High-Frequency Attenuation of the Cone ERG and ON-Response Deficits in X-linked Retinoschisis

Abstract

purpose. Individuals with X-linked retinoschisis (XLRS) show a comparatively greater reduction of the ON response than the OFF response of the electroretinogram (ERG) of the cone system. At high temporal frequencies, they also show a marked attenuation of the flicker ERG that has been attributed to an abnormal cone photoreceptor response. The purpose of this study was to determine whether the high-frequency response attenuation contributes to the abnormal ERG ON response in XLRS.

methods. Light-adapted ERGs were recorded from three patients with XLRS and from three control subjects, by using rapid-on and rapid-off sawtooth flicker to emphasize ON and OFF responses, respectively, and by using low-pass sawtooth flicker, from which the high temporal frequencies had been removed to mimic the high-frequency attenuation in XLRS.

results. For the control subjects, removing the high stimulus frequencies reduced the amplitude of the b-wave component of the ON response but had little effect on the amplitude of the d-wave component of the OFF response. In the patients with XLRS, the b-wave component of the ON response was already diminished using the full sawtooth stimulus, and removing the higher stimulus frequencies had no further effect. Patients’ ERG responses to the 16-Hz stimulus fundamental alone were also abnormal, in that an initial response component normally present in the ERG was absent.

conclusions. The overall pattern of findings indicates that two factors contribute to the preferential ON-response deficit in XLRS: first, a high-frequency attenuation of the cone photoreceptor response that effectively produces a low-pass stimulus for the postreceptoral pathway and that affects the ON response more than the OFF response and, second, a relatively greater attenuation of the ON- than of the OFF-bipolar cell response that is evident in the aberrant response to the sawtooth fundamental.

X-linked retinoschisis (XLRS) is a hereditary, juvenile-onset vitreoretinal degeneration. ¹ It is characterized by microcystic, schitic changes within the macula, and in approximately 50% of patients, by a peripheral retinal schisis, as well. ¹ The schisis occurs at the level of the nerve fiber and ganglion cell layers of the retina, ^{2 3 4} and it has been proposed that defective, degenerating Müller cells are the primary cause. ⁴ One of the typical functional characteristics of patients with XLRS is an abnormal brief-flash electroretinogram (ERG) of the rod and cone systems, in which the a-wave amplitude is normal or near normal, but the b-wave amplitude is reduced substantially, resulting in a reduced b-wave-to-a-wave ratio. ^{5 6 7 8 9 10} It has been assumed that this relative b-wave reduction results from Müller cell dysfunction, ^{6 8} under the customary notion that the b-wave of the ERG reflects the response of Müller cells to the activity of bipolar cells. 

However, recent studies have prompted a re-evaluation of this view of the underlying pathophysiology of XLRS. First, the RS1 gene that is mutated in XLRS is expressed in photoreceptor and bipolar cells but not in Müller cells, ^{11 12} so that the exact relationship between the gene defect and the Müller cell abnormalities that have been observed in XLRS is uncertain. Second, patients with XLRS have a relatively greater attenuation of the ERG ON response than OFF response of the cone system, resulting in a reduced b-wave-to-d-wave ratio. ^{13 14} Therefore, the relatively reduced b-wave amplitude of the brief-flash ERG of the cone system just described probably represents an ON-response defect rather than a Müller cell abnormality, per se. This is consistent with recent studies that have shown that Müller cells make little direct contribution to the ERG b-wave. ^{15 16}  

The exact explanation for the preferential ON-response defect in XLRS is not entirely clear. One possibility is that it represents dysfunction of the depolarizing (ON) bipolar cells (DBCs). In the primate cone system, the initial portion of the b-wave component of the ON response represents primarily the activity of DBCs, with the latter portion of the b-wave modulated by the response of hyperpolarizing bipolar cells (HBCs). ¹⁷ The initial portion of the d-wave component of the OFF response represents the activity of HBCs in combination with the offset of the cone photoreceptor response. ¹⁷ Therefore, it is likely that at least part of the significantly reduced b-wave-to-d-wave ratio of the cone system in patients with XLRS is due to a preferential response attenuation within the DBCs. 

It is possible, however, that an abnormal temporal response of the cone photoreceptors also contributes to the ON-response deficit in XLRS. It has been reported recently that patients with XLRS have a marked attenuation of the ERG response of the cone system at high temporal frequencies. ¹⁸ This high-frequency attenuation has been attributed to a cone photoreceptor defect, ¹⁹ based on an examination of nonlinear beat frequencies in the ERG, using an approach introduced by Burns et al. ²⁰ In this approach, ERGs are recorded in response to a stimulus that consists of the sum of two sinusoidal waveforms. The ERG response to this stimulus contains a“ difference frequency” that is generated by a retinal nonlinearity and that has a temporal frequency equal to the difference between the two input frequencies. Pairs of sinusoidal stimuli that differ by a small constant value are presented, and the amplitude and phase of the difference frequency is measured as a function of the input frequencies. 

