September 2009
Volume 50, Issue 9
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Retina  |   September 2009
Supernormal ERG Oscillatory Potentials in Transgenic Rabbit with Rhodopsin P347L Mutation and Retinal Degeneration
Author Affiliations
  • Takao Sakai
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Mineo Kondo
    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.
  • Toshiyuki Koyasu
    From the Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
  • Keiichi Komeima
    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 September 2009, Vol.50, 4402-4409. doi:10.1167/iovs.09-3458
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      Takao Sakai, Mineo Kondo, Shinji Ueno, Toshiyuki Koyasu, Keiichi Komeima, Hiroko Terasaki; Supernormal ERG Oscillatory Potentials in Transgenic Rabbit with Rhodopsin P347L Mutation and Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2009;50(9):4402-4409. doi: 10.1167/iovs.09-3458.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine the properties of the retina of a rhodopsin P347L transgenic (Tg) rabbit model of retinal degeneration by electroretinography (ERG).

methods. Full-field ERGs were recorded in 12- to 48-week-old wild-type (WT) and Tg rabbits. The a-wave was analyzed by the a-wave fitting model of Hood and Birch. The stimulus–response function of the b-wave was analyzed by the Michaelis-Menten equation. Oscillatory potentials (OPs) were extracted by digital filtering after subtracting the a-wave. OPs were also recorded before and after an intravitreal injection of l-2 amino-4-phosphonobutyric acid (APB), cis-2,3 piperidine dicarboxylic acid (PDA), γ-amino butyric acid (GABA), or tetrodotoxin citrate (TTX).

results. All the ERG components of Tg rabbits decreased progressively with age with the a-wave more affected than the b-wave, and the OPs were most preserved. Of interest, the summed OP amplitudes of the Tg rabbits were significantly larger than those of WT rabbits when they were 12 weeks of age. The changes in the amplitudes of the OPs after intravitreal injections of APB, PDA, or GABA in Tg rabbits did not differ significantly from those of WT rabbits. However, injection of TTX resulted in a significantly larger amplitude reduction of the OPs in Tg (65.3%) than in WT (28.6%) rabbits.

conclusions. The significantly larger OPs in Tg rabbits resulted from alterations in the inner retinal neurons. The greater effect of TTX on the OP amplitudes in Tg rabbits suggests that the supernormal OPs in Tg rabbits may be related to secondary changes in the spiking neurons of the inner retina after photoreceptor degeneration.

