- Email: [email protected]
Early receptor potential of the isolated frog (Rana pipiens) retina
Vkojl Res. Vol. 7, pp. 837-845. Pergamon Press 1967. Printed in Great Britain.
EARLY RECEPTOR POTENTIAL ISOLATED FROG (RANA PIPIENS)
OF THE RETINA’
E. BRUCE GOLDSTEIN* Department of Psychology,Brown University,Providence,Rhode island 02912 (Received 23 June 1967) VISUAL early receptor potential (ERP) is a fast, biphasic response that precedes the ERG. It was first recorded by BROWNand IViuRAKAhlr (1964) with intra-retinal microelectrodes, and by CONE(1964) with cornea1 wick electrodes. The initial positive phase (Al) has no latency on a psec scale (CONE, 1967) and reaches its peak in about 0.5 msec or less at room temperature. The negative phase (R2) reaches its peak in l-2 msec or less. These extremely short latencies support the contention that the ERP is generated at or near the receptors (CONE, 1964, 1965; BROWN,et al. 1965). There is evidence supporting the idea that the ERP is closely related to visual pigment bleaching. CONE (1964) has shown that in the albino rat the amplitude of R2 is directly proportional to the percent of rhodopsin bleached by a short flash. PAK and CONE(1964) have obtained a similar result for the faster positive phase, Rl; PAK and EBREY (1966) state that the amplitude of the ERP of the all cone retina of the ground squirrel may be linearly related to the number of visual pigment molecules excited or bleached by a flash. CONE (1964) has also noted that the ERP recorded from the intact rat eye is reduced in amplitude by light adaptation, but subsequently increases in amplitude in the dark at a rate comparable to the rate of pigment regeneration. CONE(1964), and PAK and CONE(1964) found that the action spectra of Rl and R2 in the rat closely correspond to the rhodopsin absorption spectrum and the albino rat scotopic b-wave sensitivity. They therefore concluded that the ERP is generated by a mechanism closely linked to rhodopsin. Thus, it has been found that the ERP is closely related to the action of a specific visual pigment. However, the investigations concerned with the relation between the ERP and the bleaching of specific pigments have been carried out in eyes dominated by either rods or cones (CONE,1964; PAKand CONE,1964; PAKand EBREY,1966). Some investigators have recorded ERP’s from mixed retinae (BROWNand MURAKAMI,1964; BROWN,WATANABE~~~MURAKAAII, 1965; BROWN,1965; CONE,1964; PAK, 1965); however, none have attempted to relate the ERP to specific pigments in a retina containing both rods and cones. The purpose of the following experiments was to determine which pigment or pigments are involved in ERP generation in the mixed retina of the leopard frog, Rana pipicns. This was done by measuring both the recovery of ERP amplitude during dark adaptation and the spectral sensitivity of the ERP in the isolated frog retina. THE
1 This research was supported by Public Health Service grants NB 05256 to T. P. Williams, NB 01453-08 lo L. A. Riggs, and a Public Health Service Predoctoral Research Fellowship to the author. 2 Present address: The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138. 837
838
E. BRUCEGOLDSTEIN METHODS
All responses were recorded with cotton wick Ag-A&I electrodes. Three types of preparations were used. The frog ERP was nrorded from isolated retinae (i.e. retinae separated from the pigment epitheliumchoroid complex); the rat ERP, from whole excised eyes; and the frog ERG, from excised opened eyes. All animals were dark adapted for a minimum of 12 hr at room temperature prior to the experiment, and all dissections were performed under dim red light. The isolated frog retina was prepared by decapitating the frog and enucleating the eye. The upper hemisphere of the eye was then removed by cutting around the ora serrata. The retina was carefully teased away from the pigment epithelium under Ringer’s solution with lens extracting forceps. It was then removed from the eye cup by grasping the optic nerve between the pigment epithelium and the retina and by pulling to detach the nerve. The retina was placed receptor side down on a cotton pad that had been soaked in Ringer’s solution; one electrode was placed under the cotton and the other touched the vitreal side of the retina. The rat ERP was recorded between the back of the eye and the cornea, and the frog ERG was recorded between the back of the eye and the retina. During the experiments, the preparations were placed on a constant temperature block. This block was kept at constant temperature by circulating water through it with a Haake constant temperature bath. Temperature was measured with a thermister probe connected to a Yellow Springs Instruments telethermometer. Light from a Honeywell 65C electronic flash gun was focused on the preparation through a single lens and illuminated the entire retina. The flash duration was 1.3 msec (i.e. the time necessary for 90 per cent of the Rash energy to dissipate). The intensity of the stimulus flash was controlled by placing neutral density filters between the flash gun and the focusing lens, and the wavelength was controlled by interference filters with bandwidths of 6-15 run. The energy output of the flash gun at the interference filter’s wavelengths of peak transmission was measured with a Type 3 Weston Photronic Cell calibrated by the US Bureau of Standards, Photovoltaic and electrical artifacts were eliminated by appropriate shielding. The electrical responses were amplified by a Tektronix type FM 122 low-level preamplifier which was set for a 0.2-10,ooO Hz response range for the ERP and 0.2-50 Hz for the ERG. Responses were displayed on a Tektronix 561A oscilloscope with a 3A3 differential amplifier and a 2B67 time base. Pictures of the responses were recorded with a Polaroid scope camera. In the dark adaptation experiments, a dim tesrjlash,J which evoked a response of 30-70 PV, was presented to a dark adapted isolated frog retina. The amplitude of this response, measured from the peak of RI to the peak of R2 was designated the “dark adapted response”. This test flash bleached about 1-3 per cent of the rhodopsin in the retina. An intense white bleachingflash which bleached about 75 per cent of the rhodopsin was then presented. This bleaching flash was followed by test flashes identical to the one presented to the dark adapted retina, at times of 05, 1.5, 3, 6, 10, 15, 20, 25, and 35 minutes after the presentation of the bleaching flash. The amplitudes of the responses generated by these test flashes were expressed in terms of percent of the dark adapted response. The spectral sensitivity of the dark adapted retina was measured by determining the energy necessary to generate a constant response of 20-25 PV at each of five wavelengths (420, 490, 548, 590, and 630 nm). A series of flashes consisting of one flash of each wavelength was presented. In half of the experiments the flashes were presented in ascending order (420,490,548,590,630 run) and in the other half, in descending order (630,590,548,490,420 MI). For each series the intensity presented at each wavelength was adjusted so that approximately equal responses were elicited for all wavelengths in the series. After completing a series, the intensity was either increased or decreased by an equal amount at each wavelength and the series was run again. Presentation of the flashes in this manner tends to equate the effects of any progressive changes in the preparation, e.g. deteriorating preparation, bleaching, etc., at all of the wavelengths, leaving the relative spectral sensitivity comparatively unchanged. Flashes were separated by at least one minute. The spectral sensitivity of the recovered ERP was determined in the same manner as the dark adapted sensitivity, except that measurements were not begun until 17 minutes after presentation of a bleaching flash. In order to determine the rhodopsin concentration in the retinae at various times after presentation of the bleaching flash, rhodopsin was extracted from the retinae with hexadecyltrimethylammonium bromide at 0, 10, 20, or 30 minutes after the Rash. The optical density of the extract was then measured at 502 run with a Beckman DU spectrophotometer and Gilford Model 220 absorbance indicator. The density change caused by completely bleaching the extract was then measured. This density change was compared with the corresponding density change of the rhodopsin extracted from the unbleached retina of the other eye of the frog. The rhodopsin concentration of the bleached retina could then be expressed as a percent of the concentration in the unbleached eye. BAECK,et al. (1965), GOLDSTEIN (1968), and ZEWI (1939) have found that (within 5 per cent) the eyes of a frog contain equal amounts of rhodopsin. 3 Tesr flash and bleaching /lash refer to Rashes which bleach about l-3 per cent and 75 per cent of the rhodopsin in the retina, respectively. This definition is used throughout this paper.