This difference-frequency approach was used in a previous study ¹⁹ to evaluate the likely source of the high-frequency attenuation of the flicker ERG in XLRS, using a logic described by Burns et al. ²⁰ If the high-frequency attenuation of the ERG in XLRS occurs before the site of the retinal nonlinearity that generates this difference frequency, then the difference frequency would be reduced in amplitude when either or both of the input frequencies are in the high-frequency range. However, if the amplitude attenuation at high frequencies occurs at or after the site of the nonlinearity, then the difference frequency would have a normal amplitude across pairs of input frequencies, because the input to the nonlinearity would have a normal amplitude. In agreement with the first alternative, the difference-frequency function in patients with XLRS was found to show a selective high-frequency attenuation that was identical with the high-frequency attenuation of the response fundamental. ¹⁹ Therefore, a major determinant of the loss of ERG amplitude at high temporal frequencies in XLRS is a response attenuation that occurs before the site of the nonlinearity that generates the difference frequency. Current evidence indicates that this nonlinearity is located in the outer retina, at or before the site of the convergence of signals from the different spectral classes of cones. ²¹ This convergence site is thought to occur at the synapse between cone photoreceptors and postreceptor neurons. ²² Therefore, the abnormal difference frequency in XLRS is presumed to represent an abnormal cone photoreceptor response. 

This high-frequency response attenuation at the presumed photoreceptor level in XLRS could affect the properties of their ERG ON and OFF responses, as follows. The sawtooth stimuli that were used by us previously ¹³ to elicit ERG ON and OFF responses included high as well as low harmonic temporal frequencies, with the amplitude decreasing in proportion to harmonic frequency, according to the following relationship:  

\[x_{t}{=}\ \frac{2A}{{\pi}}\ (\mathrm{sin}\ {\omega}_{\mathrm{o}}t{+}\ \frac{1}{2}\ \mathrm{sin}\ 2{\omega}_{\mathrm{o}}t{+}\ \frac{1}{3}\ \mathrm{sin}\ 3{\omega}_{\mathrm{o}}t{+}\ \frac{1}{4}\ \mathrm{sin}\ 4{\omega}_{\mathrm{o}}t{+}{\ldots})\]

where x is the amplitude of the sawtooth waveform at time t, A is the amplitude of the sawtooth stimulus, and ω_o = 2π/T, where T is the period. The rapid-on and rapid-off sawtooth waveforms used to elicit the ON and OFF responses, respectively, differed only in a 180° phase shift of the temporal frequency components. 

It might be expected that the higher harmonics of a sawtooth stimulus would make little contribution to the ERG response because of their relatively low amplitude and that an attenuation of the ERG response to these harmonic components by retinal disease would have little effect on ON and OFF responses. However, the temporal response function for the flicker ERG of visually normal subjects is strongly band-pass, so that response amplitude increases with increasing temporal frequency up to approximately 40 Hz. ^{19 20} Therefore, even though the higher stimulus harmonics of a sawtooth waveform have a smaller amplitude than the low stimulus harmonics, the greater responsiveness of the retina to these higher frequencies could have a significant role in shaping the ERG response to sawtooth stimuli. 

Further, it is possible that a relative attenuation of the higher stimulus harmonics due to retinal disease would have a greater effect on the ERG ON response than the OFF response. In a recent study of the temporal response properties of the generators of the ERG of the primate retina, ²³ it was reported that the peak of the response function for the DBCs occurred at a higher temporal frequency than the peak of the response function for the HBCs, so that the DBCs had a greater response amplitude at high temporal frequencies. Thus, based on these data, the higher harmonics of a sawtooth stimulus would be expected to make a greater contribution to the response of the DBCs than of the HBCs. Given that DBC activity is a primary determinant of the b-wave component of the ON response and HBC activity is a primary determinant of the d-wave of the OFF response, ¹⁷ it is possible that an effective attenuation of high stimulus temporal frequencies before the level of the bipolar cells would affect the b-wave component of the ON response more than the d-wave component of the OFF response. 

