The Uniform Field and Pattern ERG in Macaques with Experimental Glaucoma: Removal of Spiking Activity

Abstract

purpose. To determine whether the uniform field and pattern ERGs that are reduced in macaque eyes with experimental glaucoma have the same inner-retinal origins.

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, 6μ M) 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° × 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.

The pattern electroretinogram (PERG) is the voltage change recorded at the cornea that occurs in response to contrast reversals of pattern stimuli. This response to pattern stimuli relies to a great extent on the functional integrity of retinal ganglion cells and their axons. ^{1 2} Glaucoma is a disease characterized by progressive degeneration of the optic nerve and loss of retinal ganglion cells. ^{3 4 5} Findings from clinical studies have differed on which components of the PERG are maximally affected by glaucoma (for reviews see ^{Refs. 6} and 7) and how well those changes correlate with the other clinical findings in glaucoma (e.g., ^{Refs. 8 9 10 11 12 13 14} ). However, some recent studies ¹⁵ have shown the PERG to be a very sensitive test for detecting early functional changes in glaucoma (also see ^{Refs. 6} and 7 for reviews). 

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 neurons ^{17 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 studies ^{18 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 cells ^{34 35} and possibly interplexiform cells ³⁶ 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  

Methods

Subjects

ERGs were recorded from 14 adult rhesus monkeys (Macaca mulatta) ranging in age from 5 to 10 years. Six animals (OHT-6, -9, -11, -25, -27 and -28) had monocular experimental glaucoma and 2 others (TTX-1 and TTX-2) received intravitreal injections of tetrodotoxin citrate (TTX). The remaining 6 animals provided normative data from one eye. All experimental and animal care procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were reviewed and approved by the Institutional Animal Care Committee of the University of Houston. 

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 electrodes ³⁹ 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 shown ⁴¹ 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 . 

Perimetry

The six animals with experimental glaucoma primarily served as subjects for other studies of behavioral perimetry, and we made ERG recordings only after they had developed visual field defects. The mean and pattern SDs of the visual field sensitivity measured around the time of final recordings for the experimental eyes of the monkeys in the present study are shown in Table 1 . The perimetric testing of monkeys has been described previously. ⁴²  

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. 

Results

Normal Transient Responses of Uniform Field and Pattern ERG

A transient ERG response is one for which the retina is in a resting state before each stimulus transition and returns to its resting state before the next stimulus transition occurs. Such a response can be achieved with stimuli that are stepwise modulated at low temporal frequencies. For a uniform field that alternates between bright and dark states, this allows the separation of responses to light increments and decrements. For a grating pattern where the bright and dark bars are interchanged, every transition will produce the same response. Figure 1 illustrates typical transient ERG responses from the control eye of one monkey (OHT-25) to luminance modulations of a uniform field at 1.7 Hz and contrast reversals of grating stimuli at 3.4 rev/sec. For the uniform field ERG, as illustrated in Figure 1 (top row), luminance increments elicited a- and b-waves that were followed by a negative potential of slow time course. This slow negative potential developed a maximum amplitude at 98 ± 6 msec (N = 5) after light increment. Luminance decrements elicited a d-wave that was generally followed by a slow negative potential of low amplitude, also peaking around 100 msec. The time course of these slow negative potentials was similar to the PhNR of inner-retinal origin that we have previously described after onset (PhNR_on) and offset (PhNR_off) of discrete long flashes in the photopic flash ERG. ²⁰ Thus, in the present study we will call these slow potentials PhNRs, and in the sections describing the effects of experimental glaucoma and TTX, we will determine how similar these potentials are to the PhNRs elicited by single flashes in our previous study. ²⁰  

The normal PERG responses elicited by contrast reversal of 0.1 and 3 cpd square-wave grating stimuli are illustrated in the middle and bottom rows of Figure 1 . These responses consist of an early negative trough, followed by a positive peak, and finally by another negative trough of much slower time course, which shows an almost complete recovery to baseline before the next contrast reversal. The triphasic shape of the transient PERG in macaques is consistent with descriptions of human PERGs. The early positive and slower negative potentials have usually been called P₅₀ and N₉₅, where the subscripts denote the approximate times in milliseconds after each pattern reversal that the two potentials reach their maximum amplitudes. ³⁷ However, the vertical dashed lines in Figure 1 make it easy to see that the times of the various peaks and troughs, marked for the 0.1-cpd pattern response, were somewhat more delayed at the higher spatial frequency. Thus, the exact timing of these peaks and troughs are spatial-frequency dependent. 