Early Receptor Potential of the Isolated Frog (Rar~n pipie)rs) Retina
839
RESULTS
The results of the curves under Bin Fig. 380, 490, and 630 nm bleaching flash. These
dark adaptation experiments are shown in Figs. 1 and 2. The four 1 show the increase in the amplitude of responses generated by white, test flashes as a function of time after presentation of the intense curves indicate that there is a large increase in amplitude at 23 “C for
FIG. 1. A: Responses of isolated frog retinae to ten white test flashes. No bleaching flash was presented. Average of five experiments. Average range = k8.5. B: ERP recovery in isolated frog retinae at 23°C for 380 (O), 490 (A), 630 (o), and white (e) test flashes. The points are the means of the following number of experiments for each condition: 380 = 6; 490 = 4; 630 = 4; white = 8. Average ranges: 380 = &15; 490 = i7; 630 = &7; white = A12.5. C: Regeneration of rhodopsin in the isolated frog retina at 23’C. The points are rhodopsin concentrations determined by extraction. The concentration is expressed in relative units (left ordinate) with the concentration in the dark adapted eye being set at 100. The concentration can also be read as “Percent regeneration” from the scale on the right ordinate. Percent regeneration = lOO(0, - DO)/(Dd - D,) where D, = pigment density at time X, Do = density immediately after the bleaching flash (t = 0), and Dd = the density in the dark adapted eye. Points are the means of the following number of experiments for each time: 0 min, 7; 10 min, 3; 20 min, 3; 30 min, 4. Brackets indicate ranges. The dashed line is a plot of percent rhodopsin regeneration vs. time, adapted from Z~wr’s (1939) measurements on the isolated frog retina.
Fig. 2. ERP recovery in isolated frog retinae at 8’C. White test flash. Mean of 5 experiments. Average range = &13.
E. BRUCE GOLDSTEIN
840
all four types of test flashes during the first IO-15 min after the bleaching flash. The amplitude increases from about 40 per cent of the dark adapted amplitude at 0.5 minutes to 70-85 per cent at 10 min. A similar increase in ERP amplitude was observed by BROWN (1965) in the isolated retina and the excised opened eye of the toad. Photographs of the responses recorded before the bleaching flash and at 0.5 and 35 min after the bleaching flash are shown in Fig. 3. All of the responses have the characteristic ERP waveform; therefore, it is unlikely that the “photoproduct responses” described by ARDEN and IKEDA (1965), ARDEN et al. (1966a, b), CONE(1967), and PAK and BOES(1967) are present either before or after bleaching.
I 5ouv
+-i 2mrrc
FIG. 3. ERP responses of isolated frog retinae to 380, 490, 630, and white test flashes given before the bkaching Bash (the dark adapted response) and at 0.5 and 315min after the bleaching f&h. The responses to the test flashes at 10 and 15 min are not shown; howmr, their amplitudes and waveforms are approximately the same as those obtained at 35 min. Temperature = 23°C. The effect of pigment bleaching on the dark adaptation curves was minimized by using test flashes that bleached only a small amount of visual pigment. Curve A of Fig. 1 shows the amplitudes of the ERP’s generated when test flashes were presented without presenting the bleaching flash. The slight decrease in response amplitude is probably due primarily to pigment bleaching. The recovery of the ERP in the dark after presentation of an intense bleaching flash also occurs when the temperature of the retina is lowered to 8°C. This result is shown in Fig. 2. The recovery under these conditions takes 20-25 min to reach its maximum compared to the IO-15 min necessary at 23°C. GOLDSTEIN (1968) has also shown that recovery occurs when a continuous bleaching light is used in place of the brief bleaching flash used in the previous experiments.4 In order to determine the relationship between rhodopsin concentration and the rapid recovery of the ERP, rhodopsin concentration was measured as a function of time after the bleaching flash. These results are shown in Fig. 1 (Curve C). These data indicate that little 4 See note added in proof.
Early Receptor Potential of the Isolated Frog (Rann pi@ns) Retina
841
rhodopsin regeneration occurs over a 30 rnin period. This agrees with ZEWI’S(1939) data for isolated retinae (dashed line) and with the results of KSINE (1879) and BAUMANN (1965). This lack of substantial rhodopsin regeneration in the presence of the large recovery in ERP amplitude suggests that rhodopsin regeneration is not responsible for the ERP recovery. Regeneration of rhodopsin by photoreversal (cf. MATTHEWSet al., 1963; WIr_LIAh& 1964) can also be eliminated as a cause of recovery since the test flashes are too dim to regenerate an appreciable amount of rhodopsin. Also, good recovery is measured with the 630 nm test flash, and, since the photoproducts absorb little light of this wavelength (DARTNALL, 1953), little photoreversal can occur. Thus, photoreversal or rhodopsin regeneration cannot cause the recovery. However, it is possible that ERP recovery is caused by the regeneration of cone pigments. It is well established that the cone pigments regenerate much faster than rod pigments (WALD, 1959), and the time course of the recovery compares favorably with the rate of regeneration of cone pigments itt vitro (WALD et al., 1954-U) and in vim (RUSHTON,1957). Spectrul
serisitivify
In order to determine more directly which pigments are responsible for the frog ERP, spectral sensitivity functions for “dark adapted” and “recovered” retinae were determined. A “recovered” retina is one that has been bleached with a bleaching flash and then has
FIG. 4. Spectral sensitivites of the frog and rat. All points and sensitivity curves are expressed in terms of log reciprocal energy units. Each point is the mean of six experiments. Average rangeat eachpoint = kO.12 log units. Upper points: Spectral sensitivity of the dark adapted and recovered ERP recorded from the isolated frog retina. Criterion response = 20-25 pV. Temperature = 23°C. The line is GORDON’S (1967) photopic ERG curve for the frog. The opened circles are EBREYand CONE’S(1967) data for the ERP sensitivity of the excised frog eye. Lower points: Spectral sensitivity of the ERP recorded from the excised eye of the albino rat at 23°C and the sensitivity of the ERG recorded from the excised opened eye of the frog ar 10°C. A criterion amplitude of 15-20 PV was used for the rat, and the criterion was 15 FV for the frog. The solid line is DODT and Ecm’s (1961) sensitivity curve for the dark adapted rat h-wave. The dashed line is the absorption spectrum of rhodopsin (Amax = 502 nm) with a maximum density of 050 (DARTNALL,1953). The opened squares are CONE’S(1964) data for the ERP sensitivity of the albino rat.