The purpose of the present study was to determine whether the response attenuation at a presumed cone photoreceptor level in XLRS contributes to the preferential reduction of the ERG ON response. This possibility was evaluated by comparing ERG responses to standard sawtooth stimuli with ERG responses to low-pass filtered sawtooth stimuli, from which all but the fundamental and second harmonic components had been removed, to mimic the high-frequency attenuation of the ERG in XLRS. ERG responses to the stimulus fundamental frequency were also measured. The results demonstrate that two factors contribute to the ON-response deficits of patients with XLRS: first, a relative attenuation of the high temporal frequency components of the stimulus, presumed to occur at the photoreceptor level, ¹⁹ and, second, a postreceptoral impairment that appears to affect the DBC system more than the HBC system. 

Materials and Methods

Subjects

Three unrelated male patients with XLRS participated in the study. These patients had also participated in prior studies of the ERG in XLRS. ^{13 18} Their ages and visual characteristics are given in Table 1 . The patients had the typical symptoms and signs of XLRS, including reduced visual acuity, a reduced b-wave-to-a-wave amplitude ratio that was most apparent in the ERG response of the dark-adapted eye to a maximal ²⁴ white stimulus, and microcystic lesions within the fovea that had a radial, spokelike appearance. Two (patients 1 and 3) also had peripheral schisislike changes, predominantly in the inferior temporal quadrant. Peripheral visual field restrictions corresponded to the clinically observed peripheral regions of schisis. One (patient 3) had a sheenlike appearance at the posterior pole, primarily temporal to the macula, similar to that described previously in some patients with XLRS. ^{25 26} Patient 1 was using topical medication for increased intraocular pressure but had no glaucomatous field loss. Although a blood sample was obtained from one (patient 2), molecular genetic information was not available for any of the patients. 

The results from the patients with XLRS were compared with those of three control subjects with normal vision, ages 28, 36, and 50 years. For the analysis of the temporal response properties of the flicker ERG, the results from the three patients with XLRS were compared with those of eight control subjects: the three just listed plus five subjects, ages 24, 25, 30, 32, and 38 years. The control subjects had best corrected visual acuity of 20/20 or better in the tested eye, clear ocular media, and normal-appearing fundi on ophthalmic examination. The study adhered to the tenets of the Declaration of Helsinki and was approved by the institutional review board of the University of Illinois at Chicago. Informed consent was obtained from all subjects after the nature and possible consequences of the study had been explained to them. 

Stimuli and Instrumentation

The stimulus consisted of full-field flicker that was presented against a rod-desensitizing adapting field. Three stimulus waveforms were used: sawtooth flicker, low-pass sawtooth flicker, and sinusoidal flicker. The stimulus waveforms for sawtooth and low-pass sawtooth flicker are illustrated in Figure 1 for a temporal frequency of 16 Hz, which was the primary focus of this study. Each cycle of rapid-on sawtooth flicker (Fig. 1 , top left) consisted of an abrupt increment in luminance, to emphasize an ON response, followed by a linear decrease in luminance. Each cycle of rapid-off flicker (Fig. 1 , top right) consisted of an abrupt decrement in luminance, to emphasize an OFF response, followed by a linear increase in luminance. The two waveforms have the same time-average luminance, which is indicated by the horizontal lines in the figure. The low-pass sawtooth waveforms are illustrated at the bottom of Figure 1 . These waveforms consisted of the sum of the fundamental and second harmonic of the sawtooth, with relative amplitudes given by equation 1 . They were presented in both rapid-on (left) and rapid-off (right) phases. 

ERG recordings were made at sawtooth stimulus frequencies of 4, 8, and 16 Hz, all at maximum amplitude. ERG responses were also measured at a temporal frequency of 16 Hz for the low-pass sawtooth stimuli and for the sawtooth fundamental alone, which was in sine phase and had an amplitude relative to that of the sawtooth as given by equation 1 . In addition, ERG responses were measured to sinusoidal flicker that was presented at temporal frequencies of 8, 16, 32, 64, and 96 Hz, at maximum amplitude and in sine phase. 

The stimuli were provided by two optical channels, each with a light source consisting of a 300-W tungsten halogen bulb, each housed within a projector (Eastman Kodak, Rochester, NY) and each with infrared blocking filters. One channel provided the temporally modulated light. The other channel provided a steady, rod-desensitizing adapting field. The light source for the temporally modulated channel was powered by a custom-built, regulated DC power supply. The achromatic stimuli were presented within an integrating sphere (Oriel, Stratford, CT) and the light from the two optical channels was combined with a “y” fiber-optic light guide (Oriel). 

