XV ANIMAL TESTING

Electroretinogram

.

The electroretinogram

As described in previous chapters, the electroretinogram (ERG) recorded from the corneal surface of the eye represents the massed response of the retina to light stimulation. The ERG is easily recorded from the mouse eye with minor modifications of the general methodologies that have been employed for humans for many years. The basic methodology for recording the ERG in the mouse has been described in detail elsewhere.34 The solid curve shown in figure 81.1 is an example of an ERG recording from a normal C57BL/6J mouse in response to a bright flash of light. The major components of the response are the a-wave, which is the first negative corneal potential, and the b-wave, which is the first positive corneal potential. ERGs to bright flashes presented in the dark also contain a high-frequency oscillatory component on the ascending limb of the b-wave, collectively called the oscillatory potentials (OPs). An example record of dark-adapted OPs obtained from a normal C57BL/6J mouse are shown in the lower left panel of figure 81.2. After the onset of steady illumination, the relatively fast a- and b- waves are followed by a slower positive-going c-wave (not shown in figure 81.1). Other components of the ERG not shown in figure 81.1 will be described below.

The cellular origins of ERG components

Under dark-adapted conditions, the leading edge of the a- wave is generally associated with rod photoreceptor activity.13,14,39 The b-wave is associated with the combined activity of depolarizing bipolar cells and bipolar cell–dependent K+ currents affecting Muller cells.18,30,32,42,51,58 The cellular origin of the OPs is not completely understood, although they are likely generated by amacrine cells and other inner retinal cells interacting with bipolar and ganglion cells.19,25,35,54,55 The c-wave of the ERG is a corneal positive potential recorded across the retinal pigment epithelium (RPE) and results from an increase in the RPE’s transepithelial potential.16 Because of the technical difficulties in recording the c-

wave, it has not found general use in the mouse, although a recent studies have demonstrated its potential usefulness as an analytic tool (see, e.g., Wu et al.56).

Contributions from amacrine and ganglion cells have also been identified in the scotopic threshold response (STR), which is a negative-going potential in the dark-adapted ERG that is present at threshold and with dim illumination.49 The STR has been recorded from human, primate, cat, and rat retinas, but so far, there are no publications demonstrating such recordings from the mouse (for rats, see Bush, Hawks, and Sieving,5 and Sugawara, Sieving, and Bush53).

Rodand cone-mediated ERGs

The mouse retina is dominated by rod photoreceptors with a peak sensitivity at 510 nm, corresponding to the spectral absorption characteristics of rhodopsin. Estimates of cone percentages in the mouse retina range from 1% to 10%, with most studies suggesting that approximately 3% of photoreceptors are cones.7,29,47 Morphologically, the cones of the mouse are indistinguishable from those of higher mammals.7 Molecular biological, histological, and flicker electroretinographic results have established that mice have two cone photopigments: one peaking near 350 nm (UV-cone pigment) and a second near 510 nm (midwave [M]-cone pigment).23,24,53 ERG techniques for isolating the action spectra and absolute sensitivities of the UV-cone and M- cone driven signals have been described.27 The properties of the cone driven light-adapted murine ERG have also been described,9,37 as have regional variations in cone function.6

ERG responses obtained to dim flashes of light after a period of dark adaptation are generally presumed to derive from rod photoreceptors. Care must be exercised to ensure sufficient time to completely dark-adapt rods, as some recent studies suggest differences in the time necessary to achieve a fully dark-adapted state. Overnight dark adaptation is usually sufficient for most standard inbred strains. ERG responses obtained after a period of light adaptation are generally presumed to be driven by cone photoreceptors.

oscillatory potentials (OPs)

b-wave

flash onset

peak-to-peak

a-wave

50 V

25 ms

F 81.1 Representative ERG response to a bright flash obtained from a normal adult C57BL6 mouse. (See text for details.)

When the light stimulus does not emit a significant amount of ultraviolet (UV) light, then the response to a light flash on a rod-saturating background is mediated primarily by the middle-wavelength-sensitive cone (M-cone). Light sources with broad emission spectra are required to isolate the UVsensitive cone. As with humans, the period of light adaptation suppresses the contribution from rods, thereby yielding a cone-dominated signal. Further, presenting stimuli at a temporal frequency above the critical fusion frequency (CFF) for rods is an additional method that is sometimes used to isolate a cone response. In a recent study in our own laboratory, the rod CFF was found to be 6–7 Hz (Nusinowitz, unpublished data).

A note of caution should be added to the above discussion. Responses under a variety of conditions that theoretically suppress the contribution from rods do not always derive from cone photoreceptors. ERG studies in a mouse model of Leber’s congenital amaurosis (LCA) caused by mutations in the gene encoding RPE65, a protein vital for regeneration of the visual pigment rhodopsin in the retinal pigment epithelium, produce a severe retinal phenotype in which residual function is usually attributed to cones in the light-adapted state. However, in an elegant study, the rod system was shown to be the source of vision in the RPE65deficient mouse, not cones, even under conditions that would normally completely suppress rods.45

Representative rodand cone-mediated ERGs from a normal C57BL/6J mouse are shown in the upper left and right panels of figure 81.2. Each trace shows the response to a different light intensity, which is varied in 0.3 log unit steps. The b-wave of the rod-mediated response increases in amplitude, and implicit times (time from flash onset to peak of b-wave) are shortened with increasing intensity (compare

the heavy solid curves). The b-wave amplitude versus intensity (I-R) series for the rod-mediated responses is summarized in the inset. Note that the b-wave amplitude saturates at the highest intensities. The I-R series can be fitted with a Naka-Rushton function to obtain the maximum saturated b-wave amplitude, Vmax; the semisaturation intensity, k; a measure of sensitivity; and the ERG threshold intensity. In contrast, cone-mediated responses collected under the conditions employed in our laboratory increase in amplitude but have relatively constant timing (compare the heavy solid curve in the upper right panel of figure 81.2). The I-R series for the cone responses are shown in the inset. At flash intensities beyond those used to generate the data shown, cone amplitudes also saturate, thereby allowing fits with the NakaRushton equation as for rod-mediated function.