Electrophysiological assessments of human patients and animal models of retinitis pigmentosa (RP) are valuable for understanding the pathophysiology of RP because layer-by-layer retinal function can be evaluated objectively. The photoreceptor components of the electroretinogram (ERG) are usually most severely impaired in patients with RP, even at the early stages, 1 2 3 4 because this disease primarily affects the photoreceptor–retinal pigment epithelium complex. 1 2 3 4 5 6 7 ERG studies have also shown that not only the photoreceptors, but also the bipolar cells can be affected in patients with RP. 8 9 10 11 However, there have been reports of an unexpected preservation or even an enhancement of the postreceptoral ERG components in some animal models of RP. These components include the b-wave, 12 13 14 the scotopic threshold response (STR), 15 the negative component of the photopic ERG, 16 and oscillatory potentials (OPs). 17 The exact mechanism(s) underlying these secondary functional changes in the postreceptoral neurons after the photoreceptor degeneration has not been fully determined. 
We have recently succeeded in generating a rhodopsin P347L transgenic (Tg) rabbit by using bacterial artificial chromosome (BAC) transgenesis. 18 We have shown that the rod function of Tg rabbits was reduced at an early age, whereas the cone function was relatively well preserved. This sequence of alterations is similar to those in human patients with RP with the rhodopsin P347L mutation. 19 20 However, we did not analyze the properties of all the ERG components quantitatively and did not compare them with age-matched wild-type (WT) rabbits. 
The purpose of this study was to investigate the properties of the ERGs of our Tg rabbits. We analyzed the a- and b-waves, and the OPs which originate from inner retinal neurons 21 22 23 24 25 until ∼48 weeks of age. All ERG components of Tg rabbits progressively decreased with increasing age, and the a-wave was more severely impaired than the b-wave. Of note, the OP amplitudes of Tg rabbits were larger than those of WT rabbits when they were 12 weeks of age. The results of pharmacologic studies suggest that the functional changes in the TTX-sensitive spiking neurons of the inner retina contribute to the supernormal OPs in young Tg rabbits. 
Materials and Methods
Animals
The experiments were performed on 31 Tg and 31 WT New Zealand White rabbits. The ages of the rabbits ranged from 12 to 48 weeks. Thirty animals were used for full-field ERG recordings without any treatment and 32 for pharmacologic studies. This study was conducted in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. All protocols were approved by the Institutional Review Board of Nagoya University Graduate School of Medicine. 
Our techniques for generating of Tg rabbits has been described in detail. 18 Briefly, a rabbit BAC clone that included the entire rhodopsin gene was identified, and a C-to-T transition at the codon of proline 347 was performed by BAC recombineering. 26 27 28 This transition in exon 5 of the rabbit rhodopsin gene resulted in a proline-to-leucine substitution at codon 347. After the BAC modification in Escherichia coli, the linearized BAC Tg construct was purified and injected into rabbit embryos at the pronucleus stage. 
Southern blot analysis showed that 12 of 80 newborn rabbits were transgene positive, and 10 of the 12 survived. These 10 founders were bred with WT rabbits, and six founders were shown to have transmitted the transgene. We mainly reproduced and investigated line 7, which had the highest level of transgene expression and the most severe photoreceptor degeneration as determined histologically. 18  
ERG Recordings
Animals were dark adapted for 60 minutes and then anesthetized with an intramuscular injection of 25 mg/kg ketamine and 2 mg/kg xylazine. ERGs were recorded with Burian-Allen bipolar contact lens electrodes (Hansen Laboratory, Iowa, City, IA). The animals were placed in a Ganzfeld bowl and stimulated with stroboscopic stimuli (model SG-2002; LKC Technologies, Gaithersburg, MD). The stimulus strength at the cornea was 2.2 log cd s m−2 (photopic units). Sixteen steps (0.5 log unit steps) of stimuli ranging from −5.3 to 2.2 log cd s m−2 were used to elicit the scotopic ERGs. The photopic ERGs were then recorded on a rod-suppressing white background of 3.3 log scot td, and seven steps of stimuli ranging from −0.8 to 2.2 log cd s m−2 were used. Signals were amplified, band-pass filtered between 0.3 to 1000 Hz, and averaged by using a computer-assisted signal analysis system (MEB-9100, Neuropack; Nihon Kohden, Tokyo, Japan). 
Fitting Model to a- and b-Waves
To evaluate photoreceptor function, the leading edge of the rod and cone a-waves was fitted to a curve by the Hood and Birch modification 2 of the Lamb and Pugh model (Fig. 1A) 29 :  
\[\mathrm{P}3(i,\ t)\ {=}\ {\{}1\ {-}\ \mathrm{exp}{[}{-}i\ {\cdot}\ S(t\ {-}\ t_{\mathrm{d}})^{2}{]}{\}}Rm\ \mathrm{for}\ t{>}t_{\mathrm{d}}\]
In this equation, i is the flash energy (log phot cd s m−2); t d is the time delay; t is the time after the flash onset; S is the sensitivity; and Rm is the maximum response amplitude. This model yields values for Rm, S, and t d. For more details, see the Appendix. 
The stimulus-response function of the rod and cone b-waves was fitted by the Michaelis-Menten equation (Fig. 1B) 30 31 :  
\[V\ {=}\ V_{\mathrm{max}}\ {\cdot}\ 1/(I\ {+}\ K)\]
where V is the amplitude of the b-wave; V max is the maximum amplitude of the b-wave; I is the flash energy (log phot cd s m−2); and K is the flash energy that elicits a b-wave amplitude of half V max (half-saturation coefficient). This model yielded values for two parameters: V max and K. For responses to higher flash energies at which substantial a-wave intrusion occurred, the photorecepter component (P3) was digitally subtracted. High-frequency OPs (85–300 Hz) were also removed by digital filtering. 
Extraction and Measurement of Photopic OPs
The OPs were extracted by using the method of Zhang et al. 23 and Akula et al. 32 To reduce the contamination of the a-wave on the early OPs, we fitted a mathematical model of the a-wave (equation 1)and digitally subtracted the a-wave component from the intact ERG (Fig. 1C , top). 
To determine appropriate band-pass filters for extracting OPs of WT and Tg rabbits, we analyzed the extracted postreceptoral component (P2) by Fast Fourier Transform (FFT). We found that the OPs of the photopic ERG of WT and Tg rabbits have one main peak (Fig. 1D) . Based on these results, we chose band-pass settings of 85 to 300 Hz for extracting the photopic OPs which are similar to those used in a recent rabbit ERG study. 22 23  
To measure each OP amplitude (O1–O4), we measured the amplitude of a specific OP from the peak and trough immediately preceding it (Fig. 1C , bottom). The implicit time was defined as the time from stimulus onset to the peak of the OP. The summed OP amplitude was also used to assess total OP function. 
Drug Applications
The drugs and intravitreal injection techniques have been described in detail. 22 24 33 34 35 The drugs were injected into the vitreous with a 30-gauge needle inserted through the pars plana ∼1 mm posterior to the limbus. The drugs (Sigma-Aldrich, St. Louis, MO; Sankyo Co., Ltd., Tokyo, Japan) were dissolved in sterile PBS and injected in 0.05 mL. The intravitreal concentrations were: 2 mM for l-2 amino-4-phosphonobutyric acid (APB); 4 mM for cis-2,3 piperidine dicarboxylic acid (PDA); 2 mM for γ-amino butyric acid (GABA); and 2 μM for tetrodotoxin citrate (TTX). 
Recordings were begun approximately 60 to 90 minutes after the drug injections, and the studies were completed within 3 hours. We used rabbit eyes that had not been previously treated. 
Statistical Analyses
Unpaired Student’s t-test with Bonferroni’s correction was used to compare the amplitudes or implicit times of each ERG component between WT and Tg rabbits. Differences were considered to be significant when P < 0.05. 
Results
Scotopic and Photopic ERGs