Temporal modulation of the test field was controlled by a ferroelectric liquid crystal (FLC) shutter (Displaytech, Longmont, CO) and driver (DR-95; Displaytech). The driver was controlled by a signal-processing board (DAS-801; Keithley, Cleveland, OH) housed within a microcomputer. The FLC shutter was driven at a constant frequency of 1 kHz and was pulse-width modulated under computer control, with the duty cycle governed by a linearized look-up table. A shutter and driver (Vincent Associates, Rochester, NY) within the second optical channel controlled the adapting field presentation. 

Luminances were calibrated with a photometer (LS-110; Minolta, Osaka, Japan). The luminance of the adapting field was 17.4 candelas (cd)/m² (2.9 log troland [td], assuming an 8-mm pupil). The maximum stimulus luminance was 393.0 cd/m² (4.3 log td) and the minimum luminance was 1.4 cd/m² (1.8 log td). In the absence of the adapting field, these luminances produced a modulation of 99%. Against the adapting field, the modulation was 91.2%. 

Procedure

The pupil of the tested eye was dilated with 2.5% phenylephrine hydrochloride and 1% tropicamide drops, and the cornea was anesthetized with proparacaine drops. The subject’s head was held in position with a chin rest and forehead bar. Subjects were light-adapted to room illumination before testing and were then adapted for 2 minutes to the rod-desensitizing adapting field. ERGs were recorded with a Burian-Allen bipolar contact lens electrode, grounded at the earlobe. Responses were acquired with a signal-averaging system (Viking IV; Nicolet, Madison, WI) that was triggered by a transistor–transistor logic (TTL) signal generated by the signal processing board and synchronized with the onset of each stimulus cycle. Amplifier band-pass settings were 0.5 to 500 Hz. 

Recordings were begun after the subjects had adapted to each waveform for approximately 30 seconds. For each condition, two 500-msec recordings were obtained to determine reproducibility. Each recording was the average of four sweeps. The two recordings were averaged off-line, so that each waveform included in the analysis consisted of the average of eight 500-msec sweeps. Although the recording epoch was 500 msec, only the first 250 msec of the waveforms are presented in the following figures, so that waveform features can be identified more easily. 

Analysis

Response amplitudes at the stimulus harmonic frequencies were derived from power spectral densities, and response phases were obtained from fast Fourier transforms, by computer (Matlab Signal Processing Toolbox; The MathWorks, Natick, MA). The amplitudes of the harmonic components that are plotted in the figures represent the full peak-to-trough amplitudes of the derived sinewave components. The phases are given in cosine phase. In addition, the amplitudes of the ON and OFF responses were obtained as follows. The amplitude of the b-wave of the ON response was measured from the a-wave trough to the b-wave peak, and the amplitude of the d-wave of the OFF response was measured from the onset of the response to the peak (see Fig. 3 for illustrations of these waveform features). The values for the eight response cycles in each 500-msec waveform were averaged. The results from the patients with XLRS and the control subjects were compared using repeated-measures analyses of variance, and post-hoc comparisons were performed with two-tailed t-tests, with a Bonferroni correction for multiple comparisons (SigmaStat; SPSS, Chicago, IL). P < 0.05 was considered to be statistically significant. 

Results

Before describing the effects of low-pass stimulus filtering on the ERG ON and OFF responses of the patients with XLRS, we present background information concerning their high-frequency response attenuation and relative ON-response deficits. 

ERG Temporal Response Function in XLRS

The high-frequency response attenuation shown by the patients with XLRS is illustrated in Figure 2 . This figure plots the log amplitude of the fundamental response to sinusoidal flicker of maximum amplitude across a range of temporal frequencies. The symbols represent the fundamental responses of the three patients with XLRS, and the hatched region represents the range of the values for eight control subjects. For the control subjects, the response function was band-pass, with a peak at 32 Hz and systematically declining amplitudes at higher frequencies, although robust responses were obtained even at a temporal frequency of 96 Hz, as has been reported previously. ^{18 20} In comparison, the response functions of the patients with XLRS were less band-pass, and showed a marked response attenuation at frequencies above 32 Hz. For two (patients 1 and 3), the response amplitude did not exceed the noise level at 96 Hz, and the response amplitude of the other (patient 2) was attenuated substantially at that frequency. Statistical analysis confirmed that there was a significant difference between the fundamental responses of the patients with XLRS and the control subjects at temporal frequencies of 32 and 64 Hz (t = 2.88 and 8.58, respectively; P < 0.01), but not at 8 and 16 Hz (t = 0.43 and 0.74, respectively; P > 0.05 [96 Hz was not included in the analysis because of the negligible response amplitudes for two of the patients]). As noted in the introduction, previous evidence indicates that this amplitude reduction at high temporal frequencies results from an attenuated cone photoreceptor response. ¹⁹  

