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1 From the Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois; 2 Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania; and the 3 F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania, Philadelphia, Pennsylvania.
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Abstract |
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METHODS. Two patients with MAR syndrome whose sera produced immunolabeling of retinal bipolar cells participated in the study. Full-field ERGs were recorded in response to brief flashes, to rapid-on and rapid-off sawtooth stimuli at a temporal frequency of 8 Hz, and to sine-wave stimuli at temporal frequencies ranging from 8 to 96 Hz. Fundamental responses to the sine-wave stimuli were evaluated within the context of a vector-summation model of the depolarizing bipolar cell (DBC) and hyperpolarizing bipolar cell (HBC) contributions to the response fundamental. VEPs were recorded to the onset of luminance increments and decrements that had contrasts of 10%, 20%, and 50%. The patients’ results were compared with those of age-similar control subjects.
RESULTS. The patients with MAR showed abnormal ERG responses to luminance increments, consisting of a marked attenuation of the initial portion of the b-wave, but their ERG responses to luminance decrements were normal in amplitude and timing. The ERG temporal response functions of the patients with MAR had normal amplitudes at frequencies of 32 Hz and higher, with a constant phase lag across these frequencies, but larger-than-normal amplitudes at frequencies below 32 Hz, and a phase lead at 8 Hz. Their VEP responses showed a marked delay to increments but only a minimal delay to decrements.
CONCLUSIONS. Within the context of the vector-summation model, the ERG findings in the patients with MAR are more consistent with an attenuation of the DBC contribution to the ERG response than with a DBC response delay. The delayed VEP responses of the patients with MAR to luminance increments may represent a late response of the OFF system to increment onset.
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Introduction |
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The basis for the ERG abnormalities in patients with MAR is not well understood at present. The selective attenuation of the b-wave of the rod and cone ERGs in patients with MAR and their abnormal cone ERG ON responses are similar to the ERG changes observed when L-2-amino-4-phosphonobutyrate (L-AP4, formerly referred to as APB) is injected intravitreally into the monkey eye.6 9 L-AP4, a glutamate analogue, blocks signal transmission from photoreceptors to depolarizing (ON) bipolar cells (DBCs).10 Therefore, it has been proposed that the selective b-wave reduction of the brief flash ERG and the abnormal cone ON response in patients with MAR syndrome similarly represent an attenuation of signal transmission specifically within the DBC pathway.4 Such a response attenuation would account for the patients’ night blindness, reduced rod b-wave amplitudes, and abnormal cone ERG ON responses.
Alternatively, the abnormal ON response of the cone ERG in patients with MAR syndrome could result from an increased latency of the DBC response, with a normal DBC response amplitude. As discussed previously,11 12 a delay of a few milliseconds in the DBC response relative to that of the hyperpolarizing (OFF) bipolar cell (HBC) system can produce an apparent ON-response deficit in the cone ERG. There is some evidence in favor of a DBC response delay as an explanation for the abnormal cone ON response of patients with MAR. For example, patients with other forms of night blindness in which there is a cone ERG deficit similar to that seen in patients with MAR have shown a delay in the visual evoked potential (VEP) response to luminance increments compared with the response to decrements, with no differential change in response amplitude.13 14 This appears more consistent with a delay in the DBC response than with a DBC response attenuation. Further, Wolf and Arden15 specifically tested for psychophysical evidence of an ON-pathway defect in patients with MAR and reported no relative elevation in the threshold for increments versus decrements, concluding that there was no evidence for an attenuated signal within the cone ON pathway. Therefore, it is possible that the ERG ON-response deficit in patients with MAR syndrome represents a delay in the DBC response relative to that of the HBC response, rather than an attenuation of signal transmission within the DBC pathway, as proposed previously.4 A response delay within the DBC pathway would not explain the night blindness of patients with MAR, but as Sieving11 has noted, it is not necessarily true that a given retinal abnormality would affect both the rod pathway and the cone ON pathway in the same way.