Basic ERG recording technique

In laboratories where mouse ERGs are currently recorded, different recording techniques (e.g., electrodes), methods of stimulating the eye (e.g., Ganzfeld versus Maxwellian view), and experimental protocols are employed (see, e.g., Green et al.,15 Marti et al.,28 Peachey et al.,38 Ruether et al.,44 Shaaban et al.,46 and Smith and Hamasaki50). In principle, the techniques for recording the ERG from the mouse are virtually identical to those used in human studies. The major components of a typical ERG system are (1) a light source for stimulating the retina, (2) electrodes for recording the signal generated by the retina in response to light, (3) a signal amplification system, and (4) a data acquisition system to accumulate, condition, and display data. Most commercial systems have integrated all of these elements into a single unit. A comparison of physiological recording systems that can be used for mouse ERGs are described in chapter 19. In addition, a comprehensive description of ERG recording methodologies can be found in Nusinowitz et al.34

Factors affecting the ERG

There are many variables that affect the ERG, and standard and consistent techniques are imperative to reduce the sometimes wide variability seen in mouse ERG recordings. Improper technique strongly affects the ERG and greatly reduces the reliability and reproducibility of data. Increased variability within ERG responses decreases the test’s ability to detect differences between strains and within strains over time, particularly when these changes are subtle. Variables that can affect the ERG include the improper use of anesthetics, variations in body temperature, insufficient dilation, inadequate light or dark adaptation, and prolonged testing, all of which can lead to a decrease in response amplitude. Location of the electrode on the eye can alter the amplitude of a signal by up to 30–40%, and a decrease in mouse body

F 81.2 Rod (upper left panel) and cone (upper right panel) ERGs obtained from a normal C57BL6/J mouse. Each trace displays the response to increasing light intensities. Upper left inset, Peak-to- peak amplitude versus retinal illuminance fitted with a Naka-Rushton function to obtain the maximum saturated b-wave amplitude, Vmax, the semisaturation intensity, k, and the rod ERG threshold. Right inset, Cone b-wave amplitude versus intensity series fitted with a

temperature by just a few degrees is associated with a virtually nondetectable ERG. Repeated flashing, commonly used in signal averaging, reduces rod-mediated (but not conemediated) ERG response amplitudes by about 20% at high flash intensities unless flash presentation rate is slowed to allow sufficient recovery of rod function. The consequences on the ERG of inappropriate control of extraneous variables are described in Nusinowitz et al.34 and Ridder et al.41

Specialized ERG recording techniques

By setting specific stimulus conditions, the ERG can be used to index the functional status of a wide range of cell types and can provide information to better understand the site and mechanisms of disease action (see figure 81.4 later in the chapter).

linear regression to derive the cone ERG threshold intensity. Lower left, A single ERG recording filtered to illustrate the major oscillatory potentials. Lower right, Representative a-wave ERG recordings to a range of flash intensities for a normal mouse. The smooth dotted curves are the fit of a rod model (see text for details) from which estimates for RmP3, the maximum saturated photoreceptor response, and S, a sensitivity parameter, were derived. (See text for details.)

Long-duration stimuli have been reported suitable for dissecting the contribution of ON and OFF bipolar cells to the photopic ERG.48 For example, while the photopic b-wave is largely generated by cones and the depolarizing ON bipolar cells, the activity of the hyperpolarizing OFF bipolar cells can limit the size and shape of the b-wave. These different components can be evaluated separately with long-duration flashes that produce distinct waveform components at flash onset and offset. While standard ERG recordings are in response to brief flashes less than 10 ms in duration, the separation of ON and OFF components requires longer flashes that are typically 100–200 ms in duration. Clinical application of the long-duration stimulus to such disease entities as congenital stationary night blindness31 and paraneoplastic night blindness1 have been reported in humans.

a-Wave analyses: Studies of activation and inactivation steps of phototransduction

Photoreceptor structure and function can be studied by analyzing the leading edge of the ERG (called the a-wave) obtained to bright flashes.3,4,8,20,21 Prior research suggests that current quantitative rod models have the potential to discriminate structural from functional abnormalities as the underlying mechanism of disease action in retinal disease (see, e.g., Birch et al.2 and Hood and Birch22). These techniques have been used extensively in the mouse.10–12,26,33,36,38,40,43,57 However, this type of a-wave analysis requires stimulus intensities that are substantially higher than are available with conventional photic stimulators. Intensities that clearly saturate the a-wave of the ERG are required. High-output xenon arc lamps and photographic flash heads can be adapted for this purpose and can provide intensities 2.0–4.0log units higher than a standard flash.

An example of recordings to high-intensity flashes is shown in figure 81.2 (lower right panel) for a normal mouse. Dark-adapted ERGs were recorded to blue light flashes up to 3.1 log scot td s in 0.3 log unit steps. The first 30 ms of each of the responses is shown in the figure. Note that the amplitude of the a-wave is fairly stable at the highest intensities and that the time to the peak of the a-wave is shortened. The leading edge of the rod a-waves was fitted with a model of the activation phase of phototransduction.22 The fit of the model to the raw data is indicated by the dotted lines. Generated by the model are three parameters: S, RmP 3, and td. S is a sensitivity parameter that scales flash energy. In general, any factor that decreases quantal catch or affects the gain at one or more of the steps involved in phototransduction will result in a reduction in the estimate of S. RmP3 is propor-

tional to the magnitude of the circulating current in the rod outer-segment membrane at the time of flash presentation.4,8,20,21 A number of factors can affect this circulating current, including the ionic driving force within the cell (perhaps determined by the number of mitochondria), the electrical resistance and/or leakage of the photoreceptor layer, immaturities in membrane proteins that mediate the permeability of the outer limiting membrane, and/or the density of light-sensitive channels distributed along the rod outer segment (ROS). The parameter td is a brief delay before response onset.

