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From the College of Optometry, University of Houston, Houston, Texas.
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Abstract |
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METHODS. ERGs were recorded from 14 anesthetized adult macaques using DTL electrodes. Six monkeys had laser-induced experimental glaucoma, and two others received intravitreal injections of tetrodotoxin (TTX, 6 µM) to block spiking activity of inner-retinal neurons. The remaining 6 animals were normal. Uniform fields and grating patterns (0.1–3 cpd) were square-wave modulated at 1.7 Hz (transient) and 8 Hz (steady state). The test field (42° x 32°) had a mean luminance of 44 cd/m2 and a contrast of 10% to 82%.
RESULTS. In normal eyes transient ERGs to uniform fields contained photopic negative responses (PhNR) after the b-wave and after the d-wave. Transient pattern electroretinograms (PERGs) at each contrast reversal showed positive (P50) potentials followed by negative (N95) potentials of time course similar to that of the PhNR. The PhNR and N95 were greatly reduced or eliminated by experimental glaucoma and by TTX. Summing responses to luminance increments and decrements of the uniform field could simulate the PERG to low spatial frequency stimuli. Further, the PERG responses to high spatial frequencies were similar to the simulation in shape but slightly delayed in time. Experimental glaucoma and TTX had similar effects on the N95 of the simulated PERG as to those on the actual PERG. However, P50 was more reduced by experimental glaucoma than by TTX, indicating a nonspiking contribution to P50. For the steady state condition, the uniform field ERG, the simulated PERG, and the actual PERG all were affected by experimental glaucoma and TTX, indicating that they contained contributions from the spiking activity of ganglion cells.
CONCLUSIONS. The changes in the uniform field and PERG responses produced by experimental glaucoma are related and are largely a consequence of reduced spiking activity of ganglion cells and their axons. These findings raise the possibility that the uniform field ERG could serve as a useful alternative to the PERG in the assessment of clinical glaucomatous neuropathy.
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Introduction |
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In contrast to the PERG, the electroretinogram (ERG) elicited by modulation of uniform field luminance has been viewed traditionally as reflecting activity of retinal neurons distal to the ganglion cells (see Ref. 16 for review) and to be relatively unaffected in glaucomatous eyes (see Refs. 6 and 7 for reviews). However, there is increasing evidence that the uniform field ERG contains contributions from inner-retinal neurons17 18 19 20 and that this measure could be reduced in patients with glaucoma.21 22 23 24 From a practical standpoint the uniform field ERG is easier to record than the PERG, because it does not require refractive correction or exact foveal placement. However, it is unclear whether the uniform field and pattern ERG responses that are reduced by glaucoma result from damage to the same generators. In the present study we investigated this issue in macaques with monocular experimental glaucoma.
The macaque model of laser-induced experimental glaucoma has been used widely to study structural damage and functional losses resulting from elevation of intraocular pressure,10 18 20 25 26 27 28 29 30 31 32 33 and several studies have reported reductions in the PERG amplitude.10 26 27 Recent studies18 20 33 have reported alterations in uniform field ERGs as well. Frishman et al.18 reported a substantial selective reduction of the negative-going scotopic threshold response (nSTR) in the dark-adapted (scotopic) flash ERG of macaques with experimental glaucoma, and Hare et al.33 found that steady state flicker responses were reduced. Most recently, Viswanathan et al.20 showed that the light-adapted (photopic) ganzfeld flash ERG in macaques contains a slow negative potential, the photopic negative response (PhNR), after the b-wave (and the d-wave for long-duration flashes), that is reduced both by experimental glaucoma and by intravitreal injection of tetrodotoxin (TTX). Whereas TTX blocks the Na+-dependent spiking activity of amacrine cells34 35 and possibly interplexiform cells36 in addition to retinal ganglion cells (and their axons), experimental glaucoma is believed mainly to compromise ganglion cell function.28 29 Thus, it is likely that PhNR reflects spiking activity of ganglion cells (and their axons).