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. 

For contrast reversals, if the PERG simply reflects the summation of responses to local luminance changes, then the simulation should approximate the actual PERG. The lowest stimulus spatial frequency that was tested in the present study was 0.1 cpd, and in Figure 2 the actual PERG response elicited to this spatial frequency is shown in the third row. A comparison of the actual PERG waveform and the simulation shows similar timing for the major trough and peak (N₉₅ and P₅₀). This finding suggests that the PERG contains large contributions from responses to local luminance changes as first proposed by Spekreijse et al. ⁴³ The minor differences in amplitudes and time course between the simulation and actual PERG waveform can be attributed at least partly to responses resulting from stray light falling on retinal regions outside the test area. Such responses would be larger for luminance modulations of a uniform field than for contrast reversals of patterned stimuli. In the present study, we used a bright surround (120 cd/m²) to minimize stray light responses from surrounding retinal areas, but the surround luminance may have been insufficient to completely suppress all such responses. 

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  

Effect of Experimental Glaucoma on Transient Responses

In these experiments we examined whether experimental glaucoma altered the uniform field and PERG responses in consistent ways. Our approach here will be first to describe the effects of experimental glaucoma on the uniform field ERGs and PERG simulations and then to compare effects on the simulation with effects on the actual PERG. The effects of experimental glaucoma on the uniform field and pattern ERGs are illustrated in Figure 3A by examples from one animal (OHT-27). 

For uniform field stimulation (Fig. 3A , top), responses recorded from the experimental eye (middle column) showed a reduction of the slow negative potential (PhNR_on) 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 PhNR_off 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 . 

A comparison of the uniform field ERG responses in Figure 4A recorded before and after TTX injections clearly shows that the PhNRs were greatly reduced after TTX; the difference record on the right is dominated by the removed PhNRs. These effects are similar to those of experimental glaucoma and indicate that the PhNRs elicited by a uniform field originate to a large extent from the spiking activity of inner-retinal neurons. After TTX the d-wave peaked slightly earlier (by 5 msec) and recovered more slowly to baseline, again similar to the effect of experimental glaucoma. However, unlike experimental glaucoma, TTX reduced the a-wave amplitude and time to peak of the b-wave (by 3 msec), showing that the effects of TTX and experimental glaucoma on the early portions of the uniform field responses are not exactly the same. In the PERG simulations, TTX almost completely eliminated N₉₅ at the same time that it decreased the time to peak of P₅₀ by 2 msec. 

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. 

For the uniform field responses in Figure 5 , the initial trough-to-peak amplitude for the experimental eye was reduced compared to the control eye. In contrast the trough-to-peak amplitude of responses after luminance decrement showed a slight enhancement over the control eye. The PERG simulations for the experimental eye showed an overall reduction in size accompanied by a more symmetrical appearance. Similar results were obtained for all five animals with experimental glaucoma. 

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. 

TTX reduced the trough-to-peak amplitudes of the PERG simulations and advanced the first positive peak by approximately 5 msec. This advance was not observed for eyes with experimental glaucoma. TTX reduced the actual PERG responses, leaving waveforms whose general shape resembled the post-TTX PERG simulation. The difference records for the simulation and the actual PERG responses also are similar in shape, but as for experimental glaucoma, the difference records were increasingly more delayed as spatial frequency was increased to 1 and 2 cpd. Taken together, the findings for experimental glaucoma and TTX indicate that a major effect of experimental glaucoma on the steady state uniform field and pattern ERG was to reduce or eliminate the contribution from spiking activity. 

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. 

Although a low spatial frequency roll-off has frequently been described for the PERG spatial frequency-response function, Figure 7A does not show such an effect. However, previous investigators ^{40 48} have reported that the low-frequency roll-off is reduced by using square- rather than sine-wave spatial and temporal luminance modulations. For the results in Figures 7A and 7B , we used square-wave luminance modulations in both spatial and temporal domains. For comparison in Figure 7C , we show average PERG responses from normal eyes of nine animals (including three who had experimental glaucoma in the other eye) to spatially sinusoidal grating stimuli; the temporal modulation was still square-wave. As shown in Figure 7C , we also evaluated the effects of stimulus contrast on these responses. The data in Figure 7C showed some low spatial frequency roll-off, confirming that the absence of the roll-off in Figure 7A was due to the use of square-wave gratings. 