ERG ON and OFF Responses in XLRS

The nature of the ON-response deficit in XLRS is illustrated in Figure 3 , which presents the ERG responses of a representative control subject (Fig. 3 , top trace in each pair) and XLRS patient 2 (Fig. 3 , bottom trace in each pair) to rapid-on (Fig. 3 , left) and rapid-off (Fig. 3 , right) sawtooth flicker at temporal frequencies of 4, 8, and 16 Hz. In the control subject, there were marked asymmetries between the responses to rapid-on and rapid-off stimuli. The ERG response to rapid-on flicker was biphasic, consisting of an initial negative trough and a subsequent positive peak, similar to the ERG response obtained to the onset of a standard luminance increment. By analogy with the conventional ON response, the negative trough has been labeled the a-wave and the positive peak, the b-wave. The ERG response to each cycle of rapid-off flicker was monophasic with a positive peak, similar to the ERG response to the offset of a luminance increment. By analogy with the conventional OFF response, the peak has been labeled the d-wave. For the patient with XLRS, the d-wave component of the OFF response (Fig. 3 , right) was robust and similar to normal in amplitude at each temporal frequency, although delayed slightly in implicit time. However, the ON response (Fig. 3 , left) showed a markedly attenuated b-wave amplitude, with a substantially delayed b-wave implicit time. 

Effect of Low-Pass–Filtered Sawtooth Stimuli

The primary question examined in this study is the extent to which the high-frequency response attenuation of patients with XLRS, illustrated in Figure 2 , contributes to their ON-response deficits, illustrated in Figure 3 . To address this question, we first compared the patients’ ERG responses to full sawtooth stimuli with their ERG responses to low-pass sawtooth stimuli that consisted only of the fundamental and second harmonic. In this analysis, we focused on ERG responses at a stimulus temporal frequency of 16 Hz. At this frequency, the patients’ responses were of normal amplitude to the stimulus fundamental (Fig. 2) , but the higher stimulus harmonics were within the region of the high-frequency response attenuation. Therefore, the potential effect of the response attenuation on ON and OFF responses could be assessed more readily than at lower stimulus temporal frequencies. For 16-Hz sawtooth stimuli, statistical analysis confirmed that there was a significant difference between the b-wave amplitudes of the three patients with XLRS and the three control subjects (t = 4.31, P < 0.01) but no significant difference between their d-wave amplitudes (t = 0.21, P > 0.05). 

Figure 4 presents a comparison between the mean ERG responses of the patients with XLRS (bottom waveform in each panel) and the mean ERG responses of the control subjects (top waveform in each panel) to the full sawtooth stimuli (Figs. 4A 4B) and to the low-pass sawtooth waveforms (Figs. 4C 4D) . Mean responses were plotted to facilitate a comparison between the waveform shapes. The individual subjects showed the same pattern of results that is seen in the averaged data of Figure 4 . The responses to the rapid-on sawtooth stimuli are on the left (Figs. 4A 4C) , and the responses to rapid-off sawtooth stimuli are on the right (Figs. 4B 4D) . The respective stimulus waveforms are indicated below the mean ERG responses. 

For the control subjects, removing the higher stimulus harmonics reduced the amplitude of the b-wave component of the ON response (Fig. 4A , top waveform, versus 4C, top waveform) but had little effect on the amplitude of the d-wave component of the OFF response (Fig. 4B , top waveform versus 4D, top waveform), although the OFF response became broader and there was a more prominent response component between the peaks. In the patients with XLRS, there was little difference between the ERG responses to the full sawtooth waveform and the low-pass sawtooth waveform for either the ON response (Fig. 4A , bottom waveform, versus 4C, bottom waveform) or the OFF response (Fig. 4B , bottom waveform, versus 4D, bottom waveform). In fact, the patients’ mean response to the full rapid-on sawtooth stimulus (Fig. 4A , bottom waveform) resembled the mean response of the control subjects to the low-pass sawtooth waveform (Fig. 4C , top waveform), although it was reduced overall in amplitude and delayed in implicit time. Thus, the presence of the higher stimulus harmonics in the sawtooth waveform enhanced the amplitude of the b-wave component of the ON response for the control subjects but contributed little to the ERG ON response of the patients with XLRS. Further, the presence of the higher stimulus harmonics had minimal effect on the amplitude of the d-wave component of the OFF response in either the control subjects or the patients with XLRS. 