842
E. BRUCE GOLDSTEIN
“recovered” in the dark for at least 17 min. As indicated in Fig. 1, ERP recovery is essentially completed by 17 min. after the bleaching flash. ERP sensitivity curves for dark adapted and recovered isolated frog retinae are shown in Fig. 4. The points for the dark adapted and recovered ERP’s are identical within experimental error. GORDON’S(1967) photopic ERG sensitivity curve for the frog (solid line) fits the points well. Although there are not enough points to precisely determine the wavelength of maximum sensitivity, it appears that the curve peaks somewhere between 560 and 590 nm. This agrees with the results of LIEBMAN’S(1967) microspectrophotometry experiments. He found that one of the frog cone pigments absorbs maximally at 580 nm. EBREYand CONE’S(1967) data for the sensitivity of the whole excised frog eye are also plotted in Fig. 4 and agree with the results of the present experiments. Although all the curves in Fig. 4 are plotted in terms of log relative sensitivity, it should be noted that the absolute sensitivity of the dark adapted and recovered frog ERP is the same to within 0.1 log units at each wavelength. This result would be expected from the large magnitude of the recovery which occurred during dark adaptation. If the long wavelength of maximum sensitivity and the broadness of the frog ERP sensitivity curves were due to artifacts caused by factors such as faulty calibration of the flasher or transmission outside the main bandpass of the interference filters (cf. WALD, 1945), then other sensitivity curves, determined using the same equipment and procedures, would also be broadened and shifted towards longer wavelengths. In order to test for this possibility, the spectral sensitivities of the rat ERP and frog ERG were determined. The points in Fig. 4 indicate that the rat ERP sensitivity peaks at 500 nm and agrees with CONE’S (1964) rat ERP sensitivity and DODTand ECHTE’S (1961) determination of dark adapted rat b-wave sensitivity. The spectral sensitivity of the frog ERG also peaks at about 500 nm and agrees with the absorption spectrum of rhodopsin with a maximal density of 0.50.5 Thus, the rat ERP and frog ERG sensitivity curves are not broadened or shifted to longer wavelengths. It is, therefore, concluded that the frog ERP sensitivity curves are correct. These curves suggest that the sensitivity of the ERP recorded from dark adapted and recovered isolated frog retinae is mediated by a cone mechanism, or mechanisms, which result in maximum sensitivity at about 560-590 nm. DISCUSSION The previous results indicate that the cones may play a major role in generating the ERP in the isolated frog retina. Although it is difficult to determine the exact contributions of rod and cone pigments to the ERP, it appears that the contribution of the rod pigments is quite small. This is a surprising result because the rod pigments make up about 90 per cent of the total concentration of visual pigments in the frog’s retina (LIEBMAN, 1967)6. CONE (1967), BRWDLEYand GARDNER-MEDWIN (1966), and PAK and EBREY(1965) have suggested 5 The frog ERG sensitivity should actuall-y correspond to the slightly broader absorption spectrum of rhodonsin with a maximum density of@75 since this is the density of rhodopsin in the frog% rods (DENTON and W&LIB, i9S; D~MJW,19Sgi DAWNALL, 1962). This dis&pancy cot&l b due to e~perim&tal error or it is possible that the rhodopsin concentration in the frogs used in these cxp&rnents could kve been low due to prolonged storage at low temperatures. However, since the frog ERG sensitivity is actually xurroHIt=r than what would be predicted theoretically, this discrepancy cannot exptain the very broad frog ERP sensitivity curves. 6 SAXIW(1954) has found that in the common frog, Ranu temporaria, 45 per cent of the receptors are cones. However, the cones are much smaller than the rods; therefore, cone pigments account for only 10 per cent of the visual pigment concentration in the retina (LIEBUN, 1967).
Early Receptor Potential of the Isolated Frog (Rarta pipiens) Retina
843
that the ERP is generated by the direct action of the visual pigments. Since the frog retina is dominated by the rod pigment, rhodopsin, it would be expected that the action of this pigment would dominate the ERP in this retina. However, this is clearly not the case; the cones appear to contribute an electrical signal far out of proportion to their visual pigment content. The mechanism that is responsible for this high level of cone activity is not clear. It is unlikely that a neural mechanism would operate to inhibit or enhance the rod or cone system since the visual pigments are located distal to synapses at which neural effects might OCCUI (cf. DOWLING,1967). It is possible that there are basic differences in the chemical properties and/or the distribution of charges in the pigments contained in the rods and cones. These differences may make it possible for the cone pigments to generate a much larger signal than the rod pigments. However, even if large differences do exist between rhodopsin and the cone pigments, it seems unlikely that these differences would account for the almost total absence of rod responding. It should be noted that the spectral sensitivity of the ERP of the live frog has not yet been determined. It is possible that the cone-dominated response found in the present experiments occurs only in the isolated retina or whole excised eye (cf. Fig. 4). Besides rhodopsin and the cone pigments, the frog’s retina also contains a pigment in the “green rods” (DENTONand WYLLIE, 1955; DARTNALL,1967). This pigment absorbs maximally at 433 nm, far from the maximal sensitivity of the ERP. Therefore, the role of this pigment in generating the ERP is probably quite small. DENTONand WYLLIE(1955) have suggested, however, that the green rods also contain a photostable pigment absorbing in the range 560-680 nm. If this pigment mediates the responses recorded in the present experiments, it would be necessary to hypothesize a mechanism other than pigment bleaching to explain the large adaptation effects observed in these experiments. Furthermore. this pigment does not absorb appreciably at 520 nm, a wavelength at which the ERP sensitivity is still quite high. The rapid recovery of the ERP during dark adaptation in the absence of significant rhodopsin regeneration indicates that cone pigments may be regenerating in the isolated retina. Thus, there may be differences between the mechanisms of rod and cone pigment regeneration or between the environments inside the rods and cones. The enzymes necessary for catalyzing rhodopsin regeneration are contained in the pigment epithelium; however. the catalysts for cone pigment regeneration may be contained in or near the receptors. There appears to be no published work bearing on the question of cone pigment regeneration in the isolated retina. The crucial test for the hypothesis of cone regeneration would be. microspcctrophotometric experiments on single cones. If cone pigments do, in fact, regenerate in the isolated retina, the cone pigments of a mixed retina could be isolated by simply bleaching all of the visual pigment in the isolated preparation and then allowing time for the cone pigments to regenerate. Nore added in proof-Recovery also occurs when a second bleaching flash is presented at some time, 1. after the first bleaching flash. For example, in a typical experiment the response to a white test flash at r=35 min was 82 (dark adapted response= 100). A second bleaching flash was presented at t=35,5 min and a test flash at 36 min generated a response of 32. Recovery then proceeded again until at 70.5 min the response generated by a test flash had an amplitude of 73. The second recovery was slower than the initial recovery. The ERP reached its maximum amplitude lo-15 min after the first flash. However, it took about 20 min for the recovery following the second flash to reach completion. Acknowledgemenfs-I wish to thank T. P. WILLIAMSfor his advice throughout this investigation, L. A. RICKS for his helpful suggestions, and G. WALD for the use of his Weston cell and his criticism of the manuscript. I also thank thank R. A. CONE and P. STEWART for reading preliminary drafts of the manuscript. c
E. BRUCEGOLDSTEIN REFERENCES hDEN,
G. B. and IKEDA,H. (1965). A new property of the early receptor potential of the rat retina. Nature, fond. 288, 1100-l 101. ARDEN,G. B., IKEDA,H. and SIEGEL,I. M. (1966a). Effects of light-adaptation on the early receptor potential. Vision Res. 6,357-372. ARDEN,G. B., IKEDA,H. and Stzoet, 1. M. (1966b). New components of the mammalian receptor potential and their relation to visual photochemistry-. Vision Res. 6, 373-384. BAECK,I., DONNER,K. 0. and Rauzza, T. (1965). The screening effect of the pigment epitheiium on the retinal rods in the frog. Vision Res. 5, 101-I 11. BAUMANN,C. (1965). Die Photosensitivitat des Sehpurpurs in der isolierten Netzhaut. Vision Res. 5, 425-434. BRINDIPY,G. S. and GARDNER-MEDWIN, A. R. (1966). The origin of the early receptor potential of the retina. J. Physioi, Lond. 182, 185-194. BROWN,K. T. (1965). An early potential evoked by light from the pigment epithelium-choroid complex of the eye of the toad. Nature, Lond. 287, 1249-1253. BROWN, K. T. and MURAKAMI.M. (1964). A new receptor potential of the monkey retina with no detectable latency. Nature, Lond. 281, 626-628.
BROWN,K. T., WATANAB~Z, K. and MURAKAMI, M. (1965). The early and late receptor potentials of monkey cones and roda. ColB Spring Harb. Symp. quant. Biol. 30, 457-482. CONE. R. A. (1964). Early receptor potential of the vertebrate retina. Nature, Lond. 264, 736-739. CONE, R. A. (1965). The early receptor potential of the vertebrate eye. Cofd Spring Harb. Symp. quant. Biol. 30.483-491. CONE, R.