Based on a recent study,16 it should be possible to distinguish between a response delay and a response attenuation within the cone DBC pathway in patients with MAR by analyzing their ERG responses to sinusoidal flicker within the framework of a vector-summation model of the primate ERG. According to this model,16 the fundamental of the ERG response evoked by sinusoidal stimulation is the vector sum of the massed fundamental responses of the cone photoreceptors, DBCs, and HBCs. The amplitudes and phases of the response components of the model were derived from the monkey retina by means of pharmacologic isolation. At temporal frequencies near 32 Hz (the frequency typically used clinically), the model predicts that a reduction in the DBC response amplitude alone would have little effect on the amplitude of the fundamental of the ERG response but would produce a substantial phase lag. Conversely, a delay in the DBC response relative to the responses of the photoreceptors and HBCs would increase the fundamental response amplitude at 32 Hz compared with normal, but would introduce only a minimal phase lag.
The vector-summation model also predicts that a DBC response attenuation and a DBC response delay would have quite different effects on the shape of the temporal response function at temporal frequencies below 32 Hz. The normal ERG temporal response function has a peak near 32 Hz and a minimum near 10 Hz. The response minimum has been attributed to a relative cancellation between the out-of-phase responses of the DBCs and HBCs at this frequency.16 If there were an attenuation of the DBC contribution to the ERG, then the model predicts that the fundamental response would be enhanced compared with the normal response at frequencies near 10 Hz. This would result in a flattened temporal response function across the lower frequency range. Conversely, the vector-summation model predicts that a delayed DBC response would have little effect on response amplitudes at frequencies near 10 Hz but would increase response amplitudes at higher frequencies. This would result in a more strongly bandpass temporal response function over the frequency range from 10 to 32 Hz. (Illustrations of quantitative predictions of the model for a DBC response attenuation and a DBC response delay across a range of temporal frequencies are presented in Fig. 6 .)
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Stimuli and Instrumentation
Brief-flash ERGs were measured in response to achromatic (xenon) strobe flashes that were presented in a ganzfeld (Nicolet, Madison, WI). ERG responses to sinusoidal and sawtooth stimuli were measured with instrumentation that has been described previously.18
In brief, the stimulus consisted of achromatic full-field flicker that was superimposed on an achromatic rod-desensitizing adapting field, both presented within an integrating sphere (Oriel, Stratford, CT). The flickering stimulus and adapting field were provided by two separate 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. The light from the two optical channels was combined with a "y" fiber-optic light guide (Oriel) that was introduced into a side port of the integrating sphere.
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 temporal 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 stimulus for the VEP was based on that of Zemon et al.,19 and has been described previously.13 In brief, the stimulus consisted of a 12° x 12° grid of squares, presented on a computer monitor against a background of 1.5 log cd/m2 and controlled by a stimulus presentation and data acquisition system (Venus; NeuroScientific Corp., Farmingdale, NY). The squares were each 0.3° in width and were 0.4° apart. Each stimulus cycle consisted of 200 ms of the incremental squares (luminance higher than the background) followed by 800 ms of the background alone, which was followed in turn by 200 ms of the decremental squares (luminance lower than the background) and another 800 ms of the background alone. This stimulus cycle was repeated continuously until the requisite number of sweeps had been obtained (described later). Stimuli of 10%, 20%, and 50% Weber contrast were used, with luminances controlled by a linearized look-up table.
Procedure
For all recordings, 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. ERG responses were recorded using a signal averaging system (Viking IV; Nicolet). For the brief-flash ERG, responses to flashes of 0.9 log cd /m2·sec were recorded from the test eye with a monopolar Burian-Allen contact lens electrode, with a forehead electrode as the reference and an earlobe as the ground, after 10 minutes of light adaptation to a rod-desensitizing adapting field of 1.3 log cd/m2. The flashes were presented at 1-second intervals, and responses to four flashes were averaged.