Scotopic and photopic ERGs elicited by different stimulus strengths from 12-, 24-, and 48-week-old WT (blue) and Tg (red) rabbits are shown in Figure 2 . The amplitudes of both the scotopic and photopic ERGs of the Tg rabbits were smaller than those of the WT rabbits. In addition, there was a progressive decrease in the amplitudes with increasing age. The scotopic ERGs were more severely affected than the photopic ERGs in Tg. At 48 weeks, the scotopic b-waves at lower stimulus strengths (−5.3 to −1.3 log cd s m−2) were nearly nondetectable, and at the maximum stimulus strength, the scotopic ERG was almost the same as the photopic ERG in Tg rabbits. This indicated that the rod function was nearly extinguished at 48 weeks in Tg rabbits, and the cones were relatively well preserved. 
Analyses of a- and b-Waves
The a-wave that was fitted to a curve by the Hood and Birch 2 method showed that the rod maximum amplitude (Rm) of Tg rabbits was already reduced by 0.60 log units (% reduction, 75%) at 12 weeks, and by 1.01 log units (90%) at 24 weeks (Fig. 3A , Table 1 ). At 48 weeks, the maximum amplitude of rod a-wave of all Tg rabbits was <10 μV, which was too small to apply the fitting model. In contrast, the cone Rm of Tg rabbits was reduced by only 0.26 log unit (45%) at 12 weeks and by 0.43 log unit (63%), even at 48 weeks of age (Fig. 3A , Table 1 ; see also the Appendix). We also found that not only the Rm, but also the transduction sensitivity (S) were abnormal in both rod and cone photoreceptors of Tg rabbits, especially at 24 weeks and older (Table 1)
The b-wave analysis using the Michaelis-Menten equation 30 31 showed that both the maximum amplitude (V max) and half-saturation coefficient (K) of the rod b-wave was significantly abnormal in Tg rabbits at 24 weeks and later, and the difference between two types of rabbits increased with increasing age (Fig. 3B , Table 1 ). In contrast, there was no significant difference in the cone b-wave V max between Tg and Wt rabbits at 12 to 48 weeks, although the K was already reduced significantly at 12 weeks (Fig. 3B , Table 1 ). 
These results indicated that the function of both the rod and cone components of the Tg rabbits decreased with age, and the rod components was more severely affected than the cone components. 
Supernormal OPs in Tg Rabbits
We next analyzed the OPs, which are believed to originate from inner retinal neurons including the amacrine and ganglion cells. 21 22 23 24 25  
The photopic ERGs elicited by the maximum stimulus strength of 2.2 log cd s m−2 from five WT and Tg rabbits at 12 weeks of age are shown in Figure 4A . The a-wave amplitudes of the cone ERGs of Tg rabbits were smaller than those of WT rabbits, whereas the b-wave amplitudes were nearly the same in the two types of rabbit (see also Figs. 2 3 ). The OPs were more prominent in the Tg than in the WT rabbits. We then extracted the OPs by digital filtering after subtracting the a-wave (Figs. 1C 1D) , and confirmed that the summed OP amplitudes in Tg were noticeably larger than those in WT rabbits (Fig. 4B) . The summed OP amplitude was 90.5 ± 22.5 μV (mean ± SD, n = 5) in Tg rabbits which was significantly larger than the 51.4 ± 13.0 μV in WT rabbits (P = 0.009). At 24 to 48 weeks, the summed OP amplitudes of Tg were not significantly different from those of WT rabbits (Fig. 3C , Table 1 ). 
We also compared the amplitude of the individual Ops (O1–O4) between the WT and Tg rabbits, because there are reports suggesting that each OP wavelet may have different retinal origins. 21 22 36 We found that the amplitude for the later three OPs (O2–O4) in Tg rabbits were significantly larger than the corresponding OPs in the WT rabbits. However, the amplitude of O1 was not different between the two types of rabbits (Fig. 4C)
The implicit times of the OPs did not differ between the two types of rabbits for all the OPs (Fig. 4D)
We also compared the summed OP amplitudes of scotopic OPs between WT and Tg rabbits when they were 12 weeks old, but there was no statistically significant difference (WT, 86.2 ± 16.3 μV; Tg, 73.6 ± 18.9 μV, mean ± SD, P = 0.25). 
Comparisons of Amplitudes of Each ERG Component
The differences in the log maximum amplitude between WT and Tg rabbits for all ERG components (rod a-wave, cone a-wave, rod b-wave, cone b-wave, and cone OPs) are shown in Figure 5 . In these graphs, the values of Tg minus WT are plotted. A minus value means that the amplitude in WT is larger than that in Tg rabbits. As expected, the differences became smaller for all ERG components with increasing age, and the rod components were more severely affected than the cone components in Tg rabbits. We also found that and the a-waves were more affected than the b-waves, and OPs were better preserved than the a- and b-waves in Tg rabbits. Tg rabbits had supernormal OPs amplitude when they were 12 and 24 weeks of age. 
Effect of Pharmacologic Agents on OPs of WT and Tg Rabbits
Finally, to determine what types of retinal cells or circuits are involved in the enhanced OP amplitudes in Tg rabbits, we studied the effect of various pharmacologic agents on the OPs of WT and Tg rabbits when they were 16 weeks of age. Before the drug injections, we confirmed that the intravitreal injection of PBS alone did not change the amplitude and waveform of photopic OPs for two rabbits (data not shown). 
APB is a mGluR6 agonist that blocks synaptic transmission between photoreceptors and ON bipolar cells, leaving the OFF pathway intact. 37 Intravitreal injection of APB reduced all photopic OPs almost completely in both WT and Tg rabbits (Fig. 6A 6B) . These changes indicated that the postsynaptic ON-pathway plays an important role in the origin of the photopic OPs 22 24 38 39 in WT and Tg rabbits. 
PDA is an antagonist of AMPA/KA class ionotropic glutamate receptors and blocks the light driven response of OFF-bipolar cells, horizontal cells, and many amacrine and ganglion cells. The ON bipolar cells were relatively unaffected. 33 40 An intravitreal injection of PDA also greatly reduced the amplitude of the photopic OPs, 22 24 but to a lesser extent than APB (Fig. 6A) . The degree of amplitude reduction by PDA was not different in the WT and Tg (Fig. 6B)rabbits. 
GABA is a neurotransmitter released from retinal amacrine cells and horizontal cells. 41 As reported in other studies, 21 GABA suppressed the OPs in both WT and Tg rabbits (Fig. 6A) . Again, the degree of reduction of the OPs by GABA was not different between the WT and Tg rabbits (Figs. 6B)
TTX blocks voltage-gated sodium channels, and thus blocks action potentials produced by ganglion cells and certain classes of amacrine cells. 42 43 44 45 46 47 48 An intravitreal injection of TTX resulted in the reduction in the amplitude of the photopic OPs (Fig. 6A) . As reported, 22 the effect of TTX was greater for the later OPs (O3 and O4) in both WT and Tg rabbits (Fig. 6A) . We found that the degree of amplitude reduction by TTX was very different between the two types of rabbits: the amplitude reduction by TTX was only 28.6% ± 10.2% in WT, but was 65.3% ± 2.0% in Tg rabbits (mean ± SD, n = 5, Fig. 6B ). This difference was statistically significant (P < 0.001). As a consequence, the summed OP amplitudes in Tg rabbit after TTX (25.5 ± 6.6 μV) became smaller than those of WT rabbits (37.7 ± 3.0 μV). The OP waveforms after TTX from the five animals are superimposed in the bottom panel of Figure 6A . These results suggested that the functional changes in the TTX-sensitive spiking neurons may be related to the enhanced photopic OPs in our young Tg rabbits. 
Effect of TTX on the Photopic ERG b-Wave in WT and Tg Rabbits
It has been reported that TTX reduces the photopic ERG b-wave in rats, 46 47 48 indicating that voltage-gated sodium channels normally boost the photopic b-wave amplitude. We thought that if the amplitude reduction of the photopic ERG b-wave by TTX was larger in Tg than in WT rabbits, it could suggest that the supernormal OPs of Tg rabbits are caused by enhanced input from bipolar cells to the inner retina. We studied the effect of TTX on the amplitude of photopic ERG b-wave in our rabbits, and found that TTX reduced the amplitude of the photopic ERG b-wave for both WT and Tg rabbits. However, the degree of amplitude reduction by TTX was not different between WT (60.0% ± 7.3%, n = 5) and Tg (54.6% ± 13.9%, n = 5, P = 0.55) rabbits. 
Discussion