To confirm these relationships quantitatively, we measured the b-wave and d-wave amplitudes of each subject in response to both the full and low-pass–filtered sawtooth stimuli. The mean differences in amplitude between the ERG responses to the full- and low-pass filtered sawtooth stimuli are shown in Figure 5 . For the control subjects, low-pass stimulus filtering resulted in a significantly greater reduction in the amplitude of the b-wave than of the d-wave (t = 3.06, P < 0.05). Further, low-pass stimulus filtering resulted in a significantly greater reduction in b-wave amplitude for the control subjects than for the patients with XLRS (t = 5.22, P < 0.001). However, there was no differential effect of low-pass stimulus filtering on d-wave amplitude for the control subjects versus patients with XLRS (t = 0.40, P > 0.05). 

Most relevant to the present study is that the patients with XLRS showed no significant difference between their responses to the full and low-pass sawtooth stimuli for either the b-wave of the ON response (t = 0.16, P > 0.05) or the d-wave of the OFF response (t = 1.00, P > 0.05). This analysis therefore confirms that the removal of the higher harmonics from the sawtooth stimulus had no significant effect on the ERG responses of the patients with XLRS. That there was no difference between the ERG responses of the patients with XLRS to the full and low-pass sawtooth stimuli indicates that the attenuated b-wave amplitude of the ON response resulted, at least in part, from an effective low-pass filtering of the sawtooth stimulus by a response attenuation at an early retinal site, presumed from previous evidence to be at the level of the photoreceptors. ²¹  

ERG Response to the Sawtooth Fundamental in XLRS

If effective low-pass filtering at an early retinal site is the sole explanation for the attenuated b-wave component of the response to rapid-on flicker in XLRS, then the patients’ responses to the stimulus fundamental alone should be normal in shape, because there would be no high-frequency components of the stimulus to be attenuated. However, in a previous report, ¹⁸ we observed that the ERG waveforms of patients with XLRS in response to sinusoidal stimuli were abnormal at a range of temporal frequencies that included 16 Hz. This is illustrated for the present stimulus conditions in Figure 6 . This figure compares the mean ERG waveform of the three control subjects with the mean ERG waveform of the three patients with XLRS, in response to the fundamental component of the 16 Hz sawtooth. The mean response of the patients (thick trace) did not have the initial component in each cycle that was present in the normal response (thin trace), so that the patients’ mean waveform was more sinusoidal in shape than that of the control subjects, similar to results reported previously. ¹⁸  

The comparison between the ERG responses of the patients with XLRS and control subjects to the sawtooth fundamental is illustrated further in Figure 7 . This figure plots the amplitudes (Fig. 7 , top) and phases (Fig. 7 , bottom) of the harmonics of the ERG responses to the 16-Hz sawtooth fundamental. The symbols represent the results for the individual patients with XLRS, and the hatched regions represent the ranges for the three control subjects. For the control subjects (Fig. 7 , top), the response consisted primarily of components at the fundamental and second harmonic frequencies. The second harmonic was approximately half the amplitude of the response fundamental and was generated by retinal nonlinearities. ²⁰ For the patients with XLRS, the second harmonic component was markedly attenuated relative to the response fundamental, so that the ERG response was dominated by the fundamental. In addition, the phases of the fundamental and second harmonic components of the patients’ responses (Fig. 7 , bottom) were more negative than those of the control subjects, representing a phase lag (the phases of the higher harmonics were not plotted in this figure because they were inconsistent across subjects because of the low amplitudes of the responses). The implication of the patients’ abnormal responses to sinusoidal stimulation is considered in the Discussion section. 

Discussion

The purpose of this study was to determine whether a high-frequency attenuation of the ERG in XLRS, presumed to occur at the level of the cone photoreceptors, ¹⁹ contributes to the preferential ON-response defect that is observed in this disorder. This question was addressed by comparing ERG responses to full sawtooth stimuli with ERG responses to low-pass–filtered sawtooth stimuli from which the higher temporal frequencies had been removed. In the control subjects, the removal of the high stimulus temporal frequencies reduced the amplitude of the b-wave component of the ON response significantly (Fig. 4C versus 4A), but had little effect on the amplitude of the d-wave component of the OFF response (Fig. 4D versus 4B ). In comparison, the patients with XLRS showed no significant difference between their responses to full- and low-pass–filtered sawtooth waveforms for either the ON or OFF response (Figs. 4 5) . Further, the patients’ responses to a full rapid-on sawtooth waveform were similar in shape to the ERG responses of the control subjects to a low-pass rapid-on sawtooth waveform (Fig. 4) . 