The kinetics of recovery to bright flashes can be studied using a two-flash technique. Recovery cannot be measured directly in the ERG because of the intrusion of postreceptor components. However, recovery can be inferred from the amplitude of the a-wave response to a second saturating test flash. An example of rod a-wave responses to a test flash at varying interstimulus intervals (ISIs) following a bright conditioning flash is shown in figure 81.3 (left panel). The test flash response in isolation is shown as the tracing labeled baseline. The other tracings show the a-wave response to the same test flash but with different ISIs ranging from 50 to 250 ms. Note that the a-wave amplitude increases as the ISI is elongated, consistent with rod functional recovery. Repetition of this two-flash paradigm with variations of the interval (ISIs) between the first and subsequent saturating flash allows determination of the recovery time course for a given conditioning flash intensity and Tc, the critical delay before the onset of recovery. Examples of normalized a- wave amplitudes to a test flash at varying ISIs are shown in figure 81.3 (right panel) for a dim and a bright conditioning flash (first flash). Note the faster recovery to baseline for the dim conditioning flash. We have previously reported that

patients with retinitis pigmentosa and a Pro23His rhodopsin mutation not only had a decrease in the gain of activation but also had significantly slower recovery times to bright saturating flashes.2 More recently, we have used this technique to demonstrate a slowed photoreceptor recovery following an intense bleach in albino mice with a MET450 variant in RPE65.34 A modification of this technique can be used to obtain the full time course of the rod response in vivo to test flashes of subsaturating intensity.17

Interstrain differences in ERG parameters for normal inbred mouse strains

Standard inbred mouse strains provide a stable genetic background for the study of specific genes and their role in retinal degeneration. However, little has been published about retinal function across (normal) inbred strains without known retinopathy when tested with a standardized protocol and at the same age. In our laboratory, we characterized retinal function in normal inbred mouse strains using the ERG to provide normative data for a broad range of physiological parameters. These data are intended to provide a standard against which transgenic and knock-out mice on similar backgrounds can be evaluated.

G T P We recorded intensityresponse series to evaluate both rodand cone-mediated function (examples are shown in the upper panels of figure 81.2). Rod-mediated ERGs were recorded to brief flashes of short-wavelength (W47A; 8max = 470 nm) light presented to the dark-adapted eye. Cone-mediated responses were obtained with white flashes on a rod-saturating background. The Naka-Rushton parameters, Vmax, the saturated b-wave amplitude, k, retinal sensitivity, and rod ERG threshold were derived from the intensity-response series. Oscillatory potentials (OP) were recorded in the dark-adapted eye using bright flashes of white light. Amplitude and timing were determined for each of the first four major OPs, as shown in figure 81.2. Flash intensity was extended to record rodmediated photoresponses in the mouse as shown in the lower left panel of figure 81.2. Rod photoresponse parameters were derived from the fit of a rod model to the leading edge of the a-wave. From these photoresponses, we derived RmP3, the maximum saturated photoreceptor amplitude, and S, photoreceptor sensitivity, as previously described.33 Finally, cone-mediated maximum amplitude and ERG threshold were also determined.

S S ERGs were recorded from 11 normal inbred mouse strains (mean age 13 ± 3 weeks). The strains investigated were C57BL/6J, NZB/BINJ, A/J, C57BL/6Jc2J, BALB/cJ, NZW/LacJ, AKR, CBA/CaJ, DBA/2J, DBA/1j, and LP/J. All mice were obtained from the Jackson Laboratory.

R -M R The results for the NakaRushton analysis of the b-wave intensity-response series are shown in figures 81.4 and 81.5. Across all strains, Vmax, the saturated b-wave amplitude, ranged from 194 mV (± 77) to 374 (± 99) mV. The albino strains on average produced the highest-amplitude signals, with marginally significant differences across strains (Vmax for albino strains = 314 ± 90 mV, P = 0.061). The two black strains on average produced amplitudes that were lower than those of the albino strains but that were not significantly different from those of either the albino or agouti strains (Vmax for black strains = 280 ± 63 mV, P = 0.395). However, the agouti, or mixed coat, strains produced saturated amplitudes with wide variation across the strains (Vmax for brown/agouti strains = 278 [± 99] mV, P = 0.0002). Multiple comparisons suggested that the largest difference among the agouti strains occurred for the CBA and LP/J strains (P < 0.01).

The retinal sensitivity parameter, k, derived from the Naka-Rushton fit, also varied across strains, ranging from 0.0015 ± 0.008 to 0.032 ± 0.033 scotopic td s (lower panel of figure 81.4). Again, the albino strains produced the lowest values of k (highest sensitivity), with significant variation across strains (k for albino strains = 0.0062 ± 0.008), P < 0.0001), the agouti strains producing the lowest sensitivity (k for brown/agouti strains = 0.0195 ± 0.009, P = 0.32) and the black strains producing intermediate sensitivity (k for black strains = 0.0166 ± 0.008, P = 0.10). In general, the parameters Vmax and k were loosely correlated, with the higher saturated amplitudes also producing higher sensitivity values. Finally, ERG threshold intensity and k were highly correlated, as shown in figure 81.5.

O P An analysis of the oscillatory potentials is shown in figure 81.6. The upper panel shows the summed amplitudes (OPsum) of the four dominant components of the ERG waveform. The lower panel shows the individual amplitudes for each of the four components. OPsum amplitudes were significantly different across the three coat colors. OPsum amplitude was 56.5 ± 35.0, 35.5 ± 13.0, and 79.8 + 28.0 mV for the black, albino, and brown/agouti strains, respectively (P < 0.0001). Surprisingly, the albino strains produced the lowest OP amplitudes despite generating the highest Vmax amplitudes. Statistically significant differences within the black and agouti strains were present, but no such differences were observed across the albino strains. Finally, the timing of OP peak components was not significantly different across all strains (P > 0.05).

a-W P As previously described,33 the leading edge of the rod ERG was fitted with a rod model to derive parameters of photoreceptor structure and function. The parameters, RmP3, the photoreceptor saturated amplitude, and S, photoreceptor sensitivity, were calculated. The results are shown in figure 81.7. An analysis of a-wave revealed that

F 81.4 Naka-Rushton parameters derived from rod-mediated intensity versus b-wave amplitude response series. Upper panel, Mean

(± 1 standard error) saturated b-wave amplitudes across strains. Lower panel, Mean (± 1 standard error) retinal sensitivity across strains.

F 81.6 Upper panel, Summed oscillatory potentials (± 1 standard error) across strains. Lower panel, Mean amplitude

(± 1 standard error) of each of the first four major oscillatory potentials.

F 81.7 Rod photoreceptor structure and function. Upper panel, Saturated a-wave amplitude (± 1 standard error) across

strains. Lower panel, Mean (± 1 standard error) values of photoreceptor sensitivity, S, across strains.