Our results demonstrated that the a- and b-waves of the ERGs of Tg rabbits decrease progressively with increasing age, and the rod components were more affected than the cone components. These changes are characteristic of animal models of RP and human patients with RP. 1 2 3 4 In addition, the degree of amplitude reduction was greater for the a-wave (photoreceptor component) than for the b-wave (bipolar cell component). Such a difference in reduction in the amplitude of the a- and b-waves has been previously reported, 12 13 14 and may be explained by the difference in the stimulus-response function of the a- and b-waves. A past study using a computational model of the a- and b-waves indicated that there should be relatively little change in V max with changes in Rm, because the photoreceptor response are linear over the range of the b-wave V-log I function. 49  
The most interesting finding in this study was the enhanced amplitudes of the OPs in young Tg rabbits. The amplitudes of summed OPs of 12-week-old Tg were 1.76 times (0.25 log unit) larger than those of WT rabbits of the same age. At this age, the maximum cone a-wave amplitude of Tg rabbits was significantly smaller than that of WT rabbits, and the maximum cone b-wave amplitude of Tg rabbits was about the same amplitude as that of WT rabbits. The supernormal amplitude of the OPs cannot be explained simply by the buffering effect of inner retinal neurons 12 13 and suggest that there are most likely secondary functional changes in the inner retinal neurons. 
Banin et al. 17 have reported similar ERG findings in their pig RP model with a rhodopsin mutation. They observed supernormal photopic OPs and abnormal cone b-wave waveforms even at early stages of retinal degeneration when the photoreceptor physiology was still completely normal. They suggested that the rod photoreceptor degeneration may cause the functional changes in the inner retina of the cone circuitry even at early stages of retinal degeneration. Although they did not measure the amplitude of the OPs quantitatively, the pattern of the changes of the OPs in their pig model were very similar to those in our Tg rabbit: the early OP (O1) in the Tg did not differ significantly from that of WT, but the later OPs (O2–O4) were clearly larger in Tg (see Fig. 2ein Ref. 17 and our Fig. 4 ). It is very interesting that the same point mutation near the C-terminal of rhodopsin gene, P347L, can cause similar secondary functional changes in the inner retina in two different species of RP models: the pig and the rabbit. 
To determine what types of retinal cells or circuits might contribute to the supernormal OPs in young Tg rabbits, we injected different various pharmacologic agents into the eye of WT and Tg rabbits. Our results in WT rabbits were consistent with the conclusions of earlier reports that the OPs originate mainly from third-order neurons of the inner retina, predominantly in the ON-pathway. 21 22 23 24 36 A direct contribution from cone ON- and OFF-bipolar cells to the rabbit cone OPs must not be large, because APB or PDA alone resulted in a severe amplitude reduction of the OPs. We also found that whereas the degree of the ERG reduction after APB, PDA, and GABA did not differ between Tg and WT rabbits, TTX application resulted in dramatic differences in the degrees of amplitude change in the two types of rabbits. The degree of amplitude reduction was significantly greater in Tg (65.3%) than in WT (28.6%) rabbits. As a result, the summed amplitude of the OPs in Tg became significantly smaller than that of WT after TTX. These results suggest that the functional changes in the TTX-sensitive retinal neurons, may contribute to the abnormally large OPs in young Tg rabbits. 
A possible mechanism for supernormal OPs in our Tg rabbits is the changes in the input from ON/OFF bipolar cells to the OP generator in the inner retina. First, we compared the activity of cone ON bipolar cells (cone V max) between WT and Tg rabbits at 12 weeks, but there was no significant difference (Table 1 , Fig. 3 ). Next we compared the activity of cone OFF bipolar cells/horizontal cells between WT and Tg by measuring the change in the maximum a-wave after PDA. The degree of amplitude reduction of the a-wave after PDA in Tg was not significantly different from that of WT (see also the Appendix). Finally, we also found that the changes in the photopic ERG b-wave by TTX were not different between Tg and WT rabbits. Thus, we could not detect any changes in the input from the ON/OFF bipolar cells in our Tg rabbits. We are currently planning to analyze functional changes in the ON/OFF bipolar cells of Tg rabbits in more details using the vector modeling of flicker ERG. 50  
What kinds of further studies are needed? First, we could not determine which of two types of retinal cells, amacrine cells or ganglion cells, were the main contributors to the supernormal OPs in Tg rabbits. To determine this, photopic OPs should be recorded from Tg rabbits several weeks after optic nerve section to exclude the contribution of retinal ganglion cells. Second, we did not determine the exact mechanism of the supernormal OPs in Tg rabbit. The possible hypotheses include; an increase in the number of inner retinal neurons after the photoreceptor degeneration, 51 changes in the feedback communications, changes in some type of humoral factors which are released from degenerating neurons or glia, 52 transformation of neural phenotypes in the inner retina, changes in the resistance associated with photoreceptor loss, or synaptic rewiring in the inner retinal neurons after the photoreceptor degeneration. 53 54 55 Further anatomic and functional studies using immunohistochemistry, 47 ultrastructure, or single-unit response recording 56 in the inner retinal neurons of Tg rabbits may add more information on the exact mechanism of this phenomenon. 
Finally, the question arises as to whether similar ERG changes occur in patients with RP. We recently studied all components of macular cone ERGs in patients with RP at relatively early stages 57 and noted that macular OPs were better preserved than the a- and b-waves. We also found that one patient with RP had supernormal macular OP amplitudes: surprisingly, her summed OP amplitude was larger than those of any of the 43 normal subjects (Fig. 4B in Ref. 57 ). These macular ERG data raise a possibility that similar secondary functional changes can occur in some patients with RP, at least in some retinal areas. These functional changes in the inner retina may have important implications for future treatment strategy for patients with RP including cell transplantation 58 and retinal prosthesis. 59  
Appendix 1
Fitting Model for the Cone a-Wave
In this study we used the a-wave fitting model of Hood and Birch 2 to evaluate rod and cone photoreceptor function. However, several pharmacologic experiments have indicated that the photopic a-wave includes a PDA-sensitive, postreceptoral component in primates. 60 61 62 We investigated the change in the a-wave amplitude before and after PDA in WT rabbits and found that the maximum a-wave amplitude decreased 13% to 25% after PDA (n = 3), suggesting that the activities of postreceptoral neurons significantly contribute to the cone a-wave in rabbits. Thus, we assumed that in the method of OP extraction, the component of not only the cone photoreceptors, but also the component of OFF bipolar cell/horizontal cells were subtracted. In addition, we also supposed that when the Michaelis-Menten fit to the b-wave amplitude measurements was used, the ON bipolar cell component was mainly analyzed, since the negative component (some combination of cone and OFF cone bipolar/horizontal cell component) has been already subtracted. 
Recently, Hood and Birch 63 have shown that the cone a-wave is better fitted with a filtered version of equation 1in humans. They showed that a Michaelis-Menten version of the equation gives a better fit to the cone a-wave. 
Amplitude Reduction of Photopic a-Wave of Tg Rabbits
One question regarding the reduction in the photopic a-wave in young Tg rabbits is whether this reduction is a reflection of cone contributions to the photopic ERG or whether it is a reduction in the OFF bipolar/horizontal cell component. We compared the effect of PDA on the maximum a-wave amplitude between WT and Tg rabbits at 16 weeks and found that the degree of amplitude reduction after PDA did not differ between WT (6.4–0.5 μV, n = 3) and Tg (6.1–7.5 μV, n = 3; P = 0.23). These results indicate that the amplitude reduction of the photopic a-wave of young Tg rabbits is mainly due to the decreased cone photoreceptor activity. 
 