A. (1967). Early receptor potential: photoreversible charge displacement in rhodopsin. Science,
N. Y. 155,1128-1131.
DARTNALL,H. J. A. (1953). The interpretation of spectral sensitivity curves. Brit. med. Bull. 9, 24-30. DARTNALL,H. J. A. (1962). The photobiology of visual processes, in 7he Eye, Vol. 2, Davson, H., ed., 321533, Academic Press, New York. DARTNALL,H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. DEMON, E. J. and WYLLIE,J. H. (1955). Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiof., Loud. 127, 81-89. D~DT, E. and Ecxrz, K. (1961). Dark- and light-adaptation in the pigmented and white rat as measured by the ERG threshold. J. Neurophysiol. 24, 427-445. DoNNER, K. 0. (1958). The spectral sensitivity of vertebrate retinal elements, in Visual Problems of Colour, 539-566, H.M. Stat. Office, London. D~WLING, J. E. (1967). The site of visual adaptation. Science, N. Y. 155, 273-279. Eattar, T. G. and Co=, R. A. (1967). Melanin, a possible pigment for the photostable electrical responses of the eye. Nature, Land. 213,360-362. GOLDSIW[N, E. B. (1968). Early receptor potential of the isolated frog (Rana pipiens) retina. Doctoral dissertation, Brown Univ., Prov., R.I. GORDON, J. (1967). ERG spectral sensitivity of the frog, Rana pipiens. Master’s thesis, Brown Univ., Prov., R.I. KUHN, W. (1879). Chemische Vorgange in der Netzhaut, in Hermann’s Hanab. Physiof. 3, Leipzig, 235. bEl$blAN, P. A. (1967). Vision Res.. in preparation. MA’I?HEWS,R., HUBBARD, R., BROWN,P. K., and WALD, G. (1963). Tautomeric forms of metarhodopsin. J. gen. Physiol. 47,215-240.
PAK, W. L. (1965). Some properties of the early electrical response in the vertebrate retina. Cold Spring Harb. Symp. quant. Biol. 30,493-499.
PAK, W. L. and Bees, R. J. (1967). Rhodopsin: responses from transient intermediates formed during its bleaching. Scicnee, N. Y. 158,1131-1133. PAK, W. L. and CONE,R. A. (1964). Isolation and identification of the initial peak of the early receptor potential. Nature, Lond. 204,436-438. PAK,W. L. and EBRZY,T. G. (1965). Visual receptor potential observed at sub-zero temperatures. Nature, Land. 208, 484-486. PAK, W. L. and E~EY, T. G. (1966). Early receptor potentials of rods and cones in rodents. J. gen. Physiof. 49,11991208. RUS~N, W. A. H. (1957). Physical measurement of cone pigment in the living human eye. Nature, Lond.
179, 571-573. (1954). The development of visual cells. Annls Acad. Sci. fenn. Ser. A, 23, l-95. (1945). The spectral sensitivity of the human eye. J. opt. Sot. Am. 35, 187-l%. WALD,G. (1959). The photoreceptor process in vision, in Handb. Physiul., 671-692, Sect. I, Vol. I, edited by J. FIELD, Am. Physiol. See., Washington, D.C.
SAXEN, L. WALD, G.
Early Receptor Potential of the Isolated Frog (Ram
pipiens)
845
Retina
G., BROWN, P. K., and SMITH, P. H. (1954-55). lodopsin. J. gen. Physiol. 38, 623-681. WILLIAMS,T. P. (1964). Photoreversal of rhodopsin bleaching. J. gen. Physiol. 47, 679-689. ZEWI, M. (1939). On the regeneration of visual purple. AC/U. Ser. Sci. /em. N.S. B, l-57. WALD,
Abstract-The decreases recovers in 10-15
amplitude
of the early receptor potential
when the retinal
(ERP) of the isolated
visual pigments are bleached; however, SO-90 per cent of that prior to bleaching.
in the dark to minutes at 25°C and 20-25 minutes chromatic (380, 490, or 630 nm) test flashes this period of rapid ERP recovery, and the 560-590 nm. These results suggest that the generated by cone pigments.
the amplitude This recovery
frog retina subsequently is completed
at 8”C, and is observed when white or monoare used. Little rhodopsin regenerates during spectral sensitivity of the ERP peaks at about ERP recording from the isolated frog retina is
R&urn&-L’amplitude du potentiel initial de rkcepteur (ERP) de la retine isolee de grcnouille dtiroit quand les pigments visuels de la r&tine se dCcolorent; dependant, I’amplitude se retablit ensuite dans I’obscuritt B 80-90 pour cent de sa valeur avant dtcoloration; cette rCcupCration est compiite en 10-15 minutes g 23°C et 20-25 minutes B 8’C, et s’observe avec les &lairs tests utilist%, blancs ou monochromatiques (380, 490 ou 630 MI). La rhodopsine se r&?ntre peu pendant cette tiriode de rtiuperation rapide de I’ERP, et le maximum de sensibilitb spectrale de I’ERP se situe vers 560-590 nm. Ces rtsultats suggtrent que I’ERP enregistrt dans la rCtine isoltke de grenouille est engendrt pas les pigments des cbnes. Zusammenfassung-Die Amplitude des friihen Rezeptorpotendals (ERP) der isolierten Froschnetzhaut nimmt ab, wenn die Netzhautpigmente gebleicht werden. Im Dunklen erholt sich die Amplitude jedoch wieder auf 80-90 prozent des urspriinglichen Wertes. Diese Erholuna ist bei 23°C in IO-15 Minuten abneschlossen und bei 8°C in 20-25 Minuten. Sie wird beobachtet, wenn weiRe oder monoc&omatische (380, 490 oder 630 nm) Testblitze verwendet werden. WIhrend dieser Periode der schnelien ERP-Erholung wird nur sehr wenig Rhodopsin regeneriert. Die Spektralempfindlichkeit ist maximal bei etwa 560-590 nm. Diese Ergebnisse weisen darauf hin, dal3 das ERP-Potential der isolierten Froschnetzhaut durch Zapfenpigmente erzeugt wird. AMMkiTyAa paHHer0 peUenTopHor0 nOTeHUuana (PPn) H30nHpOBaHHOti CCT’IaTKU JD?ryIUK&i yMeHbUIaeTC5l, KOrAa 3pHTeJIbHble ITkirMeHI’bI CeT’faTKU BbIUBeTaKIT, OAHaKO, el-0 aMnJUiTyAa BOCCTaHaBJIliBaeTCII 3aTCM npH lTOCJleAyIOLUefi TeMHOBOk aAanTaUHH A0 80-90 lTpOUeHTOB t242nepBOHaWJlbHOti BcJlUWHbl. 3TO BOCCTaHOBJleHkie 3aKaHrasanocb B TeqeHkie lo-15 MRH npri 23°C u 20-25 MBH npu 8°C li Ha6nmnanocb B Tex cnyrarrx, Korna ynOTpe6nRnaCb 6enart HnMMOHOXpOMaTWzCKaR (380, 490 EinM 630 HM) TeCTOBaR BCllbILUKa. B Te’ieHUc 3TOr0 nepHOAa 6blcTpOro BOCCTaHOBJEHHR PPn PereHepHpOBWO ManO POAOnCMHa U MaKClrMyM CneKTpaJlbHOii YyBCTBHTenbHOCTB HaXOAHlnCII OKOJlO 560-590 HM. 3TU pe3yJlbTaTbI 3aCTaBJlRH)T AyhIaTb, ‘IT0 PPn, 3anUCbIBaeMblti OT Ii3OJlMpOBaliHO~ CeT’IaTKM Jl5iry!JK&i, reHepl4pyeTCR KOIl60’iKOBblMM nHl-MCHTaMM.