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. 

The effects of TTX on response amplitude and phase are illustrated in Figures 8C and 8D , respectively. Similar to experimental glaucoma, TTX nearly eliminated the responses to the higher spatial frequencies and markedly reduced the responses to the lower spatial frequencies. However, the amplitudes of the simulations showed little change after TTX. Consistent with the observations for the original waveforms, TTX advanced the phase both of the simulated and actual PERG responses, and the phase shift of the 0.1-cpd response was comparable to that for the simulation. 

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. 

Discussion

Origin of ERG Potentials Reduced by Experimental Glaucoma

In the present study, we found that both uniform field and pattern ERGs were altered by experimental glaucoma. Further, the changes in the uniform field ERGs were predictive of the changes in the PERG responses, especially for responses to pattern stimuli at low spatial frequency, indicating that the affected responses were of similar origin. We also found that the effects of experimental glaucoma were quite similar to those of TTX, indicating that the responses reduced by experimental glaucoma originated largely from nerve spike activity. Although TTX blocks spiking activity in amacrine and ganglion cells (and possibly interplexiform cells), only the ganglion cells are believed to be affected by experimental glaucoma ^{18 28 29 33 49 50} (but see ^{Ref. 51)} . Therefore, a significant proportion of the uniform field and pattern ERG reduced by experimental glaucoma probably must have originated from the spiking activity of ganglion cells (and their axons). 

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 cells ²⁰ 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 astrocytes ⁵³ 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 P₅₀ ⁴⁵ ) 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 analysis ⁵⁷ 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 reports ²⁰ 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 cells ⁶³ 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 responses ⁶⁴ ). 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 coworkers ^{1 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 cat ⁶⁶ 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 coworkers ^{1 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 glaucoma ²⁴ 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 glaucoma ^{77 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. 

 Supported by Alcon Research LTD, Fort Worth, Texas; Research Grant R01EY06671 (LJF) and Core Grant P30 EY07751 from the National Eye Institute, Bethesda, Maryland; and an Ezell fellowship (SV) from the American Optometric Foundation, Rockville, Maryland.

 Submitted for publication December 28, 1999; revised April 11, 2000; accepted April 19, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Intraocular Pressures and Visual Field Indices of Control and Experimental Eyes of Animals with Experimental Glaucoma Obtained Around the Time of ERG Recordings

Animal	Eye	Time Since 1st Laser Application (months)	Intraocular Pressure (mm Hg)	Visual Field Indices (dB)*
								MD, †	CPSD, ‡
OHT-6	Control	31	14
	Experimental		55	−13.5	8.7
OHT-9	Control	7	10
	Experimental		48	−9.2	2.4
OHT-11	Control	6	11
	Experimental		47	−26.8	11.3
OHT-25	Control	2	13
	Experimental		39	−6.0	7.2
OHT-27	Control	6	13
	Experimental		42	−8.3	4.9
OHT-28	Control	8	16
	Experimental		52	−6.6	4.3

*  For all animals visual field indices were significant at P < 0.5.

†  Mean deviation (MD) is the mean elevation or depression of the overall field compared to the normal reference field. ⁸¹

‡  Corrected pattern standard deviation shows the degree to which the shape of the measured field departs from the normal, age-corrected reference field after correcting for intra-test variability. ⁸¹

Figure 1.

 

Transient ERG responses from the normal eye of animal OHT-25 to luminance modulation of a uniform field (top) at 1.7 Hz and contrast reversal of 0.1- (middle) and 3- (bottom) cpd grating stimuli at 3.4 rev/sec.

Figure 2.

 

Simulation of a transient PERG from the uniform field ERG to luminance modulations at 1.7 Hz. Top row: the uniform field ERG from the normal eye of animal TTX-1 to a luminance increment followed by a decrement; second row: responses from the same eye reversed in phase. The traces in the first and second rows have been reduced to half the amplitude of the original responses. The waveform in the third row is the algebraic sum of the above responses and represents the PERG simulation. Fourth and fifth rows: the actual PERG response elicited to a 0.1- and 1-cpd grating stimuli from the same eye. In subsequent figures, the stimulus marker will illustrate only the increment followed by decrement phase (top trace), although both phases occur at once for pattern reversals.

Figure 3.