Responses to sinusoidal and sawtooth stimuli were recorded in a separate session. Subjects were light adapted to room illumination before testing and were then adapted for 2 minutes to a rod-desensitizing adapting field of 1.2 log cd/m2. The left eye was tested in all subjects. Recordings were made with a bipolar Burian-Allen contact lens electrode grounded at the earlobe. The signal-averaging system was triggered by a transistor-transistor logic (TTL) signal generated by the signal-processing board (DAS-801; Keithley) and synchronized with the onset of each stimulus cycle. ERG recordings were made at sine-wave temporal frequencies of 8, 16, 32, 64, and 96 Hz, with the sine waves presented at maximum amplitude and in sine phase. Recordings were also made at a sawtooth stimulus frequency of 8 Hz, at maximum amplitude and in both rapid-on and rapid-off phase. Each cycle of rapid-on sawtooth flicker 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 consisted of an abrupt decrement in luminance, to emphasize an OFF response, followed by a linear increase in luminance. These sawtooth stimulus waveforms are illustrated in Figure 2 . The maximum luminance of the sinusoidal and sawtooth stimuli was 2.6 log cd/m2 and the minimum luminance was 0.1 log cd/m2. In the absence of the adapting field, these luminances produced a modulation of 99%. Against the adapting field, the modulation was 91.2%.
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Monocular VEPs were recorded in a dimly lit room. The tested eye was chosen at random, except that the nonamblyopic left eye of patient 2 was stimulated. Subjects viewed the display through the best optical correction in a trial frame, with the untested eye occluded. Responses were recorded from an electrode positioned 3 cm above the inion (Oz), with a vertex electrode as reference and Fz as ground. For the control subjects, two blocks of 50 sweeps each were averaged at 10% stimulus contrast, and two blocks of 25 sweeps each were averaged at 20% and 50% contrast. For the patients with MAR, three blocks were acquired for each condition to determine reproducibility, and peak latencies were measured separately for each block.
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Results |
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As noted previously,4 5 the abnormal b-wave responses of the brief-flash ERG of the patients with MAR, seen in Figure 1 , represent a selective ON-response defect. This is illustrated in Figure 2 , which presents the ERG responses of the two patients with MAR and a representative control subject to rapid-on (emphasizing an ON response) and rapid-off (emphasizing an OFF response) sawtooth stimuli at a temporal frequency of 8 Hz. The patients’ responses to the rapid-on sawtooth (Fig. 2 , left) were abnormal, in that the b-wave was markedly attenuated and delayed and did not rise above the prestimulus level, in contrast to the control responses. However, both patients with MAR showed robust d-wave responses to the rapid-off sawtooth (Fig. 2 , right). The amplitudes of the patients’ d-waves were within (55.3 µV, MAR 1) or just beyond (72.7 µV, MAR 2) the normal range (34.9–71.6 µV), and their d-wave implicit times (23 ms, MAR 1; 24 ms, MAR 2) were within the normal range (21–25 ms). Furthermore, the shapes of the patients’ d-wave responses were within the range of variation exhibited by normal subjects.18
Abnormal ON responses were also apparent in the patients’ ERG responses to 8- and 16-Hz sinusoidal stimuli, as illustrated in Figure 3 . At these two temporal frequencies, control subjects typically show two response peaks per stimulus cycle.20 These peaks have been labeled in Figure 3 as "ON" and "OFF" responses, respectively, to indicate that they corresponded to the regions of increasing and decreasing luminance in the sinusoidal stimulus. By comparison, the ERG responses of the patients with MAR showed only one peak at each frequency, corresponding to an OFF response, with only a minor inflection in the waveform at the time of the normal ON response.
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The filled circles (Fig. 6 , left) indicate the fundamental response amplitude (top) and phase (bottom) to be expected if there were a complete attenuation of the DBC contribution to the ERG response fundamental. The filled triangles (Fig. 6 , right) represent the fundamental response amplitude (top) and phase (bottom) if the DBC response was of normal amplitude but was delayed by 5 ms compared with the response of the cone photoreceptors and HBCs (other DBC delays yielded a similar pattern of results). The amplitude and phase functions for the patients (Fig. 5) were more similar in shape to the predicted effect of a response attenuation within the DBC pathway (Fig. 6 , left) than with a DBC response delay (Fig. 6 , right). That is, the amplitude functions of the patients were relatively flat across the frequency range from 8 to 32 Hz, and their phase functions had a phase lead at 8 Hz and a phase lag at the higher temporal frequencies. These results support the hypothesis that the ERG ON-response deficits in patients with MAR syndrome represent an attenuation of signal transmission within the retinal ON pathway.
