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Visual pigments and the early receptor potential of the isolated frog retina
Vi&r Rcs. Vol. 8, pp. 953-963.
Pergamon Press 1968. Printed in Great Britain.
VISUAL PIGMENTS AND THE EARLY RECEPTOR POTENTIAL OF THE ISOLATED FROG RETINA1 E. BRUCEGOLDSTEIN TheBiological Laboratories, Harvard University, Cambridge, Massachusetts 02138 (Received 18 March 1968)
THE EARLY receptor potential (ERP) has been identified as a response that is generated by the direct action of light on visual pigments in the outer segments of the photoreceptors I, 1964; CONE, 1964, 1965, 1967). Much evidence in support of (BROWN and MURAICAM this conclusion has come from work on the rod-dominated eye of the albino rat. CONE (1964) and PAK and CONE (1964) have shown that the action spectrum of the rat ERP corresponds to the absorption spectrum of rhodopsin. CONE (1964) found that the amplitude of the ERP is linearly related to the amount of rhodopsin bleached by a brief gash. He also found that, following a bleaching exposure to light, the amplitude of the rat ERP increases in the dark at a rate comparable with the rate of pigment regeneration. If, by irradiating the rat eye with a light that bleaches the rhodopsin, the ERP is eliminated, it returns when the rhodopsin is photoregenerated (from long-lived intermediates of bleaching) with a blue flash (ARDEN,etal., 1966; CONE, 1967). All these results indicate that in the albino rat the ERP is closely linked to rhodopsin, the dominant visual pigment of the rat retina. In the frog however, a different situation exists. Although at least ninety percent of the visual pigment in the frog retina is rhodopsin (LIEBMAN,personal communication), this pigment appears to play only a small role in generating the ERP. GOLDSTEIN(1967a,b) has shown that after a light adaptation that bleaches most of the rhodopsin and reduces the ERP amplitude, the ERP recovers in the dark long before there has been an appreciable regeneration of rhodopsin. Furthermore, the action spectrum of the ERP of the isolated frog retina is maximally sensitive at CQ.580 run, whereas a response mediated by rhodopsin should be maximally sensitive at about 502 nm. Thus, the frog ERP arises predominantly from a pigment whose spectrum lies far to the red from rhodopsin, presumably the iodopsin of the cones (GOLDSTEIN,1967a,b). LIEBMANand ENTINE (1967) have measured the absorption spectrum of frog iodopsin by microspectrophotometry of single cones and find that it absorbs maximally at about 570-580 nm. In the present experiments, the retina was irradiated with red light which selectively bleached the long-wavelength cone pigment. This adaptation shifted the maximum sensitivity of the ERP to shorter wavelengths, indicating that pigments other than the longwavelength pigment also generate an ERP. The relationship between the ERP and rhodopsin was also investigated by comparing the ERP of dark adapted retinas with that of retinas in which almost all the rhodopsin had been bleached. * This research was supported by a National Science Foundation grant to George Wald and a US Public Health Service Postdoctoral Research Fellowship to the author. 953
E. BRUCEGOLDSTEI~~
954
METHODS AND MATERIALS All animals were dark adapted for a minimum of 12 hr at room temperature prior to the cxperimcnts, and all dissections were performed under dim red light. The frog retina was dissected out by carefully teasing it away from the pigment epitheiium under Ringer’s solution. Retinas so isolated, rather than the whole eye or live animal, were used in all experiments in order to eliminate the electrical response generated by the pigment epithelium-choroid complex (cf. BROWN,1965; EBREYand CONE, 1967) and to block rho&p&t regeneration (cf. ZEWI, 1939; GOLDSTUN,1967b). The retina was placed receptor side down on a moist cotton pad. Eiectrical contact was through cotton wick Ag-AgCl electrodes, one under the cotton pad, and the other in contact with the vitreal side of the retma. During the ex~riments the cotton pad and retina were placed on an aluminum block kept at a constant temperature of 23°C by circulating water through it from a Haake constant-tem~rature bath. Temperature was measured with a