VIII OTHER PROTOCOLS FOR RECORDING OF ERG AND SLOWER POTENTIALS, TECHNICAL ISSUES, AND AUXILIARY TESTING TECHNIQUES

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T (ERP) was first discovered by Brown and Murakami,3 who were recording a local electroretinogram (ERG) with an electrode placed within the photoreceptor layer of the monkey retina and were stimulating with a very bright light. What they saw (figure 40.1) was a rapid response having no detectable latency, which they called the early receptor potential, or early RP, followed by the a-wave of the ERG. The polarity of the ERP was the same as that of the a-wave of the ERG, which in their experiments was positivegoing. They then showed that the ERP was remarkably resistant to anoxia, suggesting that it was produced not by activation of the transduction cascade, but rather by some other movement of charge within the photoreceptor.

Richard Cone4 subsequently demonstrated that the ERP could be detected with the same configuration of electrodes and amplifier used to measure the ERG. The polarity of the major component of the ERP was again the same as that of the a-wave, but when the active electrode was placed at the cornea or in the vitreous, both the ERP and the a-wave were negative-going (figure 40.2). Cone also demonstrated that in the rat, the spectral sensitivity of the ERP matched that of the rod pigment and that the amplitude of the major component of the ERP increased nearly linearly with the intensity of the stimulus and saturated at about the light level required to bleach all of the pigment in the photoreceptor. These observations showed that the ERP is produced directly by the photopigment, probably by the movement of charge within the rhodopsin molecule that is triggered by the changes in conformation produced by bleaching.

Once it became feasible to make intracellular recordings routinely from vertebrate photoreceptors, it was possible to show that the electrical events that are responsible for the ERP can be produced in both rods15 and cones11 and that they have properties in single receptors identical to those originally inferred from measurements of whole retina. In most recordings, the ERP can be seen to have two components, usually called R1 and R2 (figure 40.3). The R1 component is an initial depolarization with a latency less than 0.5 ms5 or perhaps even smaller.19 The R2 component is hyperpolarizing, develops with a longer latency, and is usually considerably larger in amplitude. It is this R2 component that is responsible for the signal that Brown and Murakami originally recorded (see figure 40.1).

Since the ERP can be detected intracellularly from single photoreceptors as a change in membrane potential (as in

figure 40.3) or as a membrane current from cells that have been voltage clamped,10,12,13 we may conclude that it is caused by the movement of charge with some component perpendicular to the plane of the plasma membrane. Because the size of the ERP is proportional to the number of pigment molecules bleached by the stimulus, it is apparently produced by changes in the conformation of the rhodopsin molecule that move either charged amino acids or associated bound charges (such as H+) across the plasma membrane. A simple calculation shows that the amplitude of the R2 component can be accounted for by the movement of a single charge a distance of a few angstroms.7,10,11,13 The amplitude of the ERP would be expected to be larger in cones than in rods, since all of the photopigment in a cone is embedded in the plasma membrane, whereas for rods, only a small fraction of the rhodopsin lies in the plasma membrane or in the few basal disks that are continuous with the plasma membrane. This would explain why, in humans, the rods make a smaller contribution to the ERP of the whole retina (see, e.g., Goldstein and Berson9 and Sieving and Fishman17), even though rods are nearly 20 times more numerous than cones.

Because the R1 and R2 components have different time courses and are of opposite polarity, they are likely to be produced by different conformational changes of the rhodopsin molecule. They are easily separated from one another by reducing temperature, which blocks R2 (see, e.g., Pak and Ebrey16), and they have been extensively studied.7 The R1 component is probably produced by some change that occurs between the absorption of a photon and the formation of the pigment intermediate metarhodopsin I.5,6,16 The R2 component, on the other hand, probably reflects the movement of charge during the transition from metarhodopsin I to the active intermediate metarhodopsin II or R*. These charge movements are reversible.2,5 That is, one flash of light can be used to convert rhodopsin to metaI, producing R1, and another can then be given to photoreverse the metaI back to rhodopsin, producing a charge movement that is identical in waveform to R1 but opposite in sign.

Clinical application of ERP

The ERP has been used for the most part in basic research, to study aspects of photopigment function. Because the

Early RP

0.5 mV

2 msec

a-wave

4 msec

F 40.1 Discovery of ERP. The traces give same recording at two different sweep speeds. The records show potential recorded from the retina of a cynomolgus monkey (Macaca irus) with a tungsten microelectrode inserted into the layer of the photoreceptors. The reference electrode was placed in the vitreous. Notice that with this configuration, the a-wave is positive-going, opposite in polarity to the a-wave of the ERG recorded conventionally with a corneal electrode (see figure 40.2). Photoreceptors were stimulated with bright light from a condenser-discharge lamp. Stimulus artifact appears as break in record in upper trace. (Modified and reprinted with permission from Brown and Murakami.3)