The time course of the PhNR is reminiscent of the timing of the slow negative potential in the transient PERG that is reduced in patients with inner-retinal dysfunction.37 This observation raises the possibility that the slow negative potentials in the uniform field and pattern ERG are of the same origin. In the present study we compared the effects on uniform field and pattern ERGs in macaques of experimental glaucoma and of blockade of spiking activity with TTX. Results from this study have appeared previously in an abstract.38
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Methods |
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Animal Preparation for ERG Recordings
Animals were anesthetized with ketamine (20–25 mg/kg ·
h-1) and xylazine (0.8–0.9 mg/kg ·
h-1 intramuscularly [i.m.]), and atropine
sulfate (0.04 mg/kg) was injected subcutaneously. The depth of
anesthesia was sufficient to prevent the animals from blinking or
moving. The level of anesthesia halved the intraocular pressure (IOP)
in experimental and control eyes within the first hour, after which the
pressures declined more slowly without noticeable effect on the
ERG.18
Pupils were fully dilated to approximately 9 mm in
diameter with tropicamide (1%) and phenylephrine hydrochloride
(2.5%). The corneas were covered with gas permeable contact lenses.
The refractive error of the eyes over the contact lenses were
determined by retinoscopy, and an appropriate trial lens was used to
focus the tested eye at the viewing distance of the visual display. An
indirect ophthalmoscope (American Optical Company, Buffalo, NY)
was used to center the fovea of the tested eye on stimulus. Body
temperature was maintained between 36.5°C and 38°C with a heating
pad.
ERGs were recorded differentially between DTL fiber electrodes39 moistened with carboxymethylcellulose sodium 1%, centered across the cornea of each eye, and covered by contact lenses. A thin needle inserted under the scalp served as the ground electrode. Recording sessions lasted approximately 3 hours, after which the animals were allowed to recover. For the six normal animals and the animals that were injected with TTX, all recordings were done in a single session. Each animal with experimental glaucoma was tested in at least two different sessions.
Visual Stimulation
The white visual stimuli (color temperature 4700°K) were
generated by a personal computer–based Optima System (Cambridge
Research Systems, Rochester, Kent, UK) and displayed on an RGB monitor
(model HL7955SKF; Mitsubishi Electric Corporation, Nagasaki,
Japan) running at a frame rate of 100 Hz. Stimuli were
luminance modulations of a uniform field or contrast reversal of
grating patterns. For nine animals in early studies (including OHT-6,
-9, and -11) we used spatially sinusoidal gratings. For three animals
with experimental glaucoma in later studies (OHT-25, -27, and -28) and
for the two animals that received intravitreal TTX (TTX-1 and TTX-2),
we chose to use square-wave luminance modulations in both spatial and
temporal domains because this combination yields the largest response
amplitudes (e.g., Ref. 40)
. For both patterns, spatial frequencies
ranged from 0.1 to 3 cpd. It has been shown41
that
irrespective of the stimulus spatial frequency the PERG temporal
frequency response function shows a first peak for luminance
modulations at approximately 2 Hz (4 contrast reversals/sec
[rev/sec]) and a second peak around 8 Hz (16 contrast rev/sec). For
OHT-25, -27, and -28, and for TTX-1 and TTX-2 we chose frequencies near
the peaks: 1.7 and 8 Hz for uniform field modulations and 3.4 and 16
contrast rev/sec for the pattern stimuli. For the 9 animals that were
tested with spatially sinusoidal gratings, we used 1- and 8-Hz
modulation of the uniform field and 16 contrast rev/sec for the pattern
stimuli.
Photopic luminances (cd · m-2) of the visual stimuli were calibrated using a spot photometer (model LS-100; Minolta Camera Co., Osaka, Japan). The minimum and maximum luminances of the uniform field were 8 and 80 cd · m-2, respectively. In most experiments the bright and dark bars of the pattern had the same luminances, giving a contrast of 82%. In experiments using spatially sinusoidal gratings, the contrast was varied between 10% and 82%. In all experiments the viewing distance was 43 cm, at which the test field subtended 42° of visual angle horizontally and 37° vertically. For most of the experiments we used a bright rectangular surround (120 cd/m2) that extended 17° and 15° from the horizontal and vertical edges of the screen.
