August 2008
Volume 49, Issue 8
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Retina  |   August 2008
Recording Focal Macular Photopic Negative Response (PhNR) from Monkeys
Author Affiliations
  • Mineo Kondo
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Yukihide Kurimoto
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Takao Sakai
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Toshiyuki Koyasu
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Kentaro Miyata
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Shinji Ueno
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Hiroko Terasaki
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Investigative Ophthalmology & Visual Science August 2008, Vol.49, 3544-3550. doi:10.1167/iovs.08-1798
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      Mineo Kondo, Yukihide Kurimoto, Takao Sakai, Toshiyuki Koyasu, Kentaro Miyata, Shinji Ueno, Hiroko Terasaki; Recording Focal Macular Photopic Negative Response (PhNR) from Monkeys. Invest. Ophthalmol. Vis. Sci. 2008;49(8):3544-3550. doi: 10.1167/iovs.08-1798.

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      © 2015 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To record the photopic negative response (PhNR) of the focal electroretinograms (ERGs) from the macula of monkeys and to study the properties of the focal macular PhNRs.

methods. Focal macular ERGs were recorded from five rhesus monkeys using a modified infrared fundus camera, in which a red stimulus spot on a blue illuminated background were incorporated. The effects of different stimulus intensities and durations presented on a steady blue background of 100 scot cd/m2 on the focal macular PhNRs were investigated. Focal macular PhNRs were also recorded before and after an intravitreous injection of tetrodotoxin (TTX).

results. Focal ERG responses from a photocoagulated retinal site were recordable when the luminance of the red stimulus spot was ≤55 phot cd/m2 and was presented on a steady blue background of 100 scot cd/m2. The amplitude of the focal macular PhNR increased with increasing stimulus intensities and was larger than that of the b-wave at all stimulus intensities. The amplitude of the focal macular PhNR was largest at stimulus durations of 30 to 50 ms. An intravitreous injection of TTX essentially eliminated the focal macular PhNR.

conclusions. It is possible to record focal macular PhNRs from monkeys by using a red stimulus spot on a blue background. Investigations of focal PhNRs can be a useful method of studying inner retinal function of local areas in normal and diseased retinas.