RmP3 was not statistically different across or within strains (mean RmP3 = 266.4 ± 89.0 mV, P = 0.342). In contrast, S, the photoresponse sensitivity, differed across the three coat colors. Log S was found to be 2.43 (± 0.21), 2.84 (± 0.18), and 2.45 (± 0.16) for the black, albino, and brown/agouti strains, respectively (P < 0.0001). In addition, significant differences were found among strains for the albino and agouti strains but not among the black strains (P > 0.10).

C -M R A summary of cone-mediated responses is given in figure 81.8. Cone maximal amplitude across all strains ranged from 32.9 (± 21.0) mV to 116.5 (± 32.0) mV. Statistically different responses were not found across the black strains (77.8 ± 32 mV, P = 0.014). However, substantial differences in cone amplitude were found for the albino strains (69.6 ± 34 mV, P < 0.0001) and for the brown/agouti strains (95.4 ± 29 mV, P < 0.0001). As shown in figure 81.9, cone maximum amplitude and ERG threshold intensity were highly correlated (r = -0.93, P < 0.0001). Finally, implicit time of the major b-wave peak was not significantly different across strains (44.0 ± 5.4 ms).

R - V C -M C A comparison of maximal rodand cone-mediated amplitudes is shown in figure 81.10. In general, rodand cone-mediated

amplitudes were correlated across all strains (r = 0.60, P < 0.0001). Within individual strains, correlations ranged from +0.43 to +0.93. However, for AJ and LP/j mice, rod function and cone function were uncorrelated. This means that for these strains, cone function could not be predicted from the responses that were rod-mediated. In addition, for strains with weak correlations, a high rod amplitude did not necessarily predict a high cone amplitude. For example, in figure 81.10, the albino strain with the highest rod amplitude (Vmax) had only intermediate cone amplitudes.

General conclusions

The electrophysiological and analytical techniques based on the ERG can be powerful tools to better understand the sites and mechanisms of disease action in mouse models of ocular disease. The ERG is a commonly used technique to assess panretinal function and can be dissected to quantify and evaluate the functional integrity of different retinal layers. The ERG has been used extensively to describe the retinal phenotype in mouse models of human retinal disease and has been used to evaluate the efficacy of a broad spectrum of genetic and pharmaceutical interventions. At the present time, however, there are no internationally accepted standards for recording ERGs in mice.

F 81.8 Upper panel, Mean (± 1 standard error) conemediated b-wave maximal amplitudes across strains. Lower panel,

Mean (± 1 standard error) cone ERG threshold intensity across strains.

cone w8la amplitude (log V)

F 81.9 Correlation between cone ERG threshold and the maximal cone b-wave amplitude across strains.

cone max amplitude ( V)

F 81.10 Rodversus cone-mediated function. Each data point represents the mean response with error bars for each strain. (See text for discussion.)

While there are abundant published ERG data on mutant mice, little has been published that documents retinal function across normal inbred strains without known retinopathy. In addition, little is known about how retinal function in the normal mouse alters during the aging process. Within the context of our understanding of the cellular origins of different components of the ERG, such studies could provide insights into which cells in the retina are most susceptible to aging and disease.

As a first step, we have characterized retinal function with the ERG across a range of normal inbred strains that are commonly used in vision science. While there were many subtle differences between strains that could be dismissed as normal variability, some differences were substantial and statistically significant, suggesting differences in cellular function. Some of the differences can be relatively easily

explained. For example, the albino strains were generally more sensitive to light than the pigmented and agouti strains. These differences in sensitivity are largely due to differences in ocular melanin, which absorbs light, thereby reducing the amount of light reaching the photoreceptors. The underlying mechanisms responsible for other differences in ERG parameters are not yet understood. Ongoing work in our own laboratory is focused on determining the reliability and replicability of these differences, how ERG parameters are altered during aging, and the underlying cellular variations that could cause such differences.

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48.Sieving P: Photopic “ON” and “OFF” pathway abnormalities in retinal dystrophies. Trans Am Ophthalmol Soc 1993; 91: 701.

49.Sieving PA, Frishman LJ, Steinberg RH: Scotopic threshold response of proximal retina in cat. J Neurophysiol 1986; 56:1049.

50.Smith SB, Hamasaki DI: Electroretinographic study of the C57BL/6-mivit/mivit mouse model of retinal degeneration.

Invest Ophthalmol Vis Sci 1994; 35:3119.

51.Stockton RA, Slaughter MM: B-wave of the electroretinogram: Reflection of ON bipolar cell activity. J Gen Physiol 1989; 93:101.

52.Sugawara T, Sieving PA, Bush RA: Quantitative relationship of the scotopic and photopic ERG to photoreceptor cell loss in light damaged rats. Exp Eye Res 2000; 70:693.

53.Sun H, Macke JP, Nathans J: Mechanisms of spectral tuning in the mouse green cone pigment. Proc Natl Acad Sci U S A 1997; 94:8860.

54.Wachtmeister L: Oscillatory potentials in the retina: What do they reveal. Prog Retin Eye Res 1998; 17:485.

55.Wachtmeister L, Dowling JE: The oscillatory potentials of the mudpuppy retina. Invest Ophthalmol Vis Sci 1978; 17:1176.

56.Wu J, Peachey NS, Marmorstein AD: Light-evoked responses of the mouse retinal pigment epithelium. J Neurophysiol 2004; 91(3):1134–1142.

57.Yang RB, Robinson SW, Xiong WH, Yau KW, Birch DG, Garbers DL: Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior, J Neurosci 1999; 19:5889.

58.Xu XJ, Karwoski CJ: Current source density analysis of retinal field potentials: 2. Pharmacological analysis of the b-wave and m-wave. J Neurophysiol 1994; 72:96.

L with retinal dystrophy are increasing in importance as models of human conditions, particularly for the testing of therapeutic strategies. Spontaneously occurring retinal dystrophies are recognized in both dogs and chickens. Hereditary disease that leads to vision loss in the dog is also important because this species plays a valuable role in human life, not only as a working and service animal, tasks for which vision is required, but also as a companion animal. In addition to its use as a laboratory model for the study of retinal disease, the chicken is widely used for the study of ametropias.

This chapter will elucidate some practical aspects of canine and chicken ERGs, give examples, and show some of the changes that can be seen in inherited retinal diseases.