Signal Processing and Response Measurement
Signals were amplified (amplifier model MAS800; Cambridge Research
Systems, Rochester, Kent, UK), filtered (0.5–55 Hz), and
digitized at 1 kHz with a resolution of 0.1 µV. Responses were
averaged over 250 stimulus presentations. We used Fourier analysis to
measure the amplitude and phase of the second harmonic component of the
steady state uniform field responses and of the amplitudes and phases
of the steady state pattern responses at the temporal frequency of
contrast reversal.
Experimental Glaucoma
The IOPs in the right eyes of six monkeys were elevated by argon
laser treatment of the trabecular meshwork. As described
previously,30
after animals were anesthetized (ketamine:
20 mg/kg and acepromazine: 0.2 mg/kg i.m., and 0.5% topical
proparacaine hydrochloride), blue-green argon laser treatment spots
(50-µm spot size, 1.0 W power, and 0.5 seconds’ duration) were
applied to the trabecular meshwork using a slit-lamp delivery system
(model PC; HGM, Salt Lake City, UT). Spots were placed to produce
contiguous tissue blanching.
Intraocular Pressures
IOPs were measured with a hand-held applanation tonometer (model
HA-1; Kowa Co., Ltd., Tokyo, Japan) from monkeys anesthetized as for
the laser procedure, or in the initial few minutes of the anesthesia
regimen used for the ERG recordings. The IOPs of the control and
experimental eyes at the time of ERG recordings are shown in Table 1
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Pharmacological Blockade
One or two intravitreal injections (30–60 µl) of TTX (Sigma,
St. Louis, MO) dissolved in sterile basic salt solution and passed
through a 0.2-µm filter were made by inserting a sterile 30-gauge
needle through the sclera into the vitreal cavity. Assuming no leakage
and a 2.1-ml vitreal volume, the intravitreal concentration of TTX was
approximately 6 µM. All responses reported in this study were
measured at least 1 hour after the TTX injections and after recentering
the fovea on the stimulus display.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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PERG Simulations
In the present study we were interested in comparing the changes
in the uniform field and pattern ERGs produced by experimental
glaucoma. For a uniform field, the luminance of the whole field is
changed whenever the contrast is reversed, whereas for the contrast
reversals of the grating patterns the luminance increases over half the
field and decreases over the other half. To change the uniform field
response to a form that can appropriately be compared to the PERG, we
added the response to a luminance increment to the response to a
decrement after dividing each by 2, which is equivalent to calculating
the arithmetic mean of the luminance increment and decrement responses.
This simulation is illustrated in Figure 2
, where the top trace is the uniform field ERG response to a luminance
increment followed by a decrement, recorded from the normal eye of
animal TTX-1. The second row shows the top trace reversed in phase
(i.e., a response to decrement followed by an increment). These
responses have been scaled to half their original size. The sum of
these responses, which represents the PERG simulation, is shown in the
third row. The simulation is dominated by slow negative potentials that
have time course similar to that of the PhNRs in the original waveform,
suggesting that these slow potentials predominantly reflect the
summation of the PhNRs to luminance increments and decrements. The
simulation also contains earlier positive potentials that have peak
times similar to those of b- and d-waves derived
from the summation of early portions of the uniform field responses
after luminance increments and decrements.
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For the higher spatial frequencies used in this study (up to 3 cpd), the PERG responses still contained the major features of the simulations, but the timing of the actual PERG troughs and peaks were slightly different. As shown in the bottom row of Figure 2 and in Figure 1 , the responses to the higher spatial frequency stimuli were delayed relative to those for 0.1 cpd and the simulation. Further, the amplitude of P50 was markedly smaller in the PERG responses to higher spatial frequencies. The effects of spatial frequency on N95 amplitude were small and not consistent for the range of spatial frequencies that we used (cf. Figs. 1 and 2 with 3A ). The response delay at higher stimulus spatial frequency was not due to the degradation of retinal image contrast. We found that decreasing the stimulus contrast from 82% to 15% reduced the amplitude of the macaque PERG, but did not alter the timing (results not shown), which was consistent with previous reports on the human PERG.44 The effects of spatial frequency on the timing of N95 and P50 and P50 amplitude leads one to surmise that in addition to responses to local luminance changes, the macaque PERG also contains responses that are driven by the local luminance gradients (and in that sense they are pattern specific responses), as previously proposed for the human PERG.45
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For uniform field stimulation (Fig. 3A , top), responses recorded from the experimental eye (middle column) showed a reduction of the slow negative potential (PhNRon) after light increment. In comparison, the a- and b-waves were less markedly reduced. After light decrement, the d-wave peaked earlier (by 7 msec), and it was followed by a prolonged positive potential indicating removal of PhNRoff that normally counteracts it. Although not very obvious in the original records, the difference record on the right shows that the PhNRs after luminance increment and decrement were approximately equally reduced by experimental glaucoma. The difference record also contains small early positive potentials after light increment and decrement that reflect changes in the b- and d-waves. In the PERG simulations (second row) from the experimental eye (middle column), the slow negative potentials and to a lesser extent the earlier positive potentials were both reduced, and this again can be seen more clearly in the difference record. Similar results were obtained from the other five animals in which we induced experimental glaucoma, and the difference records for the two other animals for which we used 1.7-Hz luminance modulation appear in Figure 3B . It should be noted that before induction of experimental glaucoma, in control experiments we had ascertained that ganzfeld flash ERGs in the two eyes of the subjects were very similar. For this reason, we believe that the difference records are valid indicators of the effects of experimental glaucoma.