Figure 1.
 
Analyses of ERG components. (A) Cone ERG a-waves (solid lines) recorded from 12-week-old WT, 12-week-old Tg, and 48-week-old Tg rabbits. Responses to three brighter stimuli of 2.2, 1.7, and 1.2 log cd s m−2 are shown. Dashed lines: best fit of equation 1to the entire data set. The coefficients for the best fit are shown in each panel. (B) Cone ERG b-waves recorded from a 48-week-old WT (left) and Tg (middle) rabbits. Responses to seven flashes of −1.3 to 1.7 log cd s m−2 are shown. Right: best fit of equation 2to the amplitude of the b-waves. (C) Extraction and analysis of the OPs of the photopic ERG. To minimize the effect of a-wave contamination, the photoreceptor component (equation 1 , P3) was digitally subtracted from the intact ERG (top). The amplitude of the individual OP was defined as the difference between the peak and the trough immediately preceding it (bottom). (D) Frequency spectra of the photopic ERG P2 component in WT and Tg rabbits. The OPs were extracted by band-pass filtering of 85 to 300 Hz.
Figure 1.
 
Analyses of ERG components. (A) Cone ERG a-waves (solid lines) recorded from 12-week-old WT, 12-week-old Tg, and 48-week-old Tg rabbits. Responses to three brighter stimuli of 2.2, 1.7, and 1.2 log cd s m−2 are shown. Dashed lines: best fit of equation 1to the entire data set. The coefficients for the best fit are shown in each panel. (B) Cone ERG b-waves recorded from a 48-week-old WT (left) and Tg (middle) rabbits. Responses to seven flashes of −1.3 to 1.7 log cd s m−2 are shown. Right: best fit of equation 2to the amplitude of the b-waves. (C) Extraction and analysis of the OPs of the photopic ERG. To minimize the effect of a-wave contamination, the photoreceptor component (equation 1 , P3) was digitally subtracted from the intact ERG (top). The amplitude of the individual OP was defined as the difference between the peak and the trough immediately preceding it (bottom). (D) Frequency spectra of the photopic ERG P2 component in WT and Tg rabbits. The OPs were extracted by band-pass filtering of 85 to 300 Hz.
Figure 2.
 