The canine electroretinogram

The dog is becoming more widely recognized as an important model for human retinal dystrophies. There are a number of different spontaneously occurring hereditary retinal degenerations in the dog, and this coupled with the fact that the dog eye is very similar in size to the human eye makes the canine retinal dystrophies important for the study of retinal dysfunction and the therapeutic approaches to save vision.

Electroretinography is commonly performed on dogs by veterinary ophthalmologists to investigate retinal disease in working and companion dogs and by researchers utilizing the dog as a model for human disease. The increased utilization of the dog as a model of human disease demands that the normal canine electroretinogram (ERG) is fully characterized so that this technique can be most effectively utilized.

There are some important differences between the dog and human retina. For example, the dog does not appear to have an area with predominance of cones and does not have a fovea. The dog retina does have a region called the visual streak (or area centralis) in which there is greater ganglion cell density,58 and it is this region that is used for most detailed vision. As with humans, there are many more rods in the canine retina than cones, but the exact rod-to-cone ratio has not been reported. There are some reports describing how the distribution of cones varies across the retina

although the results vary between the publications (see the review by Miller and Murphy42). The dog has dichromatic color vision having two types of cone: one with peak spectral absorbance at 429–435 nm and the other at 555 nm.31,53 Peak spectral absorption of the canine rod photopigment rhodopsin is 508 nm.31

The following section gives examples of ERGs in the normal dog and in dogs with hereditary retinal disease.

M R C ERG Techniques used to record the canine ERG are described in chapter 83. The ERGs shown in the current chapter were recorded from anesthetized dogs (typically sedated with acepromazine, induced with thiopentone, and anesthesia maintained with halothane or isoflurane delivered in oxygen). Although anesthetic agents can alter the ERG responses,36,84 anesthesia is required to immobilize the dog. Ganzfeld flash ERGs were recorded with an LKC UTAS E-3000 electrophysiology unit (LKC Technologies, Gaithersburg, MD). Although 20 minutes of dark adaptation is recommended by the standards established by the European College of Veterinary Ophthalmologists (ECVO) ERG committee,51 we find that dark-adapting the eye for at least 45–60 minutes results in greater amplitudes in the scotopic ERG. We have also shown that if a dog is examined by indirect ophthalmoscopy or fundus photographs are taken immediately prior to the ERG session, the period of dark adaptation time must be increased to at least 60 minutes to achieve ERG amplitudes comparable to those that can be recorded after 20 minutes of dark adaptation76 (if examination with bright lights had not been performed.) Typically, the pupil is maximally dilated by topical application of tropicamide or a combination of tropicamide and phenylephrine. The pupil diameter is monitored before and after the procedure because some drugs used for premedication or anesthesia can result in pupillary constriction and some individual dogs develop miosis after anesthesia, despite repeated application of mydriatics.

The selection of recording lens can influence the amplitudes and also, to some extent, the shape of the ERG recorded from dogs. In a study comparing three different electrodes, we found that significantly higher amplitudes were recorded using either a DTL fiber electrode or an ERG-Jet lens electrode compared to the amplitudes

recorded by using a bipolar Burian-Allen lens.40 This finding differs from the results obtained in human patients, in which the Burian-Allen lens tended to result in greater amplitudes compared to the other electrodes.21 Additionally, we demonstrated that in using monopolar electrodes, choosing a consistent position of the reference electrode is very important. When we compared amplitudes obtained by using the ERGJet lens electrode and a skin reference electrode positioned 1, 3, or 5 cm caudal to the lateral canthus of the eye we found that between the three reference electrode positions, the further caudally the electrode, the greater the amplitude.40 A study of canine perfused eyes found that the greatest ERG could be recorded in that system with a corneal contact electrode when the reference electrode was placed on the posterior sclera adjacent to the optic nerve.17 The lack of a complete bony wall to the lateral orbit in the dog may mean that there is less electrical resistance between a skin electrode positioned more posteriorly and the current generated in the retina than there would be in a human with a complete bony orbit, given that bone offers greater electrical resistance than soft tissues. These studies demonstrate the importance of standardization of equipment and technique if ERGs are to be comparable.

In addition to the effect of recording technique on ERG amplitudes, the breed and age of dog are important. Dogs represent one of the most phenotypically diverse species (compare a chihuahua with a Great Dane, for example), so it is perhaps not surprising that normal ERG amplitudes can differ considerably between breeds of dog. Amplitudes also tend to decrease with age. For any study using a particular breed of dog, it is important that an appropriate breedand age-matched control is available, particularly in trying to detect the early stages of retinal disease.

M C ERG The retina of the dog is not fully developed at birth. Retinal maturation occurs over the first couple of months of age (figure 82.1). When the eyes of puppies open at between 10 and 14 days of age, the ERG is just a low-amplitude negative waveform in response to brighter flashes of light. By 4 weeks of age, the ERG waveform is adultlike with an a-wave, a b-wave, and oscillatory potentials. Maximal amplitudes are achieved by about 7 weeks of age and appear to decrease slightly after that age as the animal matures; a- and b-wave response thresholds are at their lowest intensity by a similar age (7 weeks).

C C ERG In addition to the a- and b-waves and oscillatory potentials (figure 82.1), other components of the canine ERG have been described. The scotopic threshold response (STR) can be recorded under specific recording conditions with good dark adaptation85,86 but can be reduced by the use of some anesthetic agents.84

The early receptor potential described in other species has not been reported in the dog, probably because of the technical difficulties in recording this response.48 Similarly, there are no reports of investigation of the canine M-wave. Averaging several flashes of red light has been reported to allow the separation of a cone-derived x-wave from the b-wave.50 A photopic i-wave can be seen in the dog,66 although we have found that the selection of recording electrode is important in being able to record this response. See figure 82.5C later in the chapter for an example of a canine i-wave. Consideration of the d-wave is included in the section about canine long-flash responses.

A C ERG Canine ERGs can be analyzed similarly to those of other species. a- and b-wave amplitudes and implicit times can be measured. Soctopic and photopic intensity-response curves can be plotted, and a- and b-wave thresholds can be calculated. To allow for consistent comparison, a criterion threshold value can be selected. The Naka-Rusthon formula can be applied to fit the b-wave intensity-response curve and used to derive a value for maximal rod photoreceptor response (Vbmax), and a value for retinal sensitivity (k) may be calculated (k = the intensity that gives a response of 1/2 Vbmax).