These results indicate that at least part of the preferential b-wave reduction of the ON response in XLRS results from an effective low-pass filtering of the stimulus. Based on previous evidence, ^{19 21} this low-pass filtering occurs at the level of the cone photoreceptors and provides an effectively low-pass signal to the postreceptor pathways. The exact explanation of why this effective low-pass filtering affects the ON response more than the OFF response remains to be determined. However, it may be related to the observation of Kondo and Sieving ²³ that the high-frequency cutoff of DBCs (which shape the b-wave of the ON response ¹⁷ ) is higher than that of HBCs (which shape the d-wave of the OFF response ¹⁷ ), so that a reduction in high-frequency input to bipolar cells in XLRS is more detrimental to the ERG ON response. 

Although the high-frequency response attenuation contributes to the ON-response deficit in these patients with XLRS, it cannot be the sole explanation, because their responses to the fundamental component of the 16-Hz sawtooth stimulus were also abnormal. Specifically, the patients’ responses were more sinusoidal than those of the control subjects and were missing an initial waveform component that was present in each cycle of the control response (Fig. 6) . In addition, the amplitude spectra of their ERG responses had a relatively reduced second harmonic compared with the control subjects, and the harmonic components showed a phase lag relative to normal (Fig. 7) . These results confirm those obtained previously in patients with XLRS, in which sinusoidal stimuli of higher contrast were used. ¹⁸ It is unlikely that this aberrant ERG response to the stimulus fundamental was due to an abnormal cone photoreceptor response, because it has been shown that the nonlinear difference frequency (thought to represent the cone photoreceptor response ²⁰ ) is normal in amplitude and phase at temporal frequencies near 16 Hz in patients with XLRS. ¹⁹  

Instead, it is more likely that the abnormal ERG waveform in response to the sawtooth fundamental is related to an impairment within the DBC pathway. According to a recent study of the generators of the primate ERG, ²³ a similar alteration in waveform shape can be observed after the pharmacologic elimination of the DBC component of the ERG response. That is, intravitreal injection of 2-amino-4-phosphonobutyric acid (AP4), which blocks the DBC light response, resulted in a waveform that had a more sinusoidal shape and a relative phase lag compared with the ERG of the untreated eye. The similarity between this finding and the properties of the flicker ERG in patients with XLRS (Figs. 6 7) suggests that there is a relative impairment in the contribution of the DBCs to the ERG of the cone system that is unrelated to the high-frequency response attenuation. A relative impairment within the DBC pathway could result from a change in synaptic gain between photoreceptors and DBCs, or from a response deficit within the DBCs themselves. 

The emphasis of this study was on a temporal frequency of 16 Hz, but it is likely that the high-frequency response attenuation also contributes to the preferential ON-response deficit of patients with XLRS at lower sawtooth frequencies (Fig. 3) , as follows. For stimuli of a lower fundamental frequency, the higher harmonics (i.e., >32 Hz) constitute a smaller portion of the stimulus. Yet, these higher harmonics are still likely to make a significant contribution to the ERG response to sawtooth stimuli because of the band-pass nature of the ERG temporal response function, which shows an approximately sixfold increase in amplitude as the temporal frequency is increased from 8 to 40 Hz. ¹⁹ Further, the contrast gain of the ERG can be compressive within this range of frequencies, ²⁷ so that the response to a low-contrast stimulus can be proportionally larger than the response at high contrast. Therefore, even though the higher harmonics of a low-frequency sawtooth stimulus have a relatively low amplitude, the increased responsiveness of the retina to these higher temporal frequencies probably enhances their contribution to the normal ERG response. A high-frequency response attenuation in XLRS could thus have an effect on ERG ON responses even at low sawtooth frequencies. Pilot data from a control subject and a patient with XLRS confirmed this possibility. For sawtooth stimuli at a fundamental frequency of 8 Hz, adding harmonic components beyond the fourth (i.e., >32 Hz) increased the amplitude of the b-wave component of the ON response substantially in a control subject, but had no effect on the b-wave amplitude of XLRS patient 3. 

Our results demonstrate that, in contrast to previous suggestions, ^{6 8} the abnormal ERG responses of the cone system in XLRS are not the result of Müller cell dysfunction per se, but instead represent abnormalities in the responses of the photoreceptors and DBCs. Nevertheless, Müller cell dysfunction may indirectly play a role. As noted in the introduction, there is considerable histologic evidence for Müller cell abnormalities in XLRS. ^{2 3 4} Müller cells have an important role in regulating the extracellular levels of glutamate and potassium. ²⁸ High levels of glutamate are potentially toxic to neural elements, and changes in extracellular potassium can affect neuronal responses. ²⁸ Further, it has been suggested that the golden-white fundus sheen that has been observed in some patients with XLRS ^{25 26} and that was apparent in one of ours (patient 3) results from an abnormal accumulation of extracellular potassium. ²⁵ Therefore, the abnormal neuronal responses that appear to be the source of the ERG response deficits of the cone system in XLRS may result from abnormalities in the extracellular environment, in which Müller cell dysfunction may play a key role. 