In a previous study of photopic ERG responses to long-duration red flashes, removal of PhNR in eyes with experimental glaucoma transformed the light onset responses into sustained positive potentials.20 We observed similar results for red flashes for all the animals with experimental glaucoma in the present study (data not shown). However, in the ERG responses to the white uniform field, the b-wave retained its transient nature. This was true even in severely impaired experimental eyes. For example, the insets to Figure 3A show the uniform field ERG responses to luminance modulations at 1 Hz from the control and experimental eyes of animal OHT-11. This animal showed profound visual field defects (MD, -26.8 dB; CPSD, 11.3 dB) around the time of ERG recording and massive ganglion cell loss and optic nerve degeneration in the histologic examination that was carried out soon afterward for another study.46 As illustrated in the middle inset, this animal’s PhNRs were greatly reduced, but the b-wave was still transient.
The actual transient PERG responses elicited by contrast-reversing square-wave gratings of 0.1, 1.5, and 3 cpd from OHT-27 are illustrated in the third through fifth rows in Figure 3A . Compared with the responses from the control eye (left column), the PERGs from the experimental eye (middle column) showed markedly reduced N95 and a less reduced P50 that was similar to the reduced troughs and peaks in the simulation. Because the slow negative potentials dominating the simulation were derived from the summation of PhNRs to light increments and decrements, these results clearly identify the reduction of N95 with the reduction of PhNR. Similarly, the reduction of P50 must be related to the changes in the early portions of the uniform field responses. The difference records isolate the potentials that were removed from the PERG by experimental glaucoma. The time course of the PERG difference records for the 0.1-cpd stimulus closely matches the time course of the difference record for the PERG simulations. Further the difference records for the 1.5- and 3-cpd responses, although of slightly different time course, contain the dominant features of the responses in the difference records of the simulations. Altogether, these results indicate that the responses removed by experimental glaucoma in the uniform field and pattern ERG responses are related and are likely to be of the same retinal origin. Similar results were obtained for the two other animals with experimental glaucoma (OHT-25 and -28) for whom we measured transient ERGs (see Fig. 3B ).
Effect of TTX on Transient Responses
Once we had established that the uniform field and pattern ERG
responses reduced by experimental glaucoma are likely to be the same
response, it was important to determine which retinal cells were
generating the response. If the uniform field and pattern ERG changes
in the experimental eye are a consequence of reduced activity of
retinal ganglion cells (and their axons), then it might be expected
that blocking the spiking activity of these neurons with TTX would
mimic the effects of experimental glaucoma. In fact, previous work
showed similarities in the effects of experimental glaucoma and
intravitreal TTX on the PhNR elicited with long-duration red ganzfeld
flashes.20
Therefore, in the present study we examined the
effect of TTX on the uniform field and pattern ERG. Effects of TTX on
the ERGs recorded from one animal (TTX-1) are illustrated in Figure 4A .