The photopic negative response (PhNR) is a slow, negative-going wave of the photopic electroretinogram (ERG) that appears immediately after the b-wave. This component was first identified by Viswanathan et al. 1 in 1999. They demonstrated that the PhNR was reduced in eyes of monkeys with increased intraocular pressure and reduced visual field sensitivity. In addition, they reported that the PhNR was essentially eliminated by an intravitreous injection of tetrodotoxin (TTX), 1 2 3 a selective blocker of voltage-gated Na+ channels. 4 5 6 These results suggest that the PhNR originates mainly from the spiking activity of inner retinal neurons including the retinal ganglion cells and their axons. 
In clinical studies, the PhNR has been reported to be reduced in patients with glaucoma, 7 8 9 optic nerve diseases, 10 11 12 and retinal vascular diseases that predominantly affect the inner retina. 13 14 15 We have reported that the amplitude of the PhNR is selectively reduced after macular hole surgery, indicating that there are some functional impairments in the inner retina after this type of surgery. 16 The results of these clinical studies suggest that recordings of the PhNR can provide a means for objective assessment of inner retinal function. 
To date, the PhNR has been elicited mainly by full-field stimuli in both basic and clinical studies. However, the full-field ERG is the summed response from the entire retina, and it is difficult to assess the function of localized retinal areas by full-field ERGs. There are some reports on recording the PhNR from localized retinal areas, 2 7 17 18 but the characteristics of the focal PhNR in primates is less well understood. The focal PhNR is important because many diseases, including glaucoma and optic nerve diseases, affect selective areas of the retina. Therefore, we believed that developing a technique to record focal PhNRs could be useful for both basic research and clinical applications. 
Thus, the purpose of this study was to determine whether a focal PhNR could be recorded from local areas of the monkey retina. For this, we developed a new recording system with a modified infrared fundus camera. A red stimulus spot was used on a blue illuminated background, because it has been reported recently that this color combination is most effective in eliciting large PhNRs especially at weak to moderate stimulus intensities. 3  
Methods
Stimulus and Observation Systems
Our new system for eliciting and recording focal PhNRs consisted of a modified infrared fundus camera and a stimulator that controlled the light-emitting diodes (LEDs) used for the stimulus and background illumination (Fig. 1A) . An infrared television fundus camera (model VX-10; Kowa, Tokyo, Japan) was modified to obtain a Maxwellian stimulating system (Fig. 2) . The image from this fundus camera was fed to a television monitor with a 45° view of the posterior pole of the eye (Fig. 1B) . The position of the stimulus spot on the fundus was monitored on the television screen, and could be moved by the examiner with a joystick (Fig. 1A)
A red LED (λmax= 627 nm; LXK2-PD12-S00; Philips Lumileds, San Jose, CA) was used as the stimulus source, and a blue LED (λmax = 450 nm; L450, Epitex, Kyoto, Japan) was used for the background illumination that covered a retinal area of 45° (Fig. 2) . The 15° red stimulus spot on the blue background that was photographed with a digital camera placed at the position of the monkey’s eye is shown in Figure 1C . The size of the stimulus spot could be changed from 5° to 15°; the 15° stimulus spot was mainly used in this study. 
The luminance of the blue background was fixed at 100 scot cd/m2, which is known to be high enough to suppress the rod photoreceptors. The luminance of the red stimulus spot was increased from 2 to 204 phot cd/m2, and the stimulus duration was increased from 5 to 150 ms. The stimulus intensity was also expressed in energy units (i.e., phot cd-s/m2, for brief flashes of ≤30 ms). The stimulus repetition rate was fixed at 2 Hz. 
The luminances of the stimulus and background illumination were measured at the position of the corneal surface and then converted to the value at the retinal surface. These luminances were measured with a photometer (model IL 1700; International Light, Newburyport, MA). 
Recording and Analyses
ERGs were recorded with a Burian-Allen bipolar contact lens electrode (Hansen Ophthalmic Development Laboratories, Iowa City, IA). The ground electrode was attached to the ipsilateral ear. The responses were amplified, and the band-pass filters were set at 0.5 to 1000 Hz. The ERGs were digitized at 5 kHz, and 100 to 300 responses were averaged for each recording (MEB-9100 Neuropack; Nihon Kohden, Tokyo, Japan). 
The amplitude of the PhNR was measured from the baseline to the bottom of the negative trough after the b-wave for the brief flashes (≤ 30 ms), or was measured from the positive peak of the b-wave to the negative trough after the b-wave for the long-duration flashes (≥50 ms), as in previous studies. 1 2 3 The amplitudes of the a- and b-waves were measured from the baseline to the first negative trough and from the negative trough to the next positive peak, respectively. 
Animals
Five eyes of five rhesus monkeys (Macaca mulatta) were studied. The animals were sedated with an intramuscular injection of ketamine hydrochloride (7 mg/kg initial dose; 5 to 10 mg/kg/h maintenance dose) and xylazine (0.6 mg/kg). The respiration and heart rate were monitored, and hydration was maintained with slow infusion of lactated Ringer solution. The cornea was anesthetized with topical 1% tetracaine, and the pupils dilated with topical 0.5% tropicamide, 0.5% phenylephrine HCl, and 1% atropine. All experimental and animal care procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care Committee of the Nagoya University. 
Drug Application
The drugs and intravitreous injection techniques have been described in detail. 1 2 3 17 19 20 The drugs were injected into the vitreous with a 30-gauge needle inserted through the pars plana approximately 3 mm posterior to the limbus. TTX (Kanto Chemical, Tokyo Japan) was dissolved in sterile saline, and 0.05 to 0.07 mL was injected. The intravitreous concentration of TTX was 4 μM, assuming that the monkey’s vitreous volume is 2.1 mL. 