Our evidence for multiple sources for the ERG deficits in XLRS is consistent with recent information regarding the molecular genetic basis for the disorder. As noted in the introduction, mutations in the RS1 gene are responsible for XLRS. ¹ The protein product of the RS1 gene, retinoschisin, is secreted by photoreceptors ^{11 12} and bipolar cells. ¹² Retinoschisin is thought to be involved in cell–cell interactions, particularly within the inner retina and probably involving Müller cells. ¹¹ Therefore, it is not unexpected that the gene mutation responsible for XLRS could have functional consequences at several levels within the retina. 

In conclusion, our results indicate that two major factors contribute to the preferential ON-response deficits of patients with XLRS: a high-frequency response attenuation at the level of the cone photoreceptors that effectively produces a low-pass input to the postreceptoral pathway, and a relative response deficit within the DBC system that is unrelated to the photoreceptoral abnormality. Because the integrity of the ON response depends not only on an adequate DBC response but also on the viability of the input from cone photoreceptors to the DBCs, an abnormal cone photoreceptor response should be considered as a possible contributor to the preferential ON-response deficits that have been observed in patients with other forms of retinal disease. 

 Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2001.

 Supported by National Institutes of Health Research Grant EY08301 (KRA) and Core Grant EY01792, a grant from the Campus Research Board of the University of Illinois at Chicago (KRA), a center grant from The Foundation Fighting Blindness (GAF), and an unrestricted departmental grant from Research to Prevent Blindness.

 Submitted for publication January 4, 2001; revised April 9, 2001; accepted May 15, 2001.

 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.

 

Table 1.

 

Patient Characteristics

Patient	Age	Visual Acuity	Visual Field Grade*	Fundus Grade, †
1	22	20/70	2A	2B
2	29	20/30	1	1
3	36	20/40	2B	2B

 Data are from the left eye.

*  1, No peripheral visual field restriction; 2A, peripheral visual field restriction in only one quadrant; 2B, peripheral visual field restriction in two quadrants.

†  1, Foveal schisis only; 2B, peripheral fundus changes present in two quadrants.

Figure 1.

 

Illustration of the sawtooth stimulus waveforms at a temporal frequency of 16 Hz, for a full rapid-on sawtooth (A), full rapid-off sawtooth (B), low-pass rapid-on sawtooth (C), and low-pass rapid-off sawtooth (D). Horizontal lines: time-average luminance.

Figure 2.

 

Log amplitude of the ERG fundamental response to sinusoidal stimulation for the individual patients with XLRS (symbols) at each of five stimulus temporal frequencies, with frequency plotted logarithmically. Linear values of the log amplitudes are indicated on the right y-axis. Hatched region: range of values for eight control subjects. ND, response not distinguishable from noise.

Figure 3.

 

ERG waveforms of a representative control subject (top waveform in each pair) and XLRS patient 2 (bottom waveform in each pair) to rapid-on (left) and rapid-off (right) sawtooth flicker at 4, 8, and 16 Hz (top to bottom). The stimulus waveform is illustrated below each pair of ERG responses. Arrows: peaks of a-, b-, and d-waves.

Figure 4.

 

Mean ERG waveforms of the three control subjects (top trace in each panel) and of the three patients with XLRS (bottom trace in each panel) in response to rapid-on (A, C) and rapid-off (B, D) stimuli, for either full sawtooth (A, B) or low-pass sawtooth (C, D) flicker. The stimulus waveforms are illustrated below the traces.

Figure 5.

 

Mean differences between the ERG responses to full- and low-pass–filtered sawtooth stimuli in patients with XLRS (filled bars) and control subjects (open bars), for the b-wave (left bars) and d-wave (right bars). Error bars, ±1 SEM.

Figure 6.

 

Illustration of the mean ERG waveform for the patients with XLRS (thick trace) and for the control subjects (thin trace) in response to a 16-Hz sinusoidal stimulus that had an amplitude given by equation 1 . The stimulus waveform is illustrated on the x-axis.

Figure 7.

 

Amplitudes (A) and phases (B) of the harmonics of the ERG responses of the individual patients with XLRS (symbols) to a 16-Hz sinusoidal stimulus, with an amplitude calculated by equation 1 . Amplitudes are plotted for the first four harmonics; phases are plotted only for the fundamental and second harmonics, because the phases at the higher harmonics were highly variable across subjects, as a result of the near-noise-level amplitudes. Hatched regions: ranges of values of three control subjects.

The authors thank Deborah J. Derlacki for assistance in subject testing.