VEP Responses
The VEP responses of the two patients with MAR and a representative control subject are shown in Figure 7
. This figure plots VEP responses to luminance increments (Fig. 7
, left) and luminance decrements (Fig. 7
, right) at a stimulus contrast of 50%. Two averaged responses are presented for each subject, to illustrate reproducibility. As observed previously,13
the normal VEP responses to increments and decrements each show a dominant positive peak, termed P1 and indicated by the arrows in the figure. In the control subjects, the peak latencies for P1 at 50% contrast were approximately 110 ms for both increments and decrements. (The normal ranges are indicated by the shaded regions in the figure.) In the two patients with MAR, the responses to luminance increments (Fig. 7
, left) were markedly delayed, and the response waveforms were broader than normal. The response latencies for luminance decrements (Fig. 7
, right), however, were normal (MAR 2) or just at the upper limit of the normal range (MAR 1). Of note, the patients’ responses to both increments and decrements occurred before the termination of the 200-ms stimulus and therefore did not represent responses to stimulus offset.
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Discussion |
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To determine whether the ERG ON-response abnormality represents an amplitude reduction or a response delay within the DBC pathway, we examined the ERG fundamental response to sinusoidal flicker within the context of a vector-summation model of the generators of the primate ERG.16 The patients with MAR showed a fundamental response at 32 Hz that was normal in amplitude but had a phase lag of approximately 50° relative to the normal mean (Fig. 4) . According to the vector-summation model,16 this result is more consistent with a reduction in the amplitude of the DBC response than with a DBC response delay, which would have increased the ERG fundamental response amplitude substantially but would have had little effect on the response phase (Fig. 6) .
Other features of the temporal response functions of the patients with MAR (Fig. 5) were also more consistent with an attenuated DBC response than with a DBC response delay. First, the patients’ relatively enhanced fundamental response amplitudes at the lower temporal frequencies and the flattening of their amplitude functions (Fig. 5 , top) are consistent with this explanation. That is, for normal subjects, the vector summation of nearly out-of-phase DBC and HBC components is thought to reduce the amplitude of the response fundamental at these lower temporal frequencies.16 Therefore, an attenuated DBC response would enhance the fundamental response amplitude at low temporal frequencies compared with normal, as is seen in the patients’ amplitude functions. Second, according to the vector-summation model,16 an attenuated DBC response would result in a phase advance at 8 Hz and a phase lag at higher frequencies, as is observed in the patients’ phase plot (Fig. 5 , bottom). As noted previously,4 an attenuated response within the DBC system would account not only for the abnormal cone ERG results of the patients with MAR, but also their reduced rod b-wave amplitude and their night blindness, given that rod bipolar cells are of the depolarizing (ON) type.21
Although the ERG data of the patients with MAR are more consistent with a relative attenuation of the DBC response than with a DBC response delay, the patients showed a marked delay in the VEP response to luminance increments (Figs. 7 8) . The explanation for this finding is presently uncertain, but we note that a similar type of late response to luminance increments has been observed in the light-evoked field potentials of the superior colliculus of the mGluR6-deficient mouse.22 The metabotropic glutamate receptor mGluR6 mediates synaptic transmission from photoreceptors to DBCs,23 24 so that mGluR6-deficient mice have a marked ON-pathway defect. Sugihara et al.22 suggested that the late response to luminance increments in these knockout mice may represent the response of the ON-surround of OFF-center ganglion cells to light onset. Consistent with this possibility, Massey et al.25 reported that some OFF-center ganglion cells of the rabbit retina responded to light onset with a delayed response from the antagonistic surround that was approximately 90 ms later than the response of ON-center ganglion cells to the same stimulus. We hypothesize that the delayed VEP response to luminance increments in our patients with MAR may similarly represent a late response of the ON-surround of OFF-center cells.