L -F ERG D Figure 82.2 shows a typical example of a canine long-flash ERG. The stimulus for photopic ON-OFF recordings (long-flash ERGs) were as described by Sieving.73 A stimulus of 200 cd/m2 (typically, a 150to 200-ms flash duration was chosen) was presented on a background light of 34 cd/m2. The ON response has typical a- and b-waves. Following the b-wave is a plateau that varies between slightly rising (as in figure 82.4 later in the chapter) and slightly decreasing. At the onset of the OFF response, there is often a small positive deflection that is then followed by a large negative deflection similar in amplitude to the b-wave. At the trough of the negative deflection, there is a small positive deflection followed by a large positive waveform that returns to baseline. The OFF response of the dog is thus similar to that of the rat.49 This represents a response from what Granit25 classified as an excitatory (E- type) retina rather than an inhibitory (I-type) retina as in the human and the chicken (see below). It is not clear whether the small positive wave seen at the onset of the OFF response represents the equivalent of the d-wave recorded in other species, such as primates, or whether the large negative response is the equivalent of the d-wave.

E ERG C C R D -

Canine retinal dystrophies include models for retinitis pigmentosa and for Leber congenital amaurosis. The retinitis pigmentosa models in dogs are known as the progressive retinal atrophies and occur in several different

F 82.1 Maturation of the canine ERG. The tracings are from the same dog. Panels A, B, and C show scotopic intensity series responses, while panels D, E, and F are photopic intensity

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F 82.2 Long-flash ERG from a dog. This shows the photopic long-flash ERG from a normal adult dog. This is the result of a 150-ms flash of 200 cd/m2 superimposed on a background light of 34 cd/m2. The ON response is characterized by an a- and a b- wave. There is a plateau region while the light remains on. The OFF response is characterized by a small positive deflection followed by a large negative response, similar in amplitude to the b- wave. At the trough of the negative OFF response, there is often a small positive deflection, which is followed by a larger positive waveform that passes the baseline before returning to baseline.

series responses. Panels A and D are at 2 weeks of age, panels B and E at 7 weeks of age, and panels C and F at 24 weeks of age. Arrowheads indicate the onset of flash.

breeds of dog. The progressive retinal atrophies show genetic heterogeneity. The gene mutations underlying some of the forms have been identified. Phenotypically, the progressive retinal atrophies can be divided into earlyand lateonset forms.

Progressive retinal atrophies Some of the early-onset forms are given the phenotypic description rod-cone dysplasias because photoreceptor development becomes halted during development, and this is followed by a rapid loss of rod photoreceptors with a much slower loss of cones. The first form was described in the Irish setter breed and is known as rodcone degeneration type 1 (rcd1), 9,14 the second form was described in the collie (rcd2),81 and the third form was described in the Cardigan Welsh corgi (rcd3).32 Rcd1 is caused by a point mutation resulting in a premature stop codon in the gene encoding the rod cyclic GMP phosphodiesterase beta subunit (PDE6B).16,74 The gene mutation underlying rcd2 has not been identified, and that underlying rcd3 is a 1-bp deletion in the gene encoding the rod cyclic

GMP phosphodiesterase alpha subunit (PDE6A) leading to a premature stop codon.60 The mutations in PDE6A and PDE6B are most likely functional null mutations. The rod cyclic GMP phosphodiesterase enzyme requires the presence of both alpha and beta excitatory subunits for normal activity. When one subunit is missing or is nonfunctional, phosphodiesterase activity is much reduced,62 so the substrate cyclic GMP accumulates. In similar animal models, such as the rd1 mouse, it has been shown that the accumulation of cyclic GMP and the resulting opening of an increased proportion of cyclic GMP-gated channels in the cell membrane trigger apoptosis in the rod photoreceptor.34 Cones, although genetically normal, are affected by the loss of surrounding rods and are halted in their development and then degenerate slowly.9,14,15 Figure 82.3 shows representative scotopic and photopic ERG intensity series from an rcd3- affected dog. The rcd3 dogs lack rod-mediated responses at all ages. This very-early-onset abnormality suggests, as would be expected given the lack of functional rod cyclic GMP phosphodiesterase alpha subunit, that normal rod phototransduction does not develop in the affected dogs. The developmental abnormalities of cones are reflected functionally in a significant reduction of the photopic a-wave amplitude as early as 3 weeks of age (figure 82.3F). The photopic b-wave of the affected dogs is not significantly different from that of breed-matched normal controls until later in the disease process.61 Although cone function is abnormal

from a very early age, the loss of cones is relatively slow (unpublished histological findings), and sufficient vision in good lighting conditions to allow negotiation of obstacle courses is maintained for two to four years. It is of note that only a small photopic b-wave remains in these animals, and histological examination of the retina of affected dogs in this age range that still have some residual vision reveals that they have only isolated areas with residual cone photoreceptor cells apparently with just stunted inner segments.

Of the later-onset forms of progressive retinal atrophy in dogs, one form called progressive rod-cone degeneration ( prcd ) is known to be present in several different breeds.3–5 Prcd maps to canine chromosome 9,2,24,41,72 although the actual gene defect underlying it had not been published at the time of writing. In prcd, the photoreceptors mature normally, but then there is a progressive rod-led loss of photoreceptors.6 The loss of photoreceptors is reflected in a progressive reduction in ERG amplitudes. Figure 82.4 shows an example of a dog presented for early diagnosis of prcd. The rod responses are affected first,8,67 but changes are not seen until after normal maturation and probably reflect a loss of total number of photoreceptors rather than a generalized abnormality in photoreceptor function. The shape of the darkadapted waveforms are essentially normal; however, the response threshold is elevated, and the a- and b-wave amplitudes are both reduced to a similar extent (compare figure 82.4B with figure 82.4A). The photopic responses are still

scotopic (A–D) and photopic (E–H) inensity series responses from a normal control dog (A, C, E, and G) compared to a

PDE6A mutant/rcd3 dog (B, D, F, and H) at 3 weeks (A, B and E, F) and 7 weeks (C, D and G, H) of age. Arrowheads indicate the onset of flash.

well preserved at this stage (compare figure 82.4D with figure 82.4C).