 

(A) Effect of experimental glaucoma on the transient responses recorded from animal OHT-27. Top: transient responses to luminance modulations of a uniform field at 1.7 Hz. Second row: the simulated PERG responses; third through fifth rows: the actual PERG responses elicited to contrast reversals of 0.1-, 1.5-, and 3-cpd grating stimuli at 3.4 rev/sec. Left, middle and right: responses from the control and experimental eyes and their difference records, respectively. Small insets: uniform field ERG responses to luminance modulations at 1 Hz from animal OHT-11 that had more severe visual field defects than OHT-27 at the time of ERG recording. (B) Difference records of simulated (top row) and actual PERG responses elicited to contrast reversals of 0.1 and 3 cpd grating stimuli (middle and bottom rows) at 3.4 rev/sec from animals OHT-25 and -28 (left and right columns). Note that the P₅₀ amplitude for OHT-25 for the 3-cpd stimulus was small in the control eye record shown in Figure 1 .

Figure 4.

 

(A) Effect of TTX on the transient responses recorded from animal TTX-1. Top: transient responses to luminance modulations at 1.7 Hz of a uniform field. Second row: the simulated PERG responses; third and fourth rows: the actual PERG responses to contrast reversals at 3.4 rev/sec of 0.1- and 1-cpd gratings. Responses before and after intravitreal injections of TTX (6 μM) and their difference records appear in the left, middle, and right, respectively. (B) Difference records of simulated (top row) and actual PERG responses elicited to contrast reversals of 0.5- and 1.5-cpd grating stimuli (middle and bottom rows) at 3.4 rev/sec from animal TTX-2.

Figure 5.

 

Effect of experimental glaucoma on the steady state responses recorded from animal OHT-25. Top: steady state responses to luminance modulations of a uniform field at 8 Hz. Second row: the simulated PERG responses; third through fifth rows: the actual PERG responses elicited to contrast reversals at 16 rev/sec of 0.1-, 1.5-, and 3-cpd gratings. Responses from the control and experimental eyes and their difference records appear on the left, middle, and right, respectively.

Figure 6.

 

Effect of TTX on the steady state responses recorded from animal TTX-2. Top: steady state responses to luminance modulations at 8 Hz of a uniform field. Second row: the simulated PERG responses; third through fifth rows: the actual PERG responses elicited to contrast reversals at 16 rev/sec of 0.1-, 1-, and 2-cpd gratings. The responses before and after intravitreal injections of TTX (6 μM) and their difference records appear in the left, middle, and right, respectively.

Figure 7.

 

The response characteristics derived by Fourier analysis of the steady state uniform field and pattern ERG in normal macaque. (A and B) Average amplitudes and phases (± SD) of responses from the control eyes of the 5 experimental animals (OHT and TTX, respectively) to spatially square-wave luminance gratings (82% contrast) that were square-wave modulated (contrast reversed at 16 rev/sec), plotted as a function of stimulus spatial frequency. The data on the far left before the break in the x-axis were from PERG simulations generated from uniform field ERGs. (C and D) Average amplitudes and phases (± SD), respectively, of PERG responses elicited from nine normal eyes to spatially sinusoidal gratings that were square-wave modulated (contrast reversed at 16 rev/sec), plotted as a function of spatial frequency for a range of stimulus contrasts. Phases in (D) were relative to the phase at 0.1 cpd and 82% contrast that was fixed at 0° (arrow). The noise (i.e., response to an unmodulated screen) ranged between 0.1 and 0.26 μV.

Figure 8.

 

The response characteristics derived by Fourier analysis of the steady state uniform field (8 Hz) and pattern (16 rev/sec) ERG of macaques with experimental glaucoma or injected with TTX. (A and B) Amplitudes and phases, respectively, of steady state responses plotted as a function of stimulus spatial frequency for the three animals with monocular experimental glaucoma. Open and filled symbols: responses from the control and experimental eyes, respectively. The amplitude (C) and phases (D) of the steady state responses are also shown for the two animals that received intravitreal injections of TTX (6 μM). Open and filled symbols: responses before and after TTX for the same eye. In all four plots the data on the far left are from PERG simulations. The data for the PERG simulation from the control eye of OHT-25 has been shifted to the left for better visibility. The noise (response to an unmodulated screen) ranged between 0.1 and 0.26 μV.

The authors thank Ronald S. Harwerth and Earl L. Smith, III for providing the perimetric data and for performing the laser treatments.