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The effects of TTX on the actual PERG responses to contrast reversals of square-wave gratings of 0.1 and 1 cpd (the lowest and highest stimulus spatial frequencies used for this animal) are illustrated in the third and fourth rows. For both spatial frequencies TTX eliminated N95, indicating that this potential originates entirely from spiking activity. A comparison of the actual and simulated PERGs after TTX shows that the elimination of N95 coincided with removal of the negative troughs in the simulation, and the difference records at both spatial frequencies contained the dominant features in the difference records of the PERG simulations. Similar results obtained for another animal (TTX-2) that received TTX injections are summarized by the difference records in Figure 4B . Although the effects of TTX on N95 are similar to those of experimental glaucoma, unlike experimental glaucoma, TTX hardly reduced the P50 amplitude. This result suggests that the decrease in P50 amplitude observed for experimental glaucoma is not simply a result of reduced spiking activity. However, TTX did decrease the peak time of P50 (by 6 msec for responses to 0.1-cpd gratings), which may reflect removal of early negative potentials, as illustrated in the difference record for the uniform field ERG (top right).
Effects of Experimental Glaucoma and TTX on the Steady State
Responses
A steady state ERG is one in which the retinal response to each
successive stimulus transition interrupts the response to the previous
transition, thereby preventing the retina from reaching a resting
state. Steady state ERG responses to luminance modulations of a uniform
field and contrast reversals of pattern stimuli have both been shown to
be altered by experimental glaucoma.26
33
In the present
study, we were interested in determining whether experimental glaucoma
altered the steady state uniform field and pattern ERG in similar ways.
Figure 5 illustrates the effects of experimental glaucoma (animal OHT-25) on
responses to luminance modulations of a uniform field at 8 Hz and
contrast reversal of square-wave gratings at 16 rev/sec. The major
features of the steady state ERG for control eyes are illustrated in
the left-hand column of Figure 5
. For the uniform field (top row), the
luminance increment response was dominated by a negative-going
potential followed by a prominent positive potential, and then after
luminance decrement the response dipped before being interrupted by
small positive waves. The second row shows the PERG simulation
constructed from the uniform field ERG as illustrated for the transient
ERG in Figure 2
. The PERG simulation after each contrast reversal
typically consisted of negative- and then positive-going waves. The
actual PERG responses to 0.1-, 1.5-, and 3-cpd gratings are illustrated
in the third through fifth rows. The 0.1-cpd PERG closely resembles the
PERG simulation in shape; however, at the higher spatial frequencies
the (small) positive peaks in the PERG were successively more delayed
relative to those in the simulation. These results are reminiscent of
those for the transient PERG and indicate that the steady state
response also contains luminance as well as pattern specific responses.
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Effects of experimental glaucoma on steady state PERGs were studied using square-wave gratings in OHT-25, -27, and -28 and sine-wave gratings in OHT-6, -9, and -11. As illustrated for OHT-25 in Figure 5 for square-wave gratings, PERG responses in experimental eyes were greatly reduced for the 0.1-cpd grating and essentially eliminated for the 1- and 3-cpd gratings. Results for sine-wave gratings were similar (not shown). As we observed for the simulation, the actual PERG responses for the 0.1-cpd grating also were more symmetrical. Although the responses in the PERG difference records were more delayed as the spatial frequency was increased, the general shape of the waveform resembled that of the simulation.
Next we examined the effects of TTX on steady state responses. The effects of TTX on the ERGs of one animal (TTX-2) are illustrated in Figure 6 , and results were similar for another animal. The uniform field ERG was clearly altered after TTX injections, indicating that normally these responses contain contributions from spiking activity. Similar to the effect of experimental glaucoma, TTX reduced the initial trough-to-peak amplitude and enhanced responses after luminance decrement. However, unlike experimental glaucoma where peak times were unchanged, TTX decreased the timing of the positive peak by 4 msec, indicating that effects of experimental glaucoma and TTX although similar, were not identical. Nevertheless, the overall shape of the uniform field responses removed by TTX resembled the waveform of those removed by experimental glaucoma.
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Amplitude and Phase of Normal Steady State Responses
The amplitude of steady state PERG response, determined by Fourier
analysis of the response at the temporal frequency of contrast
reversal, is known to be reduced in glaucoma patients and macaques with
experimental glaucoma (e.g., Refs. 26
and 47). The amplitude of the
second harmonic component of the steady state uniform field ERG also
can be reduced in glaucoma patients.21
However, it is
unclear whether reductions in the uniform field responses can be
identified with those observed for the steady state PERG responses. To
resolve this issue, we compared the effects of experimental glaucoma on
the second harmonic component of the uniform field ERG with its effects
on the steady state PERG.