Pe3loMe -
EARLY RECEPTOR POTENTIAL ISOLATED FROG (RANA PIPIENS)
OF THE RETINA’
E. BRUCE GOLDSTEIN* Department of Psychology,Brown University,Providence,Rhode island 02912 (Received 23 June 1967) VISUAL early receptor potential (ERP) is a fast, biphasic response that precedes the ERG. It was first recorded by BROWNand IViuRAKAhlr (1964) with intra-retinal microelectrodes, and by CONE(1964) with cornea1 wick electrodes. The initial positive phase (Al) has no latency on a psec scale (CONE, 1967) and reaches its peak in about 0.5 msec or less at room temperature. The negative phase (R2) reaches its peak in l-2 msec or less. These extremely short latencies support the contention that the ERP is generated at or near the receptors (CONE, 1964, 1965; BROWN,et al. 1965). There is evidence supporting the idea that the ERP is closely related to visual pigment bleaching. CONE (1964) has shown that in the albino rat the amplitude of R2 is directly proportional to the percent of rhodopsin bleached by a short flash. PAK and CONE(1964) have obtained a similar result for the faster positive phase, Rl; PAK and EBREY (1966) state that the amplitude of the ERP of the all cone retina of the ground squirrel may be linearly related to the number of visual pigment molecules excited or bleached by a flash. CONE (1964) has also noted that the ERP recorded from the intact rat eye is reduced in amplitude by light adaptation, but subsequently increases in amplitude in the dark at a rate comparable to the rate of pigment regeneration. CONE(1964), and PAK and CONE(1964) found that the action spectra of Rl and R2 in the rat closely correspond to the rhodopsin absorption spectrum and the albino rat scotopic b-wave sensitivity. They therefore concluded that the ERP is generated by a mechanism closely linked to rhodopsin. Thus, it has been found that the ERP is closely related to the action of a specific visual pigment. However, the investigations concerned with the relation between the ERP and the bleaching of specific pigments have been carried out in eyes dominated by either rods or cones (CONE,1964; PAKand CONE,1964; PAKand EBREY,1966). Some investigators have recorded ERP’s from mixed retinae (BROWNand MURAKAMI,1964; BROWN,WATANABE~~~MURAKAAII, 1965; BROWN,1965; CONE,1964; PAK, 1965); however, none have attempted to relate the ERP to specific pigments in a retina containing both rods and cones. The purpose of the following experiments was to determine which pigment or pigments are involved in ERP generation in the mixed retina of the leopard frog, Rana pipicns. This was done by measuring both the recovery of ERP amplitude during dark adaptation and the spectral sensitivity of the ERP in the isolated frog retina. THE
1 This research was supported by Public Health Service grants NB 05256 to T. P. Williams, NB 01453-08 lo L. A. Riggs, and a Public Health Service Predoctoral Research Fellowship to the author. 2 Present address: The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138. 837
838
E. BRUCEGOLDSTEIN METHODS
All responses were recorded with cotton wick Ag-A&I electrodes. Three types of preparations were used. The frog ERP was nrorded from isolated retinae (i.e. retinae separated from the pigment epitheliumchoroid complex); the rat ERP, from whole excised eyes; and the frog ERG, from excised opened eyes. All animals were dark adapted for a minimum of 12 hr at room temperature prior to the experiment, and all dissections were performed under dim red light. The isolated frog retina was prepared by decapitating the frog and enucleating the eye. The upper hemisphere of the eye was then removed by cutting around the ora serrata. The retina was carefully teased away from the pigment epithelium under Ringer’s solution with lens extracting forceps. It was then removed from the eye cup by grasping the optic nerve between the pigment epithelium and the retina and by pulling to detach the nerve. The retina was placed receptor side down on a cotton pad that had been soaked in Ringer’s solution; one electrode was placed under the cotton and the other touched the vitreal side of the retina. The rat ERP was recorded between the back of the eye and the cornea, and the frog ERG was recorded between the back of the eye and the retina. During the experiments, the preparations were placed on a constant temperature block. This block was kept at constant temperature by circulating water through it with a Haake constant temperature bath. Temperature was measured with a thermister probe connected to a Yellow Springs Instruments telethermometer. Light from a Honeywell 65C electronic flash gun was focused on the preparation through a single lens and illuminated the entire retina. The flash duration was 1.3 msec (i.e. the time necessary for 90 per cent of the Rash energy to dissipate). The intensity of the stimulus flash was controlled by placing neutral density filters between the flash gun and the focusing lens, and the wavelength was controlled by interference filters with bandwidths of 6-15 run. The energy output of the flash gun at the interference filter’s wavelengths of peak transmission was measured with a Type 3 Weston Photronic Cell calibrated by the US Bureau of Standards, Photovoltaic and electrical artifacts were eliminated by appropriate shielding. The electrical responses were amplified by a Tektronix type FM 122 low-level preamplifier which was set for a 0.2-10,ooO Hz response range for the ERP and 0.2-50 Hz for the ERG. Responses were displayed on a Tektronix 561A oscilloscope with a 3A3 differential amplifier and a 2B67 time base. Pictures of the responses were recorded with a Polaroid scope camera. In the dark adaptation experiments, a dim tesrjlash,J which evoked a response of 30-70 PV, was presented to a dark adapted isolated frog retina. The amplitude of this response, measured from the peak of RI to the peak of R2 was designated the “dark adapted response”. This test flash bleached about 1-3 per cent of the rhodopsin in the retina. An intense white bleachingflash which bleached about 75 per cent of the rhodopsin was then presented. This bleaching flash was followed by test flashes identical to the one presented to the dark adapted retina, at times of 05, 1.5, 3, 6, 10, 15, 20, 25, and 35 minutes after the presentation of the bleaching flash. The amplitudes of the responses generated by these test flashes were expressed in terms of percent of the dark adapted response. The spectral sensitivity of the dark adapted retina was measured by determining the energy necessary to generate a constant response of 20-25 PV at each of five wavelengths (420, 490, 548, 590, and 630 nm). A series of flashes consisting of one flash of each wavelength was presented. In half of the experiments the flashes were presented in ascending order (420,490,548,590,630 run) and in the other half, in descending order (630,590,548,490,420 MI). For each series the intensity presented at each wavelength was adjusted so that approximately equal responses were elicited for all wavelengths in the series. After completing a series, the intensity was either increased or decreased by an equal amount at each wavelength and the series was run again. Presentation of the flashes in this manner tends to equate the effects of any progressive changes in the preparation, e.g. deteriorating preparation, bleaching, etc., at all of the wavelengths, leaving the relative spectral sensitivity comparatively unchanged. Flashes were separated by at least one minute. The spectral sensitivity of the recovered ERP was determined in the same manner as the dark adapted sensitivity, except that measurements were not begun until 17 minutes after presentation of a bleaching flash. In order to determine the rhodopsin concentration in the retinae at various times after presentation of the bleaching flash, rhodopsin was extracted from the retinae with hexadecyltrimethylammonium bromide at 0, 10, 20, or 30 minutes after the Rash. The optical density of the extract was then measured at 502 run with a Beckman DU spectrophotometer and Gilford Model 220 absorbance indicator. The density change caused by completely bleaching the extract was then measured. This density change was compared with the corresponding density change of the rhodopsin extracted from the unbleached retina of the other eye of the frog. The rhodopsin concentration of the bleached retina could then be expressed as a percent of the concentration in the unbleached eye. BAECK,et al. (1965), GOLDSTEIN (1968), and ZEWI (1939) have found that (within 5 per cent) the eyes of a frog contain equal amounts of rhodopsin. 3 Tesr flash and bleaching /lash refer to Rashes which bleach about l-3 per cent and 75 per cent of the rhodopsin in the retina, respectively. This definition is used throughout this paper.