Because the TTX effect reaches its maximum at approximately 60 minutes after the drug injection, recordings were begun approximately 60 to 90 minutes after the injections, and studies were completed within 3 hours. Although the effects of these drugs are mostly reversible after a recovery period of several weeks, the results that are shown were recorded from eyes not previously treated. 
Results
Effects of Stray Light
To determine that the PhNRs we recorded were indeed focal responses, we investigated the effect of stray light on the responses with our system. First, we recorded focal ERGs using a 5° stimulus spot placed on the optic nerve head of monkeys. Different stimulus luminances (2–204 phot cd/m2) and stimulus durations (5, 10, 30, and 150 ms) were presented on a steady blue background illumination of 100 scot cd/m2. There were no detectable ERG responses (<0.4 μV) when the stimulus luminance was ≤55 phot cd/m2 for all stimulus durations. A small positive or negative response was elicited by a stimulus luminance of 84 phot cd/m2 and stimulus durations of 30 and 150 ms. The amplitudes of the response increased with increasing stimulus luminance (data not shown). 
We next examined the stray light effect by recording focal ERGs 1 month after an argon laser photocoagulation of a 15° spot in the macular area (Fig. 3A) . ERGs were elicited by stimulating the photocoagulated area with different stimulus luminances (26–204 phot cd/m2), stimulus durations (5, 10, 30, and 150 ms), and a 15° stimulus spot. The stimulus spot was presented on a steady blue background illumination of 100 scot cd/m2 in one monkey. We found that the response amplitudes were lower than the noise level (< 0.4 μV) when the stimulus luminance was ≤55 phot cd/m2 for all stimulus durations. A small positive wave (Fig. 3B , red asterisks) or negative wave (blue arrows) was recorded when the stimulus luminance was ≥84 phot cd/m2. These small responses were more prominent at stimulus durations of 30 and 150 ms and were most likely due to stray light, because the central retina within 15° had been completely photocoagulated. 
Based on these results, we concluded that stimulus luminances of ≤55 phot cd/m2 presented on a steady blue background of 100 scot cd/m2 are the optimal stimulus for eliciting focal ERGs in our system. 
Effect of Stimulus Intensity
Representative focal macular ERGs elicited by stimulus luminances of 2 to 55 phot cd/m2 for four stimulus durations of 5, 10, 30, and 150 ms are shown in Figure 4 . It is clear that the amplitude of the focal PhNRs increased with increasing stimulus luminance for all stimulus durations. 
The relationship between the stimulus intensities and the average amplitudes of the focal macular PhNRs is shown in Figure 5A . The amplitudes of the focal macular PhNR are plotted as a function of stimulus energy for brief flashes (5, 10, and 30 ms, left), and are plotted as a function of stimulus luminance for long flashes (150 ms, right). We found that when the stimulus duration was shorter than the integration time of PhNR (≤ 30 ms), the amplitude of the focal macular ERG was dependent on the stimulus energy (Fig. 5A)
One of the interesting findings was that the amplitude of PhNR was larger than that of the b-wave for all stimulus intensities and was more than double the b-wave amplitude at a stimulus duration of 150 ms at all stimulus luminances (Fig. 5B)
Effect of Stimulus Duration
We also examined the effects of stimulus duration on the focal macular PhNR of monkeys. Representative focal macular ERGs elicited by different stimulus durations of 5 to 150 ms at a constant stimulus luminance of 55 phot cd/m2 are shown in Figure 6A . The stimulus energy (phot cd-s/m2) is also indicated for the brief flashes of ≤30 ms. 
The amplitude of the focal macular PhNR increased with increasing stimulus durations when the stimulus durations were shorter than 30 to 50 ms. This increase in the amplitude is most likely due to the increase in the stimulus energy (see also Fig. 5A ). Further increases in the stimulus duration led to a slight decrease in the PhNR amplitude (Fig. 6B)
We also found that the implicit time of the PhNR was dependent on the stimulus duration. The implicit time of the PhNR was approximately 75 ms for a stimulus of 5 ms duration and became longer with increasing stimulus durations and then reached a maximum implicit time (110 ms) at approximately 50 ms duration (Fig. 6A , red dashed vertical lines). This gradual increase in the implicit time of the PhNR most likely resulted from an increase in stimulus energy and the increase in the midpoint of the stimulus. Further increases in the stimulus duration did not change the implicit time of the PhNR. 
We also found that another slow negative response (Fig. 6A , asterisks) developed after the stimulus offset for longer stimulus durations of 100 to 150 ms. This negative response was thought to be a homologue of the PhNR to the stimulus offset (PhNROff), which has been reported in studies of full-field photopic ERGs. 1 2 3 20  
Effect of Intravitreous Injection of TTX
Finally, we studied whether the focal macular PhNR recorded from monkeys changed after an intravitreous injection of TTX, which blocks voltage-gated sodium channels and prevents the generation of sodium-based action potentials. Representative waveforms of focal macular ERGs recorded before (black) and after (red) an intravitreous injection of TTX are shown in Figure 7 . The stimulus luminance was set at 55 phot cd/m2, and the responses to stimulus durations of 5 to 150 ms are shown. We found that blocking the spiking activities of inner retinal neurons by TTX essentially eliminated the focal macular PhNR, which was similar to the effect of TTX on the full-field photopic ERG. 1 2 3  
For long-duration stimuli of 100 to 150 ms, the slow negative potential that was found after the light offset (PhNRoff, Fig. 7 , asterisks) was also not present after TTX. Similar effects have been reported for full-field ERG studies. 1 2 3 20  
Although the major effect of TTX was seen in the PhNR, other ERG components of the focal macular ERG were also slightly altered after TTX. The amplitude of the a-wave became slightly smaller, and the implicit times of the b-wave were delayed. These minor changes were also very similar to those reported for full-field PhNR studies. 1 2 3  
Discussion