Dog model of Leber congenital amaurosis Leber congenital amaurosis (LCA) is an early-onset severe retinal dystrophy with vision loss in childhood.59 Similarly to retinitis pigmentosa, it shows genetic heterogeneity.28 LCA type II results from mutations in RPE65,19,26,46,75 a gene encoding a 65-kDa protein expressed in retinal pigment epithelium (RPE) that plays a role in the visual cycle.64 A spontaneously occurring dog model of LCA type II in the briard breed has been investigated.52,82,83 The original colony that was characterized and in which the mutation in RPE65 was identified was established in Sweden; hence, the name Swedish briard dog came into use, although “Swedish briard” is not a separate breed from the briard. The mutation identified in the briard dog is a 4-bp deletion leading to a premature stop codon.78 The affected dogs have a lack of dim light vision and a variable degree of daytime vision loss. Studies in mice

F 82.4 Scotopic (A and B) and photopic (C and D) intensity series responses from a prcd dog (B and D) compared to a normal control (A and C). The dog being tested had been observed to have some subtle bilateral fundoscopic changes that were not considered to be diagnostic for PRA and was referred for ERG testing. The ERGs clearly showed a marked decrease in ERG amplitudes compared to the normal control dog, showing that the test dog had a generalized retinal dysfunction. Arrowheads indicate the onset of flash.

have shown that the lack of RPE65 protein activity means that 11-cis-retinal is not recycled from the RPE to the photoreceptors.64 There is therefore a lack of formation of visual pigment and a resultant failure in normal rod phototransduction.64 In affected dogs, there is only a slow degeneration of photoreceptors, and as the animals get older, there is a progressive accumulation of retinyl esters in the RPE.82,83 The lack of phototransduction is reflected in the ERG of affected dogs,54 as can be seen in the representative ERGs in figure 82.5. Comparison of dark-adapted with light-adapted ERG waveforms in the RPE65 dog reveal that the intensitymatched responses are very similar in amplitude, suggesting that the majority of the response recorded in the RPE65 mutant dog at this age is cone-mediated; similar findings are reported in children with RPE65 mutations37 and RPE65 knock-out mice.64 An ERG study using RPE65 knock-out mice suggested that the mutant mice have a pronounced loss of UV cone function with preservation of M cone function.20 Similar studies have not been reported in dogs. However a study utilizing double knockout mice that had a lack or functional RPE65 with either a lack of functional

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F 82.5 Scotopic (A and B) and photopic (C and D) intensity series responses from an RPE65 mutant dog (B and D) compared with a normal control (A and C). Arrowheads indicate the onset of flash.

rods or a lack of functional cones showed that the major ERG response was due to a response from desensitized rods,68 although it was subsequently shown that a small cone derived response was also recordable.65

O C M This article has given only some selected examples of ERGs of dog retinal dystrophy models. There are other canine retinal dystrophy models in which the gene mutation has been described, such as X-linked PRA (due to mutations in RP GTPase regulator; RPGR mutations),87 dominantly inherited PRA (due to a mutation in the rod opsin gene),33 and achromatopsia in the cone dysplasia dog (due to a mutation in cyclic nucleotide-gated channel beta-subunit gene; CNGB3).10,68,69 There are also other canine models that have been phenotypically characterized but for which the causal gene mutation has not been identified, such as the rod dysplasia (rd ) Norwegian elkhound,7 the early retinal degeneration (erd ) Norwegian elkhound,1 and the photoreceptor dysplasia (pd ) miniature schnauzer.57 Many other naturally occurring canine retinal dystrophies remain to be characterized. The publishing of the canine genome35 and the development of tools for disease mapping mean that it is becoming progressively easier to identify the gene mutations underlying the myriad of genetic diseases that occur in pure-bred dogs. This will provide the opportunity to establish further colonies of experimental dogs for study and investigation of potential therapies for homologues of human disease.

S This article has emphasized the canine ERG in hereditary retinal disease. ERG studies in dogs are performed for other reasons, such as the investigation of ocular disease in veterinary medicine and in toxicology studies. Although the ERG of the dog has been well described in several studies, there is a need for further characterization, particularly in view of the emergence of the dog as an important model species of human diseases.

Electroretinography in the chicken

There are a few spontaneously occurring retinal dystrophies in chickens, although thus far, the gene mutation underlying only one of these diseases has been identified.

The chicken retina has a number of differences from the human retina. The retina is avascular, receiving its nutrition from the vitreous and via the choroid. Birds have a highly vascular structure called the pecten that protrudes from the surface of the optic nerve head into the vitreous. There are several theories as to the purpose of the pecten, one of which is that it is involved in supplying nutrition to the inner retina via the vitreous. The chicken has a relatively cone-rich retina compared to humans and canines. Most of the cones

contain colored oil droplets within the photoreceptor inner segments. These droplets act as spectral filters for the light that reaches the outer segments. The cones are divided into single cones and double cones. Double cones consist of a principal cone (similar in structure to a normal single cone), which contains an oil droplet, and an accessory cone, which curves around the inner segment of the principal cone and, according to some authors, only rarely contains a miniscule oil droplet,11 whereas others report that it has one or more oil droplets.79 On the basis of morphological appearance, three separate forms of double cone have been described.80 Four cone visual pigments are recognized: ultraviolet, shortwavelength, middle-wavelength, and long-wavelength. A different type of oil droplet is matched with each of the four visual pigments.11 The visual pigment in both members of the double cone is the same as that found in the long- wavelength-sensitive single cone. The reported proportion of each of the photoreceptor types in the chicken retina differs between studies. For example, Bowmaker and Knowles12 reported that double cones account for 50–60% of photoreceptors, whereas Morris47 reported that in the central retina, the ratios were 14% rods, 32% double cones, and 54% single cones and peripherally, 33% rods, 30% double cones, and 37% single cones.

M R C ERG The chicken ERGs shown in this chapter were recorded from anesthetized chickens (typically with isoflurane delivered in oxygen). The flash ERGs were recorded by using an LKC Utas 3000 electrophysiology unit (LKC) with a Ganzfeld unit. A bipolar Burian-Allen lens (Hansen Ophthalmic Development Laboratories, Coralville, Iowa) was used with the ground electrode placed in the hind leg. Conjunctival stay sutures were used to stabilize the globe and keep the recording lens in position. Hypromellose (0.25–0.5%) was used as a coupling solution for the lens. Birds have striated muscle in their iris; therefore, the pupil does not dilate with the topical parasympatholytic or sympathomimetic drugs used in mammals. Topical neuromuscular blocking drugs were used to induce mydriasis; for example, topical 1% vecuronium bromide given 20 minutes prior to the procedure.