The normal response characteristics of the uniform field and steady state pattern ERG of macaques derived by Fourier analysis are illustrated in Figure 7 . The dependence of the amplitude and phase of the responses on stimulus spatial frequency is illustrated in Figures 7A and 7B , where the data represent mean (± SD) values calculated from the control eyes of the five experimental (OHT and TTX) animals in the present study for which we used square-wave gratings. The data points on the extreme left of the plots before the breaks in the x-axis are from the PERG simulations. The amplitude of the steady state PERG decreased with increasing spatial frequency (Fig. 7A) , and the phase became more lagged (Fig. 7B) . On average the phase difference between the 0.1- and 3-cpd PERGs was 88° (equivalent to 15.2 msec at 16 contrast rev/sec), a value that is consistent with an average delay of approximately 16 msec observed for the positive peaks in the original waveforms for the five control eyes (e.g., Figs. 5 and 6 , left columns). The amplitudes and phases of the PERG simulations were reasonable values for spatial frequencies <0.1 cpd. Further, in separate experiments (results not shown) an estimate of the PERG delay for different stimulus spatial frequencies was calculated from the approximately linear relationship between response phase and temporal frequency. These estimates confirmed that the phase change with increase in stimulus spatial frequency was a delay rather than an advance.
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Figure 7D shows the response phase for the nine normal eyes plotted as a function of stimulus contrast for different spatial frequencies. For these data all phases are expressed relative to the response phase at 0.1 cpd and 82% contrast, which was fixed at 0° (see arrow). It is evident that for each spatial frequency, the phase is essentially invariant with stimulus contrast. As noted for the transient PERG, these results again show that the response delay with increasing spatial frequency seen for high-contrast stimuli is not due to degradation of retinal image contrast. For the rest of the study, a fixed high contrast of 82% was used with square-wave luminance modulations in both spatial and temporal domains.
Effects of Experimental Glaucoma and TTX on the Amplitude and Phase
of the Steady State Responses
The effects of experimental glaucoma on the amplitude and phase of
the steady state PERGs to square-wave luminance modulations and
simulations from OHT-25, -27, and -28 are illustrated in Figures 8A
and 8B
; responses from the control and experimental eyes appear as
open and filled symbols, respectively. Experimental glaucoma reduced
the amplitudes of the simulated as well as actual pattern ERGs, and the
reduction was greater for stimuli of higher spatial frequency. These
amplitude reductions were not accompanied by obvious phase changes. As
for the control eye, the phase of the actual PERG response elicited by
the 0.1-cpd grating pattern was similar to that of the simulation.
These results again indicate that experimental glaucoma had similar
effects on responses to uniform field and pattern stimuli.
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Altogether these findings on the effects of experimental glaucoma and TTX on steady state responses demonstrate the commonality and prominence of the inner-retinal contributions to both the uniform field and pattern ERG. The results also highlight the importance of spiking activity in ERG responses to the uniform fields and pattern stimuli.
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Discussion |
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These results confirm our previous finding that the PhNR reduced in experimental glaucoma originates to a large extent from the spiking activity of retinal ganglion cells20 and extend the finding to the N95 of the PERG. The slow time course and TTX sensitivity of the PhNR are reminiscent of a response in the cat ERG that can be suppressed by barium,19 an ion that blocks inward rectifying and outward K+ channels in glia.52 As previously suggested,20 an increase in extracellular [K+] in the proximal retina resulting from spiking activity of inner-retinal neurons can activate K+ buffer currents in glial cells, which in turn could give rise to the PhNR. Given the association between the PhNR and N95, the speculation of glial mediation can also be extended to the N95. It therefore follows that damage to either inner-retinal neurons or glia could reduce the PhNR and N95. In fact, changes in retinal astrocytes53 and Müller cells in glaucomatous eyes have been reported, and they may occur early in the pathologic process.54 Therefore, it is possible that the reduction in PhNR and N95 in eyes with experimental glaucoma, especially at early stages of retinal damage, may reflect some combination of neuronal damage and glial cell alterations.
