and decreased toward the peripheral retina. This finding, which agrees with those of previous reports, 1219 ' 20 can be explained by the fact that the neuron density is greatest at the fovea. We also found that on and off responses have different spatial distributions, that is, the amplitude of the d-wave is smallest in the fovea when compared with that of the b-wave, and it increases with eccentricity. The a-wave, to a lesser degree, does the same as shown in Figures 4 and 6. There are previous reports on the waveform change at different retinal locations. In pigeons 21 and cats, 22 the d/b amplitude ratio was reported to be largest in the central retina, and it tended to decrease when the stimulus area was enlarged. Possible reasons for the discrepancy between these reports and our findings may be attributable to species differences or to differences in the experimental conditions. Yamamoto and colleagues 23 recorded the focal cone ERG on and off responses in monkey retinas with a 3 pulse of laser light on a high background of 230 cd/m 2. Although they did not describe in detail the variation of the on and off responses across the retina, the amplitudes of b-waves seemed to decrease more quickly than those of d-waves with increasing eccentricity, which agrees with our results. Errico et al. 24 recorded the on and off responses of focal ERGs in humans elicited by an on-off stimulus with various spot sizes centered on the fovea. They reported that the d/b ratios tended to decrease with an increase of the stimulus size, which is contrary to our results. The reason for the discrepancy between their results and ours may be explained by several differences in the experimental conditions. Errico et al. 24 used just sufficient luminance around the stimulus area to suppress the stray light effect, but they did not use constant background illumination within the stimulus area. Although the responses may have been focal, they probably contained mixed rod and cone contributions. When the size of the stimulus is small at the fovea, recorded responses are predominantly cone responses, and when the size of the stimulus is enlarged, the response has a greater contribution from the rods because the rod cells are denser in the peripheral retina. Because the rod ERGs have on response (a- and b- waves) at light onset but do not have a positive off response at light offset, 6 the d/b ratios tend to decrease with increasing rod contribution to the response. In addition, we think that our findings are valid, at least in humans, because they were consistently found for various stimulus intensities and at different background intensities, as shown Figure 7. The photopic ERG components in the primate retina have been reported to contain the summation of contributions that originate mainly from the cone photoreceptors plus the activity of postsynaptic neurons of both the depolarizing on bipolar cells and the hyperpolarizing off bipolar cells. 5 " 8 The human photopic a-wave seems to be shaped from the activity of both the cone photoreceptors and the hyperpolarizing off bipolar a/b d/b FIGURE 6. Mean of the amplitude ratios of electroretinogram components (d-waves to b-waves [d/b] and a-waves to b-waves [a/b]) as a function of retinal eccentricity. Note that the means of both d/b and a/b ratios increase from the center to the periphery. Error bars represent 1 SEM (n = 5).

6 JOVS, March 1998, Vol. 39, No. 3 Multifocal On and Off Responses ST: 50 cd/m 2 ST: 120cd/m2 0.8n ST: 50 cd/m 2 ST: 120 cd/m " ST: 80 cd/m ST: 80 cd/m I " 0.2" BG : 40 cd/nr 0.8" 2 0.6" BG : 20 cd/m 2 BG: 10 cd/m 2 BG : 5 cd/m 2 0.8i 2 0.6" BG : 20 cd/m' BG : 40 cd/m 2 BG : 10 cd/m 2 0.4" BG : 5 cd/m 2 0.2" FIGURE 7. The amplitude ratios of d-waves to b-waves (d/b) and a-waves to b-waves (a/b) from five eccentric annuli at various stimulus intensities ( cd/m 2 ) and at various background intensities (5-40 cd/m 2 ) in one subject, (a) The d/b amplitude ratios at various stimulus intensities. The background intensity was 20 cd/m 2. (b) The a/b amplitude ratios at various stimulus intensities. The background intensity was 20 cd/m 2. (c) d/b amplitude ratios at various background intensities. The stimulus intensity was 120 cd/m 2. (d) The a/b amplitude ratios at various background intensities. The stimulus intensity was 120 cd/m 2. cells. 5 ' 7 Although the photopic b-wave is largely generated by the activity of the depolarizing on bipolar cells, the activity of the hyperpolarizing off bipolar cells can limit the amplitude and shape of the b-wave. 8 The photopic d-wave, however, seems to be shaped mainly from the activity of the cone photoreceptor cells and the hyperpolarizing off bipolar cells. 5 ' 8 Because the cellular origin of each photopic ERG component in the primate retina is complex, as mentioned above, it is difficult to explain the exact mechanism for our present results. However, pharmacologic studies 5 " 8 may add to our understanding of them. When the signal transmission from the cone to the depolarizing on bipolar cells is suppressed with 2-amino-4-phosphonobutyric acid, the b-wave is reduced, the negative a-wave becomes deeper, and the positive d-wave is enhanced. However, when the signal transmission from the cone to the hyperpolarizing off bipolar cells is suppressed with cz's-2,3-piperidine dicarboxylic acid or kynurenic acid, the a- wave becomes less negative, the b-wave becomes larger, the positive d-wave becomes smaller, and the plateau between the b- and d-waves is elevated. These two waveforms, produced by the two drugs, are comparable to those observed in the peripheral retina and central retina, respectively, in the present study. This suggests that the ratio of cone-depolarizing on bipolar cells to cone-hyperpolarizing off bipolar cells may be different in number, function, or both at different retinal eccentricities. We have not found any reports of variations in die distributions of cone on and off pathways within the primate retina. To confirm our hypothesis, it will be necessary to record focal on and off responses at different retinal locations while treatment for the primate retina is given with various pharmacologic agents. Although the exact mechanism of the present findings is unclear, we think that our findings can provide important information about the distribution of cone on and off pathways within the human retina. In addition, the slow, long-duration multifocal technique used in the present study may be useful for assessing the local activity of cone on and off pathways in patients with diseased retinas. References 1. Heck J. Der Off-Effekt im menschlichen Elektroretinogramm. Ada Physiol Scand. 1957;40: Nagata M. Studies on the photopic ERG of the human retina. Jpn JOpbthalmol. 1963;7: Biersdorf WR. Rod and cone contribution to the off-effect of the human ERG. Invest Ophthalmol Vis Sci. 1968;7:

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