N C ERG The chick retina is well developed at hatch. The ERG can be recorded from the chick embryo by the eighteenth day of incubation.55 After hatch, the ERG continues to mature over the first week or so.56 We have found that over the first 2–3 weeks post hatch, the a- and b-wave thresholds decrease. Figures 82.6A and 82.6C show representative scotopic and photopic ERG tracings, respectively, from a normal 7-day-old chick. The cone dominance of the chicken retina means that there is a significant cone component to the dark-adapted ERG, particularly

F 82.6 Representative scotopic (A and B) and photopic (C and D) intensity series responses from normal (A and C) and rge (B and D) 7-day-old chicks. There is a major cone component to the dark-adapted ERG in response to brighter flashes of light. The rge chicks have elevated response thresholds, a lack of oscillatory potentials, and enhanced b-waves to brighter flashes of light. The photopic and scotopic ERGs are very similar, indicating that rod function is more severely affected than cone function at this stage of the disease. Arrowheads indicate the onset of flash.

those in response to brighter flashes. The light-adapted responses are of proportionally greater amplitude than would be recorded from a species with a rod-dominated retina (e.g., Mears et al.39).

L -F ERG The stimulus for the photopic ON-OFF recording (long-flash ERGs) was as described by Sieving.73

and OFF responses shows that the chicken has a positive OFF response (d-wave) (figure 82.7) similar to that of

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F 82.7 Long-flash ERG of a chicken. A photopic ON-OFF response to a 150-ms flash (200 cd/m2) superimposed on a white background light of 34 cd/m2. The ON response consists of an a- and a b-wave followed by a plateau. The OFF response consists of a positive d-wave typical of the response from an I-type retina.

humans and primates (an I-type response) but unlike rats and dogs.

C ERG D The ERG changes that characterize some of the different forms of hereditary retinal degeneration have been reported in the chicken. These include retinal degeneration (rd ), retinal dysplasia and degeneration (rdd ), delayed amelanotic (dam), and retinopathy, globe enlarged (rge).

The rd phenotype is caused by a null mutation in the photoreceptor guanylate cyclase (GUCY2D) gene and is therefore a model of Leber congenital amaurosis type 1 in humans.69 The rd birds have a severe phenotype with nonrecordable ERGs under lightor dark-adapted conditions from the time of hatch.77

The rdd chicken phenotype is sex-linked and characterized by a progressive degeneration of the retina, culminating in blindness. By 3 weeks of age, homozygotes have a flat ERG, indicative of their severe loss of visual function.63 Linkage analysis mapped the rdd locus to a small region of the chicken Z chromosome with homologies to human chromosomes 5q and 9p.13

The dam chicken is characterized by a postnatal, spontaneous cutaneous amelanosis and a high incidence of blindness.22 The main ERG change observed in this phenotype is a generalized decrease in a- and b-wave amplitudes.23

Retinopathy, globe enlarged chick (rge) is autosomalrecessive18 and is due to a mutant locus on chicken chromosome one.30 Rge chicks have unusual ERG changes.44 The homozygous affected chicks have reduced vision, particularly in dim light, from hatch and lose functional vision at about one month after hatch.44 The retina slowly degenerates but has some early ultrastructural abnormalities of photoreceptor synaptic terminals.43 The ERGs are abnormal in shape from hatch, slowly deteriorate, and yet maintain relatively large amplitudes for some months after functional

vision loss.45 Examples of the affected birds’ scotopic and photopic ERGs are shown in figures 82.6B and 82.6D, respectively. a- and b-wave thresholds are elevated for both the darkand light-adapted ERGs (figure 82.8). The shape of the b-wave is abnormal, partly owing to the lack of oscillatory potentials. Interestingly, in response to very bright flashes of light, both the scotopic and photopic b-wave amplitudes of rge birds are supernormal for the first 6 or 7 weeks of age (see figure 82.8). Supernormal ERG amplitudes are reported in certain retinal dystrophies, including enhanced S-cone syndrome.29,38 In this condition, there is a lack of rods and a marked increase in numbers of S-cones.27 However, histological studies showed that rge chicks do not have marked alterations in photoreceptor ratios.43 Drug dissections of the ERG of rge chicks revealed that intravitreous APB (2-amino-4-phosphonobutyric acid) does not block this abnormal b-wave. In the normal control chick, APB almost completely eliminates the b-wave (figure 82.9). This finding suggests that the “b-wave” of rge chicks may be generated

differently from the normal chicken b-wave or from different cellular components. The true basis for the ERG changes in this interesting dystrophy remains to be elucidated.

S The chicken ERG reflects the fact that this species has a cone-dominated retina. Spontaneously occurring retinal dystrophies have been described in this species. The only one of these to be characterized at the molecular level, the rd mutation, provides a model for Leber congenital amaurosis type 1. Other chicken retinal dystrophies have been characterized to varying degrees at the phenotypic level, and the chromosomal locations of the underlying genetic mutation of some forms have been mapped. The presence of naturally occurring retinal dystrophies in chickens, coupled with the large size of the eye, the cone-dominated retina, and the ease of access and manipulation of the embryo, makes this an attractive model system for studying retinal gene function in higher vertebrates.

Intensity (log cd-s/m²)

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F 82.8 Scotopic and photopic a- and b-wave intensityresponse and implicit time plots form normal and rge chicks at 7 days of age. Mean scotopic (A) and photopic (C) a-wave amplitude and implicit times and mean scotopic (B) and photopic (D) b-wave amplitude and implicit times. In each graph, the black solid curve represents the mean intensityresponse curve of the rge birds, and the black dashed curve represents that of the control birds. The gray solid curve represents

the intensity-implicit time curve of the rge birds, and the gray dashed curves represent the a-wave implicit time curve from normal birds. Note the increased a- and b-wave threshold of the rge birds compared to control birds. The scotopic and photopic b- wave amplitudes are supernormal in response to the brighter light intensities (above 1.4 log cd s/m2). Seven control and seven rge birds’ standard error bars are shown. (* = a significant difference at P < 0.05.)