100 V

1 msec

F 40.2 ERP recorded from a 38-year-old man with ocular siderosis. The recording was made with monopolar corneal contact lens. The patient had sustained traumatic injury to his right eye by penetration of a metal fragment containing a high iron concentration. Even though the a- and b-waves of the involved eye were substantially reduced (not shown), the amplitude of the ERP in the right eye was approximately the same as that in the left (uninjured) eye. (Modified and reprinted with permission from Sieving et al.18)

R1

+5

mV

-5

-10

msec

F 40.3 Intracellular recording of ERP from turtle cone stimulated with xenon flash. The upper trace shows the waveform of the flash. Note the two components of the ERP labeled R1 and R2. (Modified and reprinted with permission from Hodgkin and O’Bryan.11)

amplitude of the ERP is proportional to pigment concentration, measurement of ERP provides a convenient way to quantitate pigment concentration in the living retina and can, for example, be used to determine the photosensitivity of the visual pigment.12,13

Because the ERP can be measured with the same apparatus that is used to measure the ERG, it is possible to record the ERP from patients in a clinical setting (see, e.g., Goldstein and Berson,9 Sieving and Fishman,17 and Walther and Hellner20). The proportionality of ERP amplitude with the amount of photopigment makes possible the use of the ERP to assess the general health of the photoreceptors. Several groups have reported, for example, a decrease in the size of the ERP in humans affected with inherited retinal disease (see, e.g., Goldstein and Berson9 and Muller and Topke14). It is unclear, however, whether measurements of ERP are more accurate or more informative in the assessment of retinal function than is the ERG or even simpler tests, such as tests of visual acuity.

There are, however, two circumstances for which the ERP could be particularly useful in a clinical setting. Suppose, for example, that a patient exhibited a decrease in visual acuity and ERG amplitude with no evidence of degeneration or other anatomical lesion. One possible explanation might be that the photoreceptor outer segments were undamaged but that the patient was experiencing some difficulty either with the mechanism of transduction by the rods and cones or with synaptic processing by interneurons in the retina. The measurement of ERP might then be useful, since if the ERP amplitude were normal, it would indicate that no impairment of distribution of photopigment in the outer segment plasma membrane had occurred. The ERP was used in just this way by Sieving et al.18 in a study of human ocular siderosis (see figure 40.2).

The ERP might also be helpful in assessing the general health of the photoreceptors in selecting patients for genetic

therapy. Imagine a future in which all of the difficulties of molecular gene therapy had been solved, so it had become possible to introduce foreign genes into the eyes of patients who had lost or were losing visual function from genetically inherited retinal degeneration. Recent experiments on a canine model for RPE65-related retinal degeneration1 suggest that such a future might arrive sooner than anyone previously thought possible.

It would, of course, be pointless to inject a novel gene into the eye of a patient for whom the photoreceptor cells had already completely degenerated. The ERP might then provide a helpful index of the morphological state of the rods and cones. This might be particularly useful for mutations that do not affect rhodopsin itself but affect some other protein that is essential for visual function.8 In such cases, the ERP may provide a simple test of the integrity of the photoreceptors when other measures such as acuity or ERG amplitude are uninformative.

REFERENCES

1.Acland GM, Aguirre GD, Ray J, et al: Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 2001; 28:92–95.

2.Arden GB, Ikeda H: A new property of the early receptor potential of rat retina. Nature 1965; 208:1100–1101.

3.Brown KT, Murakami M: A new receptor potential of the monkey retina with no detectable latency. Nature 1964; 201:626–628.

4.Cone RA: Early receptor potential of the vertebrate retina. Nature 1964; 204:736–740.

5.Cone RA: Early receptor potential: Photoreversible charge displacement in rhodopsin. Science 1967; 155:1128–1131.

6.Cone RA, Cobbs WH 3rd: Rhodopsin cycle in the living eye of the rat. Nature 1969; 221:820–822.

7.Cone RA, Pak WL: The early receptor potential. In Lowenstein WR (ed): Handbook of Sensory Physiology, vol. 1: Principles of Receptor Physiology. Berlin, Springer Verlag, 1971, pp 345–365.

8.Fain GL, Lisman JE: Light, Ca2+, and photoreceptor death: New evidence for the equivalent-light hypothesis from arrestin knockout mice. Invest Ophthalmol Vis Sci 1999; 40:2770–2772.

9.Goldstein EB, Berson EL: Rod and cone contributions to the human early receptor potential. Vision Res 1970; 10:207–218.

10.Hestrin S, Korenbrot JI: Activation kinetics of retinal cones and rods: Response to intense flashes of light. J Neurosci 1990; 10:1967–1973.

11.Hodgkin AL, O’Bryan PM: Internal recording of the early receptor potential in turtle cones. J Physiol 1977; 267:737–766.