Early Receptor Potential of the Isolated Frog (Rar~n pipie)rs) Retina
839
RESULTS
The results of the curves under Bin Fig. 380, 490, and 630 nm bleaching flash. These
dark adaptation experiments are shown in Figs. 1 and 2. The four 1 show the increase in the amplitude of responses generated by white, test flashes as a function of time after presentation of the intense curves indicate that there is a large increase in amplitude at 23 “C for
FIG. 1. A: Responses of isolated frog retinae to ten white test flashes. No bleaching flash was presented. Average of five experiments. Average range = k8.5. B: ERP recovery in isolated frog retinae at 23°C for 380 (O), 490 (A), 630 (o), and white (e) test flashes. The points are the means of the following number of experiments for each condition: 380 = 6; 490 = 4; 630 = 4; white = 8. Average ranges: 380 = &15; 490 = i7; 630 = &7; white = A12.5. C: Regeneration of rhodopsin in the isolated frog retina at 23’C. The points are rhodopsin concentrations determined by extraction. The concentration is expressed in relative units (left ordinate) with the concentration in the dark adapted eye being set at 100. The concentration can also be read as “Percent regeneration” from the scale on the right ordinate. Percent regeneration = lOO(0, - DO)/(Dd - D,) where D, = pigment density at time X, Do = density immediately after the bleaching flash (t = 0), and Dd = the density in the dark adapted eye. Points are the means of the following number of experiments for each time: 0 min, 7; 10 min, 3; 20 min, 3; 30 min, 4. Brackets indicate ranges. The dashed line is a plot of percent rhodopsin regeneration vs. time, adapted from Z~wr’s (1939) measurements on the isolated frog retina.
Fig. 2. ERP recovery in isolated frog retinae at 8’C. White test flash. Mean of 5 experiments. Average range = &13.
E. BRUCE GOLDSTEIN
840
all four types of test flashes during the first IO-15 min after the bleaching flash. The amplitude increases from about 40 per cent of the dark adapted amplitude at 0.5 minutes to 70-85 per cent at 10 min. A similar increase in ERP amplitude was observed by BROWN (1965) in the isolated retina and the excised opened eye of the toad. Photographs of the responses recorded before the bleaching flash and at 0.5 and 35 min after the bleaching flash are shown in Fig. 3. All of the responses have the characteristic ERP waveform; therefore, it is unlikely that the “photoproduct responses” described by ARDEN and IKEDA (1965), ARDEN et al. (1966a, b), CONE(1967), and PAK and BOES(1967) are present either before or after bleaching.
I 5ouv
+-i 2mrrc
FIG. 3. ERP responses of isolated frog retinae to 380, 490, 630, and white test flashes given before the bkaching Bash (the dark adapted response) and at 0.5 and 315min after the bleaching f&h. The responses to the test flashes at 10 and 15 min are not shown; howmr, their amplitudes and waveforms are approximately the same as those obtained at 35 min. Temperature = 23°C. The effect of pigment bleaching on the dark adaptation curves was minimized by using test flashes that bleached only a small amount of visual pigment. Curve A of Fig. 1 shows the amplitudes of the ERP’s generated when test flashes were presented without presenting the bleaching flash. The slight decrease in response amplitude is probably due primarily to pigment bleaching. The recovery of the ERP in the dark after presentation of an intense bleaching flash also occurs when the temperature of the retina is lowered to 8°C. This result is shown in Fig. 2. The recovery under these conditions takes 20-25 min to reach its maximum compared to the IO-15 min necessary at 23°C. GOLDSTEIN (1968) has also shown that recovery occurs when a continuous bleaching light is used in place of the brief bleaching flash used in the previous experiments.4 In order to determine the relationship between rhodopsin concentration and the rapid recovery of the ERP, rhodopsin concentration was measured as a function of time after the bleaching flash. These results are shown in Fig. 1 (Curve C). These data indicate that little 4 See note added in proof.
Early Receptor Potential of the Isolated Frog (Rann pi@ns) Retina
841
rhodopsin regeneration occurs over a 30 rnin period. This agrees with ZEWI’S(1939) data for isolated retinae (dashed line) and with the results of KSINE (1879) and BAUMANN (1965). This lack of substantial rhodopsin regeneration in the presence of the large recovery in ERP amplitude suggests that rhodopsin regeneration is not responsible for the ERP recovery. Regeneration of rhodopsin by photoreversal (cf. MATTHEWSet al., 1963; WIr_LIAh& 1964) can also be eliminated as a cause of recovery since the test flashes are too dim to regenerate an appreciable amount of rhodopsin. Also, good recovery is measured with the 630 nm test flash, and, since the photoproducts absorb little light of this wavelength (DARTNALL, 1953), little photoreversal can occur. Thus, photoreversal or rhodopsin regeneration cannot cause the recovery. However, it is possible that ERP recovery is caused by the regeneration of cone pigments. It is well established that the cone pigments regenerate much faster than rod pigments (WALD, 1959), and the time course of the recovery compares favorably with the rate of regeneration of cone pigments itt vitro (WALD et al., 1954-U) and in vim (RUSHTON,1957). Spectrul
serisitivify
In order to determine more directly which pigments are responsible for the frog ERP, spectral sensitivity functions for “dark adapted” and “recovered” retinae were determined. A “recovered” retina is one that has been bleached with a bleaching flash and then has
FIG. 4. Spectral sensitivites of the frog and rat. All points and sensitivity curves are expressed in terms of log reciprocal energy units. Each point is the mean of six experiments. Average rangeat eachpoint = kO.12 log units. Upper points: Spectral sensitivity of the dark adapted and recovered ERP recorded from the isolated frog retina. Criterion response = 20-25 pV. Temperature = 23°C. The line is GORDON’S (1967) photopic ERG curve for the frog. The opened circles are EBREYand CONE’S(1967) data for the ERP sensitivity of the excised frog eye. Lower points: Spectral sensitivity of the ERP recorded from the excised eye of the albino rat at 23°C and the sensitivity of the ERG recorded from the excised opened eye of the frog ar 10°C. A criterion amplitude of 15-20 PV was used for the rat, and the criterion was 15 FV for the frog. The solid line is DODT and Ecm’s (1961) sensitivity curve for the dark adapted rat h-wave. The dashed line is the absorption spectrum of rhodopsin (Amax = 502 nm) with a maximum density of 050 (DARTNALL,1953). The opened squares are CONE’S(1964) data for the ERP sensitivity of the albino rat.