ERGs recorded from 12-, 24-, and 48-week-old WT (blue), and rhodopsin P347L transgenic (Tg, red) rabbits. (A) Scotopic ERGs elicited by nine different stimulus strengths. (B) Photopic ERGs elicited by four different stimulus strengths.
Figure 2.
 
ERGs recorded from 12-, 24-, and 48-week-old WT (blue), and rhodopsin P347L transgenic (Tg, red) rabbits. (A) Scotopic ERGs elicited by nine different stimulus strengths. (B) Photopic ERGs elicited by four different stimulus strengths.
Figure 3.
 
Plots of maximum amplitude of each ERG components. Mean values of five animals are plotted. Bars, SEM. *P < 0.05 (unpaired t-test with Bonferroni’s correction). (A) Plots of log maximum amplitude for rod and cone a-waves (Rm) for rabbits at 12, 24, and 48 weeks of age. (B) Plots of log maximum amplitude for rod and cone b-waves (V max) for rabbits at 12, 24, and 48 weeks of age. (C) Plots of log summed OPs of cone ERG at 12, 24, and 48 weeks of age.
Figure 3.
 
Plots of maximum amplitude of each ERG components. Mean values of five animals are plotted. Bars, SEM. *P < 0.05 (unpaired t-test with Bonferroni’s correction). (A) Plots of log maximum amplitude for rod and cone a-waves (Rm) for rabbits at 12, 24, and 48 weeks of age. (B) Plots of log maximum amplitude for rod and cone b-waves (V max) for rabbits at 12, 24, and 48 weeks of age. (C) Plots of log summed OPs of cone ERG at 12, 24, and 48 weeks of age.
Table 1.
 
Summary of ERG Parameters in WT and Tg Rabbits
Table 1.
 
Summary of ERG Parameters in WT and Tg Rabbits
12 Weeks 24 Weeks 48 Weeks
WT (n = 5) Tg (n = 5) WT (n = 5) Tg (n = 5) WT (n = 5) Tg (n = 5)
a-Wave analysis
 Rod log Rm 2.23 ± 0.08 1.63 ± 0.06* 2.14 ± 0.07 1.13 ± 0.12* 2.06 ± 0.10 , †
 Rod log S 3.50 ± 0.04 3.19 ± 0.11* 3.46 ± 0.15 3.29 ± 0.09* 3.42 ± 0.06 , †
 Cone log Rm 1.73 ± 0.09 1.47 ± 0.07* 1.75 ± 0.07 1.36 ± 0.10* 1.64 ± 0.08 1.21 ± 0.23*
 Cone log S 2.97 ± 0.05 2.84 ± 0.14 2.91 ± 0.09 2.75 ± 0.07* 2.93 ± 0.10 2.68 ± 0.18*
b-Wave analysis
 Rod log V max 2.61 ± 0.07 2.11 ± 0.09* 2.58 ± 0.12 1.76 ± 0.06* 2.51 ± 0.12 1.62 ± 0.22*
 Rod log K −2.43 ± 0.15 −1.96 ± 0.42 −2.20 ± 0.21 −1.87 ± 0.13* −2.21 ± 0.20 −1.80 ± 0.18*
 Cone log V max 2.16 ± 0.10 2.12 ± 0.04 2.22 ± 0.06 2.12 ± 0.08 2.18 ± 0.07 1.89 ± 0.23
 Cone log K −0.22 ± 0.10 0.16 ± 0.08* −0.13 ± 0.14 0.32 ± 0.07* −0.11 ± 0.08 0.33 ± 0.17*
Summed OPs
 Log Σ OPs 1.71 ± 1.35 1.96 ± 1.10* 1.78 ± 1.24 1.94 ± 1.43 1.71 ± 1.10 1.56 ± 1.40
Figure 4.
 