12.Makino CL, Dodd RL: Multiple visual pigments in a photoreceptor of the salamander retina. J Gen Physiol 1996; 108:27–34.

13.Makino CL, Taylor WR, Baylor DA: Rapid charge movements and photosensitivity of visual pigments in salamander rods and cones. J Physiol 1991; 442:761–780.

14.Muller W, Topke H: The early receptor potential (ERP). Doc Ophthalmol 1987; 66:35–74.

15.Murakami M, Pak WL: Intracellularly recorded early receptor potential of the vertebrate photoreceptors. Vision Res 1970; 10:965–975.

16.Pak WL, Ebrey TG: Visual receptor potential observed at subzero temperatures. Nature 1965; 205:484–486.

17.Sieving PA, Fishman GA: Rod contribution to the human early receptor potential (ERP) estimated from monochromats’ data. In Niemeyer G, Huber C (eds): Techniques in Clinical Electrophysiology of Vision. Documenta Ophthalmologica Proceedings Series, vol. 31. The Hague, Dr. W. Junk Publishers, 1982, pp 95–102.

18.Sieving PA, Fishman GA, Alexander KR, Goldberg MF: Early receptor potential measurements in human ocular siderosis. Arch Ophthalmol 1983; 101:1716–1720.

19.Trissl HW: On the rise time of the R1-component of the “early receptor potential”: Evidence for a fast light-induced charge separation in rhodopsin. Biophys Struct Mech 1982; 8:213–230.

20.Walther G, Hellner KA: Early receptor potential recordings for clinical routine. Doc Ophthalmol 1986; 62:31–39.

T / trough ratio (L/D) (Arden ratio1) is widely accepted as useful for evaluating retinal pigment epithelium (RPE) activity since the light peak and dark trough represent mainly the changes of the RPE membrane potentials. It also depends on the photoreceptor activity and RPE-receptor attachment, because the photoreceptors and their attachment with the RPE are essential for evoking the light peak. The L/D is also changed by occlusion of the central retinal artery, which nourishes the middle and inner layers of the retina. Therefore, abnormal L/D alone does not necessarily indicate RPE disorders. So far as a photic stimulus to the photoreceptors is used, the response obtained is not solely specific to the RPE.

The ocular standing potential, which mainly comes from the transepithelial potential (TEP) of the RPE, can be changed by nonphotic stimuli. For example, hyperosmolarity,5,8,10 bicarbonate,11,12 and acetazolamide3 (Diamox) decrease the TEP in vitro and the ocular standing potential in vivo. We call these responses the hyperosmolarity response, bicarbonate response, and Diamox response, respectively. The standing potential is changed by breathing a hypoxic mixture of oxygen and nitrogen.9 These responses are recordable by conventional electro-oculographic (EOG) technique in the dark.

Figure 41.1 shows these three responses in normal human subjects. The EOG amplitude is virtually stabilized (V0) usually about in 30 minutes in the dark. Then, a hypertonic solution (e.g., 20% mannitol or Fructmanit, see legend for figure 41.1), 7% sodium bicarbonate solution (Meylon), or

Diamox is given intravenously. These procedures decrease the EOG amplitude in the dark down to the minimum (Vmin) approximately 8 to 20 minutes after the onset of administration. The amplitude of the response is defined as the percent amplitude change of the EOG:100 ¥ (V0 - Vmin)/V0. The distribution of the amplitudes of these responses in the normal subjects is approximated by the normal distribution. Thus, their normal range is the mean ± 2 SD of the amplitude in the normal subjects; 22.8% to 45.2% for the hyperosmolarity response, 15.2% to 28.6% for the bicarbonate response, and 32.1% to 52.9% for the Diamox response (the dose of each stimulant is described in the legend for figure 41.1).

The amplitudes of the hyperosmolarity response and bicarbonate response are frequently decreased in retinitis pigmentosa,15 rhegmatogenous retinal detachment even in a localized area,2 diabetic retinopathy4 (occasionally abnormal even in diabetics without visible retinopathy), angioid streaks, Stargardt’s disease-fundus flavimaculatus,14,16 vitelliform macular dystrophy (Best’s disease13), Vogt-Harada- Koyanagi disease,7 and temporarily after cataract extraction6 (especially after intracapsular extraction) (Table 41.1). The hyperosmolarity response is more frequently abnormal than the L/D in the aforementioned diseases. The Diamox response remains within the normal range in most of the diseases described above and predominantly depends on the RPE in the posterior region of the ocular fundus since this response is suppressed in patients with severe macular atrophy (e.g., advanced stage in Stargardt’s disease).14