842
E. BRUCE GOLDSTEIN
“recovered” in the dark for at least 17 min. As indicated in Fig. 1, ERP recovery is essentially completed by 17 min. after the bleaching flash. ERP sensitivity curves for dark adapted and recovered isolated frog retinae are shown in Fig. 4. The points for the dark adapted and recovered ERP’s are identical within experimental error. GORDON’S(1967) photopic ERG sensitivity curve for the frog (solid line) fits the points well. Although there are not enough points to precisely determine the wavelength of maximum sensitivity, it appears that the curve peaks somewhere between 560 and 590 nm. This agrees with the results of LIEBMAN’S(1967) microspectrophotometry experiments. He found that one of the frog cone pigments absorbs maximally at 580 nm. EBREYand CONE’S(1967) data for the sensitivity of the whole excised frog eye are also plotted in Fig. 4 and agree with the results of the present experiments. Although all the curves in Fig. 4 are plotted in terms of log relative sensitivity, it should be noted that the absolute sensitivity of the dark adapted and recovered frog ERP is the same to within 0.1 log units at each wavelength. This result would be expected from the large magnitude of the recovery which occurred during dark adaptation. If the long wavelength of maximum sensitivity and the broadness of the frog ERP sensitivity curves were due to artifacts caused by factors such as faulty calibration of the flasher or transmission outside the main bandpass of the interference filters (cf. WALD, 1945), then other sensitivity curves, determined using the same equipment and procedures, would also be broadened and shifted towards longer wavelengths. In order to test for this possibility, the spectral sensitivities of the rat ERP and frog ERG were determined. The points in Fig. 4 indicate that the rat ERP sensitivity peaks at 500 nm and agrees with CONE’S (1964) rat ERP sensitivity and DODTand ECHTE’S (1961) determination of dark adapted rat b-wave sensitivity. The spectral sensitivity of the frog ERG also peaks at about 500 nm and agrees with the absorption spectrum of rhodopsin with a maximal density of 0.50.5 Thus, the rat ERP and frog ERG sensitivity curves are not broadened or shifted to longer wavelengths. It is, therefore, concluded that the frog ERP sensitivity curves are correct. These curves suggest that the sensitivity of the ERP recorded from dark adapted and recovered isolated frog retinae is mediated by a cone mechanism, or mechanisms, which result in maximum sensitivity at about 560-590 nm. DISCUSSION The previous results indicate that the cones may play a major role in generating the ERP in the isolated frog retina. Although it is difficult to determine the exact contributions of rod and cone pigments to the ERP, it appears that the contribution of the rod pigments is quite small. This is a surprising result because the rod pigments make up about 90 per cent of the total concentration of visual pigments in the frog’s retina (LIEBMAN, 1967)6. CONE (1967), BRWDLEYand GARDNER-MEDWIN (1966), and PAK and EBREY(1965) have suggested 5 The frog ERG sensitivity should actuall-y correspond to the slightly broader absorption spectrum of rhodonsin with a maximum density of@75 since this is the density of rhodopsin in the frog% rods (DENTON and W&LIB, i9S; D~MJW,19Sgi DAWNALL, 1962). This dis&pancy cot&l b due to e~perim&tal error or it is possible that the rhodopsin concentration in the frogs used in these cxp&rnents could kve been low due to prolonged storage at low temperatures. However, since the frog ERG sensitivity is actually xurroHIt=r than what would be predicted theoretically, this discrepancy cannot exptain the very broad frog ERP sensitivity curves. 6 SAXIW(1954) has found that in the common frog, Ranu temporaria, 45 per cent of the receptors are cones. However, the cones are much smaller than the rods; therefore, cone pigments account for only 10 per cent of the visual pigment concentration in the retina (LIEBUN, 1967).
Early Receptor Potential of the Isolated Frog (Rarta pipiens) Retina
843
that the ERP is generated by the direct action of the visual pigments. Since the frog retina is dominated by the rod pigment, rhodopsin, it would be expected that the action of this pigment would dominate the ERP in this retina. However, this is clearly not the case; the cones appear to contribute an electrical signal far out of proportion to their visual pigment content. The mechanism that is responsible for this high level of cone activity is not clear. It is unlikely that a neural mechanism would operate to inhibit or enhance the rod or cone system since the visual pigments are located distal to synapses at which neural effects might OCCUI (cf. DOWLING,1967). It is possible that there are basic differences in the chemical properties and/or the distribution of charges in the pigments contained in the rods and cones. These differences may make it possible for the cone pigments to generate a much larger signal than the rod pigments. However, even if large differences do exist between rhodopsin and the cone pigments, it seems unlikely that these differences would account for the almost total absence of rod responding. It should be noted that the spectral sensitivity of the ERP of the live frog has not yet been determined. It is possible that the cone-dominated response found in the present experiments occurs only in the isolated retina or whole excised eye (cf. Fig. 4). Besides rhodopsin and the cone pigments, the frog’s retina also contains a pigment in the “green rods” (DENTONand WYLLIE, 1955; DARTNALL,1967). This pigment absorbs maximally at 433 nm, far from the maximal sensitivity of the ERP. Therefore, the role of this pigment in generating the ERP is probably quite small. DENTONand WYLLIE(1955) have suggested, however, that the green rods also contain a photostable pigment absorbing in the range 560-680 nm. If this pigment mediates the responses recorded in the present experiments, it would be necessary to hypothesize a mechanism other than pigment bleaching to explain the large adaptation effects observed in these experiments. Furthermore. this pigment does not absorb appreciably at 520 nm, a wavelength at which the ERP sensitivity is still quite high. The rapid recovery of the ERP during dark adaptation in the absence of significant rhodopsin regeneration indicates that cone pigments may be regenerating in the isolated retina. Thus, there may be differences between the mechanisms of rod and cone pigment regeneration or between the environments inside the rods and cones. The enzymes necessary for catalyzing rhodopsin regeneration are contained in the pigment epithelium; however. the catalysts for cone pigment regeneration may be contained in or near the receptors. There appears to be no published work bearing on the question of cone pigment regeneration in the isolated retina. The crucial test for the hypothesis of cone regeneration would be. microspcctrophotometric experiments on single cones. If cone pigments do, in fact, regenerate in the isolated retina, the cone pigments of a mixed retina could be isolated by simply bleaching all of the visual pigment in the isolated preparation and then allowing time for the cone pigments to regenerate. Nore added in proof-Recovery also occurs when a second bleaching flash is presented at some time, 1. after the first bleaching flash. For example, in a typical experiment the response to a white test flash at r=35 min was 82 (dark adapted response= 100). A second bleaching flash was presented at t=35,5 min and a test flash at 36 min generated a response of 32. Recovery then proceeded again until at 70.5 min the response generated by a test flash had an amplitude of 73. The second recovery was slower than the initial recovery. The ERP reached its maximum amplitude lo-15 min after the first flash. However, it took about 20 min for the recovery following the second flash to reach completion. Acknowledgemenfs-I wish to thank T. P. WILLIAMSfor his advice throughout this investigation, L. A. RICKS for his helpful suggestions, and G. WALD for the use of his Weston cell and his criticism of the manuscript. I also thank thank R. A. CONE and P. STEWART for reading preliminary drafts of the manuscript. c
E. BRUCEGOLDSTEIN REFERENCES hDEN,
G. B. and IKEDA,H. (1965). A new property of the early receptor potential of the rat retina. Nature, fond. 288, 1100-l 101. ARDEN,G. B., IKEDA,H. and SIEGEL,I. M. (1966a). Effects of light-adaptation on the early receptor potential. Vision Res. 6,357-372. ARDEN,G. B., IKEDA,H. and Stzoet, 1. M. (1966b). New components of the mammalian receptor potential and their relation to visual photochemistry-. Vision Res. 6, 373-384. BAECK,I., DONNER,K. 0. and Rauzza, T. (1965). The screening effect of the pigment epitheiium on the retinal rods in the frog. Vision Res. 5, 101-I 11. BAUMANN,C. (1965). Die Photosensitivitat des Sehpurpurs in der isolierten Netzhaut. Vision Res. 5, 425-434. BRINDIPY,G. S. and GARDNER-MEDWIN, A. R. (1966). The origin of the early receptor potential of the retina. J. Physioi, Lond. 182, 185-194. BROWN,K. T. (1965). An early potential evoked by light from the pigment epithelium-choroid complex of the eye of the toad. Nature, Lond. 287, 1249-1253. BROWN, K. T. and MURAKAMI.M. (1964). A new receptor potential of the monkey retina with no detectable latency. Nature, Lond. 281, 626-628.
BROWN,K. T., WATANAB~Z, K. and MURAKAMI, M. (1965). The early and late receptor potentials of monkey cones and roda. ColB Spring Harb. Symp. quant. Biol. 30, 457-482. CONE. R. A. (1964). Early receptor potential of the vertebrate retina. Nature, Lond. 264, 736-739. CONE, R. A. (1965). The early receptor potential of the vertebrate eye. Cofd Spring Harb. Symp. quant. Biol. 30.483-491. CONE, R.