Differences between the Effects of Experimental Glaucoma and TTX
Although we found that the effects of experimental glaucoma and
TTX were similar, they were not identical. An obvious difference can be
seen in the early portions of transient ERGs in Figures 3A and 4A
. TTX
removed more of the a-wave, (and less of
P50, see below) than experimental glaucoma. One
difference between the animals with experimental glaucoma and those
treated with TTX was that the glaucomatous eyes had some functioning
ganglion cells, whereas eyes with intravitreal TTX, for the doses we
used, had essentially no spiking activity.20
55
However,
if the different results were simply due to the degree to which
ganglion cell spiking was reduced, the difference records for the two
treatments would be scaled versions of one another, and this was not
the case for the differential effects noted above. The greater effects
on the a-wave of TTX might have occurred because amacrine
(and spiking interplexiform) cells also are affected by TTX.
The differential effects of experimental glaucoma and TTX on P50 at higher spatial frequencies are more difficult to explain. In the present study we found that for higher spatial frequency stimuli, P50 was eliminated in the glaucomatous eyes, but not in the eyes injected with TTX. Because experimental glaucoma ultimately destroys the entire ganglion cell and consequently all their responses, whereas TTX just suppresses the Na+-dependent spiking activity, the elimination of P50 by experimental glaucoma at the higher spatial frequencies might be considered as evidence that this potential originates from local potentials of retinal ganglion cells. Such a conclusion would be in agreement with a previous report that P50 in the cat is eliminated after retrograde degeneration of retinal ganglion cells after optic nerve transection.56 An inner-retinal origin of P50 also is consistent with results from current source density (CSD) analysis of the PERG in the primate retina, that indicated generators for the PERG only in the inner retina.57 However, reports from other animal studies and from clinical studies make an exclusively ganglion cell origin of P50 response at higher spatial frequencies less plausible. For instance, after optic nerve transection in the pigeon,58 P50 was preserved in spite of an almost complete disappearance of retinal ganglion cells and their axons. In human studies, relative sparing of P50 has been reported for several conditions that predominantly affect the optic nerve and retinal ganglion cells.37 59 60 For example, in patients with dominant optic atrophy, Holder et al.60 reported that early changes are noted in the pattern-evoked cortical potentials, followed by a decrease in N95, and finally alterations of P50 mainly manifest as a reduction in the latency, as we found after TTX. These findings in humans led Holder to suggest that P50 arises from retinal neurons distal to the ganglion cells, and that it is dysfunction in these neurons that leads to a reduction in P50. Thus, at present, the exact retinal neurons that generate P50 response to high spatial frequencies remains unresolved.
The origin of the P50 response to lower spatial frequencies is not as uncertain. In animals with experimental glaucoma and those injected with TTX, a prominent P50 could still be elicited for stimuli of low spatial frequencies. Similarly steady state PERG responses (believed to reflect the same process that generates P5045 ) of appreciable amplitude could be recorded at low spatial frequencies when PERG responses were unrecordable at high spatial frequencies. These results for the low spatial frequency responses agree with the findings from pigeons,58 cats,56 and human patients,61 62 in whom low spatial frequency responses were preserved even after profound loss of retinal ganglion cells subsequent to optic nerve transection. Therefore, it is likely that the residual positive potentials for the actual low spatial frequency PERG responses and PERG simulations (constructed from uniform field ERG responses) that we observed in eyes with experimental glaucoma and TTX mainly originated from generators in the distal retina. This conclusion is supported by results from CSD analysis57 that showed that both uniform field and pattern ERG responses contain contributions from the inner retina, but the uniform field flicker responses include contributions from more distal generators as well.
Comparison with Previous Studies of Uniform Field and Pattern ERG
The finding that PhNRs in the uniform field ERG responses and
especially the PhNRs were altered by experimental glaucoma and TTX is
consistent with previous reports20
of similar effects on
long-duration red ganzfeld flashes delivered on a rod-saturating blue
background. However, with the removal of the PhNR in the previous
study, the b-wave lost its transient nature, whereas in the
present study it remained transient. This difference in
b-wave duration may be due to the presence of other negative
potentials, that is, potentials originating from hyperpolarizing
bipolar or horizontal cells63
and photoreceptor responses
that might have been larger for the present stimulus conditions than
for the red flash.