Supernormal OPs in Tg rabbits. (A) Photopic ERGs (B) extracted OPs, (C) mean amplitudes of individual OPs (O1–O4) and (D) mean implicit times of individual OPs (O1–O4) for five WT and Tg rabbits of 12 weeks of age. Bars, SEM.
Figure 4.
 
Supernormal OPs in Tg rabbits. (A) Photopic ERGs (B) extracted OPs, (C) mean amplitudes of individual OPs (O1–O4) and (D) mean implicit times of individual OPs (O1–O4) for five WT and Tg rabbits of 12 weeks of age. Bars, SEM.
Figure 5.
 
Difference of the log maximum amplitude between Tg and WT rabbits for all ERG components. Values of Tg minus WT are shown. The minus values indicate that the amplitudes of the WT rabbits were larger than those of Tg rabbits.
Figure 5.
 
Difference of the log maximum amplitude between Tg and WT rabbits for all ERG components. Values of Tg minus WT are shown. The minus values indicate that the amplitudes of the WT rabbits were larger than those of Tg rabbits.
Figure 6.
 
Effects of various pharmacologic agents on the cone OPs of WT and Tg rabbits. (A) Representative waveforms of OPs before (black) and after (red) APB, PDA, GABA, or TTX. The post-TTX recordings from five different rabbits are superimposed in the bottom panel. Note that the OP amplitudes of Tg rabbits are smaller than those of WT rabbits after TTX. (B) Plots of relative amplitude reduction after drugs in WT and Tg rabbits. Data are the mean ± SEM. The numbers in the parenthesis indicate the number of animals used. The effect of TTX on the amplitude reduction of OPs in Tg rabbits was significantly larger than in WT rabbits.
Figure 6.
 
Effects of various pharmacologic agents on the cone OPs of WT and Tg rabbits. (A) Representative waveforms of OPs before (black) and after (red) APB, PDA, GABA, or TTX. The post-TTX recordings from five different rabbits are superimposed in the bottom panel. Note that the OP amplitudes of Tg rabbits are smaller than those of WT rabbits after TTX. (B) Plots of relative amplitude reduction after drugs in WT and Tg rabbits. Data are the mean ± SEM. The numbers in the parenthesis indicate the number of animals used. The effect of TTX on the amplitude reduction of OPs in Tg rabbits was significantly larger than in WT rabbits.
The authors thank Paul A. Sieving (National Eye Institute), Yozo Miyake (Shukutoku University), Michael A. Sandberg (Massachusetts Eye and Ear Infirmary), and Duco I. Hamasaki (Bascom Palmer Eye Institute) for critical comments and the anonymous reviewers for valuable suggestions. 
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Figure 1.
 
Analyses of ERG components. (A) Cone ERG a-waves (solid lines) recorded from 12-week-old WT, 12-week-old Tg, and 48-week-old Tg rabbits. Responses to three brighter stimuli of 2.2, 1.7, and 1.2 log cd s m−2 are shown. Dashed lines: best fit of equation 1to the entire data set. The coefficients for the best fit are shown in each panel. (B) Cone ERG b-waves recorded from a 48-week-old WT (left) and Tg (middle) rabbits. Responses to seven flashes of −1.3 to 1.7 log cd s m−2 are shown. Right: best fit of equation 2to the amplitude of the b-waves. (C) Extraction and analysis of the OPs of the photopic ERG. To minimize the effect of a-wave contamination, the photoreceptor component (equation 1 , P3) was digitally subtracted from the intact ERG (top). The amplitude of the individual OP was defined as the difference between the peak and the trough immediately preceding it (bottom). (D) Frequency spectra of the photopic ERG P2 component in WT and Tg rabbits. The OPs were extracted by band-pass filtering of 85 to 300 Hz.
Figure 1.
 
Analyses of ERG components. (A) Cone ERG a-waves (solid lines) recorded from 12-week-old WT, 12-week-old Tg, and 48-week-old Tg rabbits. Responses to three brighter stimuli of 2.2, 1.7, and 1.2 log cd s m−2 are shown. Dashed lines: best fit of equation 1to the entire data set. The coefficients for the best fit are shown in each panel. (B) Cone ERG b-waves recorded from a 48-week-old WT (left) and Tg (middle) rabbits. Responses to seven flashes of −1.3 to 1.7 log cd s m−2 are shown. Right: best fit of equation 2to the amplitude of the b-waves. (C) Extraction and analysis of the OPs of the photopic ERG. To minimize the effect of a-wave contamination, the photoreceptor component (equation 1 , P3) was digitally subtracted from the intact ERG (top). The amplitude of the individual OP was defined as the difference between the peak and the trough immediately preceding it (bottom). (D) Frequency spectra of the photopic ERG P2 component in WT and Tg rabbits. The OPs were extracted by band-pass filtering of 85 to 300 Hz.
Figure 2.
 
ERGs recorded from 12-, 24-, and 48-week-old WT (blue), and rhodopsin P347L transgenic (Tg, red) rabbits. (A) Scotopic ERGs elicited by nine different stimulus strengths. (B) Photopic ERGs elicited by four different stimulus strengths.
Figure 2.
 
ERGs recorded from 12-, 24-, and 48-week-old WT (blue), and rhodopsin P347L transgenic (Tg, red) rabbits. (A) Scotopic ERGs elicited by nine different stimulus strengths. (B) Photopic ERGs elicited by four different stimulus strengths.
Figure 3.
 