Our results showed that focal PhNRs can be recorded from the macular area of monkeys by using our newly developed system. In this system, a red stimulus spot was presented on a blue background, which was earlier shown to be the optimal stimulus conditions to elicit large PhNRs especially at low to intermediate stimulus intensities. 3 In addition, our system allowed us to monitor the position of the stimulus spot on the monkey’s fundus during the recordings. Our ability to record focal PhNRs from monkey eyes is important, because this will allow us to manipulate the recording conditions or alter the normal PhNR with drugs known to affect specific neural elements to study the physiological properties of the PhNRs. 
To establish a technique to record focal ERGs, it was important for us to determine the optimal combination of stimulus and background intensities. 21 22 23 24 25 26 27 28 Based on the results of trying to elicit focal ERGs from the optic nerve head and from a retinal site damaged by focal laser coagulation, we found that a 15° red stimulus spot of ≤55 phot cd/m2 presented on a steady blue background of 100 scot cd/m2, will elicit a focal ERG. For stimulus intensities >55 phot cd/m2, small responses were recorded even from the retinal site damaged by focal laser coagulation, and this response was most likely due to stray light (Fig. 3) . The stray light effect was dependent on the stimulus intensity and was greater for longer stimulus durations of 30 and 150 ms (Fig. 3B)
We studied the response characteristics of the focal macular PhNRs recorded by our system, and found the following: (1) The focal macular PhNR was a slow, negative response, with an implicit time of approximately 75 ms for short-duration stimuli and approximately 110 ms for long-duration stimuli (Fig. 6) ; (2) for long-duration stimuli, the PhNR was seen after both the onset and the offset of the stimulus (Figs. 4 6) ; (3) the amplitude of the focal macular PhNR increased with increasing stimulus intensity (Fig. 5) ; and, (4) the amplitude of the focal macular PhNR was greatly reduced after an intravitreous injection of TTX. 
These results showed that the response characteristics of focal PhNRs recorded from the monkey’s macula were very similar to those of the full-field PhNR, 1 2 3 11 and that this negative response originates mainly from the action potentials of the inner retinal neurons. 
One interesting finding in our study was that the amplitudes of focal macular PhNRs recorded by our system were large; the maximum PhNR amplitude reached 6.1 μV for a 10-ms duration stimulus and 8.3 μV for a 150-ms duration stimulus. The amplitudes of the focal PhNR recorded from the 15° macular area were relatively large when compared with the amplitudes of the full-field PhNR which were 25 to 40 μV in earlier monkey studies. 1 2 3 11 Large PhNR amplitudes in our focal ERGs can also be understood when one examines the amplitude of the b-wave and PhNR. The amplitude of PhNR was larger than that of the b-wave in all stimulus conditions and was more than twice that of the b-wave for long-duration stimuli of 150 ms (Fig. 5B)
The reason for the relatively large PhNR amplitude in the macular region in monkeys was not examined, but may be explained by two possibilities. First, the density of ganglion cells (number/mm2) is highest in the central retina of monkeys, 29 30 31 32 33 and the slope of ganglion cell number as a function of retinal eccentricity is steeper than that of cone cells in the human retina. 34 This high ganglion cell density in the central retina may contribute to the large amplitude of the macular PhNR. And second, we used a red stimulus spot on a blue background. This combination was recently shown to be optimal for eliciting maximum PhNR amplitude. Using various stimulus and background color combinations, Rangaswamy et al. 3 concluded that at weak to moderate stimulus intensities, the amplitude of PhNR is larger in response to stimuli that are relatively more cone specific. 
The amplitude of macular PhNR increased with increasing stimulus durations up to 30 to 50 ms, because of due to the increase in the stimulus energy. However, further increases in the stimulus duration led to a decrease in amplitude. The decrease in amplitude may be explained by the separation of the two PhNR components: PhNRon elicited by stimulus onset and PhNRoff elicited by stimulus offset, both of which are superimposed when a brief-flash stimuli (≤50 ms) is used. A second possibility is that this amplitude decrease may be due to factors other than the spiking activities of inner retinal neurons. As seen in Figure 7 , the longer duration stimuli tended to elicit prolonged b-waves (plateau) when the PhNR was eliminated by TTX, whereas the brief-flash responses generally leveled off at the baseline. 
There remain some critical matters that should be addressed in future studies. First, we did not show how full-field PhNR and focal macular PhNR are different with regard to the intensity–response function, duration–response function, and the effect of TTX. To determine these differences, we must compare the full-field and focal PhNRs in the same stimulus and recording conditions. Second, although we succeeded in recording focal PhNRs from the macula of monkeys, we did not examine whether there are any regional variations across the retina in the waveform or amplitude of the PhNR. We are currently comparing the focal PhNRs between upper and lower retinas, or nasal and temporal retinas by using semicircular stimulus spots. Finally, in this study we used only one type of electrode, the Burian-Allen bipolar contact lens electrode. However, it is known that the speculum of this electrode, which also acts as the reference, can pick up signals that can cancel out the signals picked up by the corneal electrode. It may be better to place the reference farther from the recording electrode (e.g., fellow eye), to maximize the response of small signals. 35 36  
In conclusion, we successfully recorded focal PhNRs from the macula of monkeys by using a red stimulus spot on a blue background. Although there are still many factors that need to be tested, we believe that examinations of the focal PhNRs can be a useful technique for studying the inner retinal function of local retinal areas in normal and diseased retinas. 
 