In the present study we found that experimental glaucoma and TTX both reduced the b-waves in transient ERGs (Figs. 3 and 4) . TTX also reduced the a-wave (Fig. 4) . In the previous study using red ganzfeld flashes, the amplitudes of these components were not consistently altered by the treatments. Importantly, in the present study we also measured responses to the red ganzfeld flashes for all experimental subjects, and the amplitudes of the a- and b-waves were not reduced relative to controls, although the a-waves after TTX were briefer in duration (the a-waves were not of photoreceptor origin; they were eliminated in other macaques by pharmacological blockade of postreceptoral responses64 ). Therefore, as noted above, the wavelength or area could be a critical factor in determining the origins of the components of an ERG response to a particular stimulus.
Our findings of PERG alterations in experimental glaucoma are in agreement with previous reports,10 26 27 though the previous studies did not report changes in the flash or uniform field flicker responses. However, in these studies only peak amplitudes and implicit times of the a- and b-waves were measured, whereas we looked specifically for changes in the PhNR. Also our finding may at first appear to contradict the inference from the studies of Maffei and coworkers1 2 that the uniform-field flicker ERGs are unaffected by the retrograde degeneration of ganglion cells after optic nerve section. However, their results show alterations over time in the luminance ERGs of eyes with optic nerve section, but compared to the PERG loss, the changes were relatively small.2
Our finding that reduced inner-retinal activity alters both uniform field and pattern ERGs agrees with findings of Vaegan and coworkers,65 who showed that pharmacological blockade of inner-retinal activity as well as optic nerve section in the cat66 alters these responses. Our study also confirms results of Trimarchi et al.,67 who reported that intravitreal TTX in cat reduces the PERG.
It has been argued that the PERG may simply reflect the summation of nonlinear responses to local luminance changes.43 68 On the other hand, Maffei and coworkers1 2 concluded that electrical sources of the luminance and pattern ERGs are largely different. The similarity of our simulation and PERG results indicate, at least for the range of spatial frequencies that we used, that generation of the N95 of the PERG is dependent on the retinal generators that produce spiking responses to local luminance changes. This is a major result of our study. However, the delayed responses to high spatial frequency stimulation and the effects on those responses by experimental glaucoma and TTX also are consistent with previous observations that the PERG reflects contributions from inner-retinal mechanisms that are pattern-specific.45 69
Clinical Relevance
Recent studies of the photopic full field flash ERG of patients
with primary open angle glaucoma24
have shown that the
sensitivity of PhNR measurements in detecting primary open angle
glaucoma compares quite favorably with previous reports for the
N95 of the PERG.15
The present
finding in macaques that the changes in the uniform field and pattern
ERG responses (especially changes in the PhNR and
N95) in glaucomatous eyes are of similar origin
strengthens the suggestions from previous studies that the full-field
ERG could serve as a useful alternative to the PERG in the assessment
of clinical glaucomatous neuropathy.
In contrast to the consistent findings for N95, studies of PERGs of patients with diseases of inner retina have differed in reported effects on P50 responses to high spatial frequency stimuli. For example, many studies of patients with primary open angle glaucoma have reported reductions in P50 amplitude (e.g., Refs. 14 23, and 70–76). However, other studies have reported no significant alterations in P50 in glaucoma77 78 79 80 and other diseases that predominantly affect the optic nerve.37 59 60 Our finding that P50 is less affected by TTX than experimental glaucoma is helpful in demonstrating a cellular mechanism for P50 that is clearly different from the N95 generator, but it does not further localize the retinal origin of this response. Thus, only when the origins of P50 are better clarified, will the potential for the PERG to discriminate pathologic effects on different retinal generators be fully realized.
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Acknowledgements |
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Footnotes |
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Submitted for publication December 28, 1999; revised April 11, 2000; accepted April 19, 2000.
Commercial relationships policy: N.
Corresponding author: Laura J. Frishman, College of Optometry, University of Houston, 4901 Calhoun Road, Houston, TX 77204-6052. lfrishman{at}uh.edu
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