A. (1967). Early receptor potential: photoreversible charge displacement in rhodopsin. Science,
N. Y. 155,1128-1131.
DARTNALL,H. J. A. (1953). The interpretation of spectral sensitivity curves. Brit. med. Bull. 9, 24-30. DARTNALL,H. J. A. (1962). The photobiology of visual processes, in 7he Eye, Vol. 2, Davson, H., ed., 321533, Academic Press, New York. DARTNALL,H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. DEMON, E. J. and WYLLIE,J. H. (1955). Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiof., Loud. 127, 81-89. D~DT, E. and Ecxrz, K. (1961). Dark- and light-adaptation in the pigmented and white rat as measured by the ERG threshold. J. Neurophysiol. 24, 427-445. DoNNER, K. 0. (1958). The spectral sensitivity of vertebrate retinal elements, in Visual Problems of Colour, 539-566, H.M. Stat. Office, London. D~WLING, J. E. (1967). The site of visual adaptation. Science, N. Y. 155, 273-279. Eattar, T. G. and Co=, R. A. (1967). Melanin, a possible pigment for the photostable electrical responses of the eye. Nature, Land. 213,360-362. GOLDSIW[N, E. B. (1968). Early receptor potential of the isolated frog (Rana pipiens) retina. Doctoral dissertation, Brown Univ., Prov., R.I. GORDON, J. (1967). ERG spectral sensitivity of the frog, Rana pipiens. Master’s thesis, Brown Univ., Prov., R.I. KUHN, W. (1879). Chemische Vorgange in der Netzhaut, in Hermann’s Hanab. Physiof. 3, Leipzig, 235. bEl$blAN, P. A. (1967). Vision Res.. in preparation. MA’I?HEWS,R., HUBBARD, R., BROWN,P. K., and WALD, G. (1963). Tautomeric forms of metarhodopsin. J. gen. Physiol. 47,215-240.
PAK, W. L. (1965). Some properties of the early electrical response in the vertebrate retina. Cold Spring Harb. Symp. quant. Biol. 30,493-499.
PAK, W. L. and Bees, R. J. (1967). Rhodopsin: responses from transient intermediates formed during its bleaching. Scicnee, N. Y. 158,1131-1133. PAK, W. L. and CONE,R. A. (1964). Isolation and identification of the initial peak of the early receptor potential. Nature, Lond. 204,436-438. PAK,W. L. and EBRZY,T. G. (1965). Visual receptor potential observed at sub-zero temperatures. Nature, Land. 208, 484-486. PAK, W. L. and E~EY, T. G. (1966). Early receptor potentials of rods and cones in rodents. J. gen. Physiof. 49,11991208. RUS~N, W. A. H. (1957). Physical measurement of cone pigment in the living human eye. Nature, Lond.
179, 571-573. (1954). The development of visual cells. Annls Acad. Sci. fenn. Ser. A, 23, l-95. (1945). The spectral sensitivity of the human eye. J. opt. Sot. Am. 35, 187-l%. WALD,G. (1959). The photoreceptor process in vision, in Handb. Physiul., 671-692, Sect. I, Vol. I, edited by J. FIELD, Am. Physiol. See., Washington, D.C.
SAXEN, L. WALD, G.
Early Receptor Potential of the Isolated Frog (Ram
pipiens)
845
Retina
G., BROWN, P. K., and SMITH, P. H. (1954-55). lodopsin. J. gen. Physiol. 38, 623-681. WILLIAMS,T. P. (1964). Photoreversal of rhodopsin bleaching. J. gen. Physiol. 47, 679-689. ZEWI, M. (1939). On the regeneration of visual purple. AC/U. Ser. Sci. /em. N.S. B, l-57. WALD,
Abstract-The decreases recovers in 10-15
amplitude
of the early receptor potential
when the retinal
(ERP) of the isolated
visual pigments are bleached; however, SO-90 per cent of that prior to bleaching.
in the dark to minutes at 25°C and 20-25 minutes chromatic (380, 490, or 630 nm) test flashes this period of rapid ERP recovery, and the 560-590 nm. These results suggest that the generated by cone pigments.
the amplitude This recovery
frog retina subsequently is completed
at 8”C, and is observed when white or monoare used. Little rhodopsin regenerates during spectral sensitivity of the ERP peaks at about ERP recording from the isolated frog retina is
R&urn&-L’amplitude du potentiel initial de rkcepteur (ERP) de la retine isolee de grcnouille dtiroit quand les pigments visuels de la r&tine se dCcolorent; dependant, I’amplitude se retablit ensuite dans I’obscuritt B 80-90 pour cent de sa valeur avant dtcoloration; cette rCcupCration est compiite en 10-15 minutes g 23°C et 20-25 minutes B 8’C, et s’observe avec les &lairs tests utilist%, blancs ou monochromatiques (380, 490 ou 630 MI). La rhodopsine se r&?ntre peu pendant cette tiriode de rtiuperation rapide de I’ERP, et le maximum de sensibilitb spectrale de I’ERP se situe vers 560-590 nm. Ces rtsultats suggtrent que I’ERP enregistrt dans la rCtine isoltke de grenouille est engendrt pas les pigments des cbnes. Zusammenfassung-Die Amplitude des friihen Rezeptorpotendals (ERP) der isolierten Froschnetzhaut nimmt ab, wenn die Netzhautpigmente gebleicht werden. Im Dunklen erholt sich die Amplitude jedoch wieder auf 80-90 prozent des urspriinglichen Wertes. Diese Erholuna ist bei 23°C in IO-15 Minuten abneschlossen und bei 8°C in 20-25 Minuten. Sie wird beobachtet, wenn weiRe oder monoc&omatische (380, 490 oder 630 nm) Testblitze verwendet werden. WIhrend dieser Periode der schnelien ERP-Erholung wird nur sehr wenig Rhodopsin regeneriert. Die Spektralempfindlichkeit ist maximal bei etwa 560-590 nm. Diese Ergebnisse weisen darauf hin, dal3 das ERP-Potential der isolierten Froschnetzhaut durch Zapfenpigmente erzeugt wird. AMMkiTyAa paHHer0 peUenTopHor0 nOTeHUuana (PPn) H30nHpOBaHHOti CCT’IaTKU JD?ryIUK&i yMeHbUIaeTC5l, KOrAa 3pHTeJIbHble ITkirMeHI’bI CeT’faTKU BbIUBeTaKIT, OAHaKO, el-0 aMnJUiTyAa BOCCTaHaBJIliBaeTCII 3aTCM npH lTOCJleAyIOLUefi TeMHOBOk aAanTaUHH A0 80-90 lTpOUeHTOB t242nepBOHaWJlbHOti BcJlUWHbl. 3TO BOCCTaHOBJleHkie 3aKaHrasanocb B TeqeHkie lo-15 MRH npri 23°C u 20-25 MBH npu 8°C li Ha6nmnanocb B Tex cnyrarrx, Korna ynOTpe6nRnaCb 6enart HnMMOHOXpOMaTWzCKaR (380, 490 EinM 630 HM) TeCTOBaR BCllbILUKa. B Te’ieHUc 3TOr0 nepHOAa 6blcTpOro BOCCTaHOBJEHHR PPn PereHepHpOBWO ManO POAOnCMHa U MaKClrMyM CneKTpaJlbHOii YyBCTBHTenbHOCTB HaXOAHlnCII OKOJlO 560-590 HM. 3TU pe3yJlbTaTbI 3aCTaBJlRH)T AyhIaTb, ‘IT0 PPn, 3anUCbIBaeMblti OT Ii3OJlMpOBaliHO~ CeT’IaTKM Jl5iry!JK&i, reHepl4pyeTCR KOIl60’iKOBblMM nHl-MCHTaMM.
Pe3loMe -