Figure 1.
 
(A) Stimulus and observation system for recording the focal PhNR. This system consists of a modified infrared fundus camera (1) and LED control box (2). The infrared fundus image can be observed on a monitor (3), and the stimulus spot can be moved with a joystick (4). (B) Infrared fundus image of the monkey retina. A 15° stimulus spot positioned on the monkey’s macula. (C) Image of the red stimulus spot on the blue background. This image was photographed by a digital camera at the position of the monkey’s eye.
Figure 1.
 
(A) Stimulus and observation system for recording the focal PhNR. This system consists of a modified infrared fundus camera (1) and LED control box (2). The infrared fundus image can be observed on a monitor (3), and the stimulus spot can be moved with a joystick (4). (B) Infrared fundus image of the monkey retina. A 15° stimulus spot positioned on the monkey’s macula. (C) Image of the red stimulus spot on the blue background. This image was photographed by a digital camera at the position of the monkey’s eye.
Figure 2.
 
Diagram of the focal PhNR recording system with fundus monitoring with an infrared fundus camera. A red LED was used for the stimulus source, and a blue LED was used for the background illumination.
Figure 2.
 
Diagram of the focal PhNR recording system with fundus monitoring with an infrared fundus camera. A red LED was used for the stimulus source, and a blue LED was used for the background illumination.
Figure 3.
 
Studies of stray light effect in our system. (A) Fundus photograph of a monkey whose macula was damaged by 15° in the central area by focal laser photocoagulation (within the white dashed line). (B) Focal ERGs recorded with a 15° stimulus spot centered on the photocoagulation. Stimulus luminance and stimulus duration were changed on a steady blue background illumination of 100 scot cd/m2. Small positive (red asterisks) or negative (blue arrows) waves were detected when the stimulus luminance was 84 cd/m2 or higher, presumably due to the effect of stray light.
Figure 3.
 
Studies of stray light effect in our system. (A) Fundus photograph of a monkey whose macula was damaged by 15° in the central area by focal laser photocoagulation (within the white dashed line). (B) Focal ERGs recorded with a 15° stimulus spot centered on the photocoagulation. Stimulus luminance and stimulus duration were changed on a steady blue background illumination of 100 scot cd/m2. Small positive (red asterisks) or negative (blue arrows) waves were detected when the stimulus luminance was 84 cd/m2 or higher, presumably due to the effect of stray light.
Figure 4.
 
Representative focal ERGs elicited from a rhesus monkey by different stimulus luminances (2–55 phot cd/m2) and different stimulus durations (5, 10, 30, and 150 ms). The amplitude of PhNR increases with increasing stimulus luminances.
Figure 4.
 