As noted in the introduction, Wolf and Arden15 concluded that there was no specific damage to the ON-pathway in patients with MAR syndrome, based on the observation that their three patients showed no threshold asymmetry for identifying letters of positive versus negative contrast, or for detecting increment versus decrement Gaussian patches. However, these tests are not likely to be entirely appropriate for assessing ON-pathway dysfunction. It has been shown that letter identification is based on a limited band of object frequencies,26 27 and within that critical band, letter stimuli contain regions of both positive and negative contrast.28 Therefore, it is not likely that this task preferentially stimulates ON versus OFF pathways. Further, the detection of briefly presented incremental Gaussian patches by their patients with MAR could have been based on stimulus offset (effectively a luminance decrement) rather than stimulus onset.
In agreement with Wolf and Arden,15 the patients with MAR syndrome in our study showed a marked loss of contrast sensitivity for large letters but had normal high-contrast visual acuity (Table 1) . Although a selective magnocellular pathway defect was invoked previously to account for this finding,15 a loss of contrast sensitivity at low spatial frequencies accompanied by normal visual acuity is also consistent with an ON-pathway defect. For example, the intravitreal injection of L-AP4 to inactivate the retinal ON pathway of monkeys greatly reduced their contrast sensitivity at low spatial frequencies but had little effect on their visual acuity.29 Therefore, an ON-pathway deficit could account for this finding in patients with MAR, as well.
Wolf and Arden15 reported that their patients with MAR had the greatest loss of sensitivity under test conditions that involved transient stimuli (i.e., temporal contrast sensitivity and low-contrast displacement thresholds). They hypothesized that this might be due to selective damage within the magnocellular pathway, although the locus of this damage was not specified. It has been reported recently that the sustained and transient properties of the visual pathway are first organized at the level of the retinal bipolar cells.30 This raises the intriguing possibility that the visual disability experienced by patients with MAR may represent the effect of an antitumor antibody on a specific class of cone ON bipolar cells: those with transient response properties. The exact mechanism by which an autoantibody might affect the function of a particular class of retinal bipolar cells remains to be resolved, however.
In conclusion, the two patients with MAR syndrome who were investigated in this study showed an ON-response deficit of the ERG of the cone system that was manifested as a selectively reduced b-wave response to brief luminance increments, an abnormal response to rapid-on (incremental) sawtooth flicker with a normal response to rapid-off (decremental) sawtooth flicker, and a reduction in the first (ON) component of the ERG response to low-frequency sinusoidal flicker. They also showed ERG responses to 32-Hz sinusoidal flicker that were of normal amplitude but had a relative phase lag. Analysis of their ERG responses to sinusoidal flicker at a range of temporal frequencies within the framework of a vector-summation model16 indicated that the ON-response deficits of the cone ERG were more consistent with an attenuation of the DBC response component than with a DBC response delay. Nevertheless, these patients showed profoundly delayed VEP responses to luminance increments with relatively normal responses to luminance decrements. The delayed VEP responses to increments may represent the response of OFF-center cells to light onset, as has been proposed for the delayed light-evoked field potentials of the superior colliculus in mGluR6 knockout mice.22
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Acknowledgements |
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Footnotes |
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Submitted for publication September 12, 2001; revised December 6, 2001; accepted December 11, 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.
Corresponding author: Kenneth R. Alexander, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor Street, Chicago, IL 60612; kennalex{at}uic.edu
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), Log amplitude and phase of the ERG response fundamental under standard recording conditions. (•), Log amplitude and phase of the ERG response fundamental if the DBC response were completely absent. (
), Amplitude and phase of the ERG response fundamental if the DBC response were delayed by 5 ms compared with the responses of the cone photoreceptors and HBCs.