F 41.1 Hyperosmolarity response (A), bicarbonate response (B), and Diamox response (C) in normal human subjects. The mean and standard deviation of the EOG amplitude as a percentage of the stabilized amplitude (so-called base value) after dark adaptation of 30 minutes are shown. The hyperosmolarity response was recorded in 50 eyes of 30 subjects, bicarbonate response in 70 eyes of 45 subjects, and Diamox response in 36 eyes of 24 subjects. To evoke the hyperosmolarity response, Fructmanit (10% fructose, 15% mannitol) was given intravenously. The solution was administered for 15 to 20 minutes at a rate of 11% of the subject’s total blood volume per hour. The total blood volume (liters) was calculated by the following formulas: 0.168H 3 + 0.05W + 0.444 in males or 0.25H 3 + 0.063W - 0.662 in females, where H is the height in meters and W is weight in kilograms. To evoke the bicarbonate response, 0.83 mL/kg of 7% sodium bicarbonate (Meylon) was intravenously given in 5 minutes. To evoke the Diamox response, 500 mg of Diamox was given intravenously in 1 minute. No light was used except for two dim miniature lamps to alternatively fixate the eye for the conventional EOG procedure. The onset of stimulant application was at 0 minutes on the abscissa.

REFERENCES

1.Arden GB, Barrada A, Kelsey JH: New clinical test of retinal function based upon the standing potential of the eye. Br J Ophthalmol 1962; 46:449–467.

2.Kawasaki K, Madachi-Yamamoto S, Yonemura D: Hyperosmolarity response of ocular standing potential as a clinical test for retinal pigment epithelium activity—Rhegmatogenous retinal detachment. Doc Ophthalmol 1984; 57:175–180.

3.Kawasaki K, Mukoh S, Yonemura D, Fujii S, Segawa Y: Acetazolamide-induced changes of the membrane potentials of the retinal pigment epithelial cell. Doc Ophthalmol 1986; 63:375–381.

4.Kawasaki K, Yonemura D, Madachi-Yamamoto S: Hyperosmolarity response of ocular standing potential as a clinical test for retinal pigment epithelium activity—Diabetic retinopathy. Doc Ophthalmol 1984; 58:375–384.

5.Kawasaki K, Yonemura D, Mukoh S, Tanabe J: Hyperosmo- larity-induced changes in the transepithelial potential of the human and frog retinae. Doc Ophthalmol 1983; 37:29–33.

6.Kawasaki K, Yonemura D, Yanagida T, Segawa Y, Wakabayashi K, Mukoh S, Ishida H, Fujii S, Takahara Y: Suppression of the hyperosmolarity response after cataract surgery. Doc Ophthalmol 1986; 63:367–373.

7.Madachi-Yamamoto S, Kawasaki K, Yonemura D: Retinal pigment epithelium disorder in Vogt-Koyanagi-Harada disease revealed by hyperosmolarity response of ocular standing potential. Jpn J Ophthalmol 1984; 28:362–369.

8.Madachi-Yamamoto S, Yonemura D, Kawasaki K: Hyperosmolarity response of ocular standing potential as a clinical test for retinal pigment epithelium activity—Normative data. Doc Ophthalmol 1984; 57:153–162.

9.Marmor MF, Donovan WJ, Gaba DM: Effects of hypoxia on the human standing potential. Doc Ophthalmol 1985; 60:347–352.

10.Mukoh S, Kawasaki K, Yonemura D: Hyperosmolarityinduced hyperpolarization of the membrane potential of the retinal pigment epithelium. Doc Ophthalmol 1985; 60:369–374.

11.Segawa Y, Mukoh S, Tanabe J, Kawasaki K: Bicarbonateinduced response from the retinal pigment epithelium for electrodiagnosis. Presented at the 26th International Symposium on Clinical Electrophysiology of Vision, Estoril, Portugal, May 23, 1988.

12.Steinberg RH, Miller SS: Transport and membrane properties of the retinal pigment epithelium. In Marmor MF, Zinn KH (eds): The Retinal Pigment Epithelium. Cambridge, Mass, Harvard University Press, 1979, pp 205–225.

13.Wakabayashi K, Yonemura D, Kawasaki K: Electrophysiological analysis of Best’s macular dystrophy and retinal pigment epithelial pattern dystrophy. Ophthalmic Paediatr Genet 1983; 3:13–17.

14.Wakabayashi K, Yonemura D, Kawasaki K: Electrophysiological analysis of Stargardt’s disease fundus flavimaculatus group. Doc Ophthalmol 1985; 60:141–147.

15.Yonemura D, Kawasaki K, Madachi-Yamamoto S: Hyperosmolarity response of ocular standing potential as a clinical test for retinal pigment epithelium activity—Chorioretinal dystrophies. Doc Ophthalmol 1984; 57:163–173.

16.Yonemura D, Kawasaki K, Wakabayuashi K, Tanabe J: EOG application for Stargardt’s disease and X-linked juvenile retinoschisis. Doc Ophthalmol Proc Ser 1983; 37:115–120.