Representative focal ERGs elicited from a rhesus monkey by different stimulus luminances (2–55 phot cd/m2) and different stimulus durations (5, 10, 30, and 150 ms). The amplitude of PhNR increases with increasing stimulus luminances.
Figure 5.
 
(A) Stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the focal macular PhNR. The PhNR amplitudes are plotted as a function of stimulus energy (cd-s/m2) for brief flashes of 5 to 30 ms in the left half and are plotted as a function of stimulus luminance (cd/m2) for a long 150-ms flash in the right half. (B) Comparison of stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the b-wave and PhNR for stimulus duration of 10 ms (left) and 150 ms (right).
Figure 5.
 
(A) Stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the focal macular PhNR. The PhNR amplitudes are plotted as a function of stimulus energy (cd-s/m2) for brief flashes of 5 to 30 ms in the left half and are plotted as a function of stimulus luminance (cd/m2) for a long 150-ms flash in the right half. (B) Comparison of stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the b-wave and PhNR for stimulus duration of 10 ms (left) and 150 ms (right).
Figure 6.
 
Effect of stimulus duration on the focal PhNR. (A) Representative focal ERGs elicited by different stimulus durations (5–150 ms) for a constant stimulus luminance of 55 phot cd/m2. Vertical dotted line: peak of the PhNR. The values of stimulus energy (cd-s/m2) are also indicated for brief flashes of 5 to 30 ms. (B) Superimposed focal ERG waveforms recorded with different stimulus durations (5–150 ms). Stimulus luminance was fixed at 55 phot cd/m2.
Figure 6.
 
Effect of stimulus duration on the focal PhNR. (A) Representative focal ERGs elicited by different stimulus durations (5–150 ms) for a constant stimulus luminance of 55 phot cd/m2. Vertical dotted line: peak of the PhNR. The values of stimulus energy (cd-s/m2) are also indicated for brief flashes of 5 to 30 ms. (B) Superimposed focal ERG waveforms recorded with different stimulus durations (5–150 ms). Stimulus luminance was fixed at 55 phot cd/m2.
Figure 7.
 
Focal ERGs before and after intravitreous injection of TTX in one monkey. The focal ERGs before (black lines) and after TTX (red lines) are superimposed. Stimulus luminance was fixed at 55 phot cd/m2, and stimulus duration was changed from 5 to 150 ms. Note that the amplitude of focal PhNR was greatly reduced after TTX. The other slow negative response to the stimulus offset (PhNROff, asterisks) was also reduced after TTX.
Figure 7.
 
Focal ERGs before and after intravitreous injection of TTX in one monkey. The focal ERGs before (black lines) and after TTX (red lines) are superimposed. Stimulus luminance was fixed at 55 phot cd/m2, and stimulus duration was changed from 5 to 150 ms. Note that the amplitude of focal PhNR was greatly reduced after TTX. The other slow negative response to the stimulus offset (PhNROff, asterisks) was also reduced after TTX.
The authors thank Yozo Miyake of Shukutoku University and Duco I. Hamasaki for discussions on the manuscript, and Masao Yoshikawa, Hideteka Kudo, and Ei-ichiro Nagasaka of Mayo Corporation for technical assistance. 
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Figure 1.
 
(A) Stimulus and observation system for recording the focal PhNR. This system consists of a modified infrared fundus camera (1) and LED control box (2). The infrared fundus image can be observed on a monitor (3), and the stimulus spot can be moved with a joystick (4). (B) Infrared fundus image of the monkey retina. A 15° stimulus spot positioned on the monkey’s macula. (C) Image of the red stimulus spot on the blue background. This image was photographed by a digital camera at the position of the monkey’s eye.
Figure 1.
 
(A) Stimulus and observation system for recording the focal PhNR. This system consists of a modified infrared fundus camera (1) and LED control box (2). The infrared fundus image can be observed on a monitor (3), and the stimulus spot can be moved with a joystick (4). (B) Infrared fundus image of the monkey retina. A 15° stimulus spot positioned on the monkey’s macula. (C) Image of the red stimulus spot on the blue background. This image was photographed by a digital camera at the position of the monkey’s eye.
Figure 2.
 
Diagram of the focal PhNR recording system with fundus monitoring with an infrared fundus camera. A red LED was used for the stimulus source, and a blue LED was used for the background illumination.
Figure 2.
 
Diagram of the focal PhNR recording system with fundus monitoring with an infrared fundus camera. A red LED was used for the stimulus source, and a blue LED was used for the background illumination.
Figure 3.
 