Plots of maximum amplitude of each ERG components. Mean values of five animals are plotted. Bars, SEM. *P < 0.05 (unpaired t-test with Bonferroni’s correction). (A) Plots of log maximum amplitude for rod and cone a-waves (Rm) for rabbits at 12, 24, and 48 weeks of age. (B) Plots of log maximum amplitude for rod and cone b-waves (V max) for rabbits at 12, 24, and 48 weeks of age. (C) Plots of log summed OPs of cone ERG at 12, 24, and 48 weeks of age.
Figure 3.
 
Plots of maximum amplitude of each ERG components. Mean values of five animals are plotted. Bars, SEM. *P < 0.05 (unpaired t-test with Bonferroni’s correction). (A) Plots of log maximum amplitude for rod and cone a-waves (Rm) for rabbits at 12, 24, and 48 weeks of age. (B) Plots of log maximum amplitude for rod and cone b-waves (V max) for rabbits at 12, 24, and 48 weeks of age. (C) Plots of log summed OPs of cone ERG at 12, 24, and 48 weeks of age.
Figure 4.
 
Supernormal OPs in Tg rabbits. (A) Photopic ERGs (B) extracted OPs, (C) mean amplitudes of individual OPs (O1–O4) and (D) mean implicit times of individual OPs (O1–O4) for five WT and Tg rabbits of 12 weeks of age. Bars, SEM.
Figure 4.
 
Supernormal OPs in Tg rabbits. (A) Photopic ERGs (B) extracted OPs, (C) mean amplitudes of individual OPs (O1–O4) and (D) mean implicit times of individual OPs (O1–O4) for five WT and Tg rabbits of 12 weeks of age. Bars, SEM.
Figure 5.
 
Difference of the log maximum amplitude between Tg and WT rabbits for all ERG components. Values of Tg minus WT are shown. The minus values indicate that the amplitudes of the WT rabbits were larger than those of Tg rabbits.
Figure 5.
 
Difference of the log maximum amplitude between Tg and WT rabbits for all ERG components. Values of Tg minus WT are shown. The minus values indicate that the amplitudes of the WT rabbits were larger than those of Tg rabbits.
Figure 6.
 
Effects of various pharmacologic agents on the cone OPs of WT and Tg rabbits. (A) Representative waveforms of OPs before (black) and after (red) APB, PDA, GABA, or TTX. The post-TTX recordings from five different rabbits are superimposed in the bottom panel. Note that the OP amplitudes of Tg rabbits are smaller than those of WT rabbits after TTX. (B) Plots of relative amplitude reduction after drugs in WT and Tg rabbits. Data are the mean ± SEM. The numbers in the parenthesis indicate the number of animals used. The effect of TTX on the amplitude reduction of OPs in Tg rabbits was significantly larger than in WT rabbits.
Figure 6.
 
Effects of various pharmacologic agents on the cone OPs of WT and Tg rabbits. (A) Representative waveforms of OPs before (black) and after (red) APB, PDA, GABA, or TTX. The post-TTX recordings from five different rabbits are superimposed in the bottom panel. Note that the OP amplitudes of Tg rabbits are smaller than those of WT rabbits after TTX. (B) Plots of relative amplitude reduction after drugs in WT and Tg rabbits. Data are the mean ± SEM. The numbers in the parenthesis indicate the number of animals used. The effect of TTX on the amplitude reduction of OPs in Tg rabbits was significantly larger than in WT rabbits.
Table 1.
 
Summary of ERG Parameters in WT and Tg Rabbits
Table 1.
 
Summary of ERG Parameters in WT and Tg Rabbits
12 Weeks 24 Weeks 48 Weeks
WT (n = 5) Tg (n = 5) WT (n = 5) Tg (n = 5) WT (n = 5) Tg (n = 5)
a-Wave analysis
 Rod log Rm 2.23 ± 0.08 1.63 ± 0.06* 2.14 ± 0.07 1.13 ± 0.12* 2.06 ± 0.10 , †
 Rod log S 3.50 ± 0.04 3.19 ± 0.11* 3.46 ± 0.15 3.29 ± 0.09* 3.42 ± 0.06 , †
 Cone log Rm 1.73 ± 0.09 1.47 ± 0.07* 1.75 ± 0.07 1.36 ± 0.10* 1.64 ± 0.08 1.21 ± 0.23*
 Cone log S 2.97 ± 0.05 2.84 ± 0.14 2.91 ± 0.09 2.75 ± 0.07* 2.93 ± 0.10 2.68 ± 0.18*
b-Wave analysis
 Rod log V max 2.61 ± 0.07 2.11 ± 0.09* 2.58 ± 0.12 1.76 ± 0.06* 2.51 ± 0.12 1.62 ± 0.22*
 Rod log K −2.43 ± 0.15 −1.96 ± 0.42 −2.20 ± 0.21 −1.87 ± 0.13* −2.21 ± 0.20 −1.80 ± 0.18*
 Cone log V max 2.16 ± 0.10 2.12 ± 0.04 2.22 ± 0.06 2.12 ± 0.08 2.18 ± 0.07 1.89 ± 0.23
 Cone log K −0.22 ± 0.10 0.16 ± 0.08* −0.13 ± 0.14 0.32 ± 0.07* −0.11 ± 0.08 0.33 ± 0.17*
Summed OPs
 Log Σ OPs 1.71 ± 1.35 1.96 ± 1.10* 1.78 ± 1.24 1.94 ± 1.43 1.71 ± 1.10 1.56 ± 1.40
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