Studies of stray light effect in our system. (A) Fundus photograph of a monkey whose macula was damaged by 15° in the central area by focal laser photocoagulation (within the white dashed line). (B) Focal ERGs recorded with a 15° stimulus spot centered on the photocoagulation. Stimulus luminance and stimulus duration were changed on a steady blue background illumination of 100 scot cd/m2. Small positive (red asterisks) or negative (blue arrows) waves were detected when the stimulus luminance was 84 cd/m2 or higher, presumably due to the effect of stray light.
Figure 3.
 
Studies of stray light effect in our system. (A) Fundus photograph of a monkey whose macula was damaged by 15° in the central area by focal laser photocoagulation (within the white dashed line). (B) Focal ERGs recorded with a 15° stimulus spot centered on the photocoagulation. Stimulus luminance and stimulus duration were changed on a steady blue background illumination of 100 scot cd/m2. Small positive (red asterisks) or negative (blue arrows) waves were detected when the stimulus luminance was 84 cd/m2 or higher, presumably due to the effect of stray light.
Figure 4.
 
Representative focal ERGs elicited from a rhesus monkey by different stimulus luminances (2–55 phot cd/m2) and different stimulus durations (5, 10, 30, and 150 ms). The amplitude of PhNR increases with increasing stimulus luminances.
Figure 4.
 
Representative focal ERGs elicited from a rhesus monkey by different stimulus luminances (2–55 phot cd/m2) and different stimulus durations (5, 10, 30, and 150 ms). The amplitude of PhNR increases with increasing stimulus luminances.
Figure 5.
 
(A) Stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the focal macular PhNR. The PhNR amplitudes are plotted as a function of stimulus energy (cd-s/m2) for brief flashes of 5 to 30 ms in the left half and are plotted as a function of stimulus luminance (cd/m2) for a long 150-ms flash in the right half. (B) Comparison of stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the b-wave and PhNR for stimulus duration of 10 ms (left) and 150 ms (right).
Figure 5.
 
(A) Stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the focal macular PhNR. The PhNR amplitudes are plotted as a function of stimulus energy (cd-s/m2) for brief flashes of 5 to 30 ms in the left half and are plotted as a function of stimulus luminance (cd/m2) for a long 150-ms flash in the right half. (B) Comparison of stimulus intensity–response curves of the mean (± SEM, n = 4) amplitudes of the b-wave and PhNR for stimulus duration of 10 ms (left) and 150 ms (right).
Figure 6.
 
Effect of stimulus duration on the focal PhNR. (A) Representative focal ERGs elicited by different stimulus durations (5–150 ms) for a constant stimulus luminance of 55 phot cd/m2. Vertical dotted line: peak of the PhNR. The values of stimulus energy (cd-s/m2) are also indicated for brief flashes of 5 to 30 ms. (B) Superimposed focal ERG waveforms recorded with different stimulus durations (5–150 ms). Stimulus luminance was fixed at 55 phot cd/m2.
Figure 6.
 
Effect of stimulus duration on the focal PhNR. (A) Representative focal ERGs elicited by different stimulus durations (5–150 ms) for a constant stimulus luminance of 55 phot cd/m2. Vertical dotted line: peak of the PhNR. The values of stimulus energy (cd-s/m2) are also indicated for brief flashes of 5 to 30 ms. (B) Superimposed focal ERG waveforms recorded with different stimulus durations (5–150 ms). Stimulus luminance was fixed at 55 phot cd/m2.
Figure 7.
 
Focal ERGs before and after intravitreous injection of TTX in one monkey. The focal ERGs before (black lines) and after TTX (red lines) are superimposed. Stimulus luminance was fixed at 55 phot cd/m2, and stimulus duration was changed from 5 to 150 ms. Note that the amplitude of focal PhNR was greatly reduced after TTX. The other slow negative response to the stimulus offset (PhNROff, asterisks) was also reduced after TTX.
Figure 7.
 
Focal ERGs before and after intravitreous injection of TTX in one monkey. The focal ERGs before (black lines) and after TTX (red lines) are superimposed. Stimulus luminance was fixed at 55 phot cd/m2, and stimulus duration was changed from 5 to 150 ms. Note that the amplitude of focal PhNR was greatly reduced after TTX. The other slow negative response to the stimulus offset (PhNROff, asterisks) was also reduced after TTX.
Copyright 2008 The Association for Research in Vision and Ophthalmology, Inc.
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