thermister probe and a Yellow Springs Instruments telethermometer. The stimulating light was a Honeywell 65C electronic flash gun with flash duration 1.3 msec. The adapting light from a General Electric 1OOWmicroscope lamp passed through a Jena KG 3 heat filter. The stimulating and adapting beams were superimposed by means of a beamsplitter, and were focused on the retina by a single lens. Both the stimulating and the adapting lights illuminated the entire retina. Their intensities were controlled by Bausch and Lomb neutral filters, and their wavelengths by BairdAtomic interference filters having half-bandwidths of 6-15 nm (stimulating light) and by a Wratten #92 filter (adapting light). Calibration of the interference filters with a Cary 11 recording spectrophotometer indicated that the rn~rn~ transmission at wavelengths outside the main bandpass was O-05 percent. The temperature rise at the retina caused by the adapting light was, at most, 0*5”C. The energy output of the flash gun at the wavelengths of peak transmission by the interference filters was measured with a calibrated Type 3 Weston Photronic Cell. Photovoltaic and electrical artifacts were eliminated by appropriate shielding. The electrical responses were amplified either by a Tektronix type FM 122 low-level preamplifier or by a Grass P5 AC preamplifier. The preamplifier bandpass was 0*2-10,000 Hz. Responses were displayed on a Tektronix 502 oscilloscope. Rhodopsin concentrations were determined by extracting the pigment with hexadecyltrimethylammonium bromide (cf. BIUDOES, 1957), and measuring the change in absorbance at 502 nm upon bleaching in a Cary 11 recording spectrophotometer. The absorbance change of the rhodopsin extracted from the experimental retina was compared with that of the rhodopsin extracted from the unbleached retina of the same frog’s other eye. The rhodopsin concentration of the experimental retina was expressed as a percentage of the concent~tion in the unbieach~ eye. BAECK,et uf. (196Jf, GOLDSTEIN (1968) and ZEWI (1939) have found that the two eyes of a frog yield equal amounts of rhodopsin within 5 percent.
RESULTS Chromatic adap’tation of the ERP Isolation of a short-wavelength component of the ERP. To determine whether the ERP of the isolated frog retina is generated by pigments other than the long-wavelength cone pigment (h,,, = 570-580 nm), the retina was irradiated with red light (X> 630 nm) which bleaches this pigment while sparing rhodopsin and pigments that absorb at shorter wavelengths. The intensity of a monochromatic test flash was first adjusted to produce a 30-60 PV response in the dark adapted retina. A single test flash of this intensity causes only a small decrease in the amplitude of the ERP generated by subsequent flashes. ERP amplitudes were measured from the peak of the cornea-positive phase of the ERP, Rl, to the peak of the negative phase, R2. To determine the course of adaptation to red Iight, two or three monochromatic test flashes were presented to the retina at various intervals after the start of the ~l~u~nati~n. Such measurements, made at 420, 490, 548, 590 and 630 nm, are shown in Fig. 1. The ERP amplitudes, expressed as percent of the dark adapted amplitude, are plotted vs. time. If only one pigment were responsible for the ERP, the course of adaptation plotted as percentage of the dark adapted response at each wavelength should be independent of
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
955
the wavelength of the test flash. 2 The results in Fig. 1, however, indicate that the extent of adaptation increases markedly with the wavelength. This suggests that some pigment in addition to the long-wavelength cone pigment must generate an ERP. The dashed line in Fig, 1 indicates that the adapting light bleaches little rhodopsin.
ADAPTATION TIME - MINUTES Chromatic adaptation of the ERP. ERP amplitude as a function of adaptation time for monochromatic test flashes presented during adaptation with red light. The number beside each curve indicates the wavelength of the test flash. All amplitudes are expressed relative to the ampiitude generated by the test Dash in the dark adapted retina. Each curve is the average of two experiments. The dashed line represents the reiative rhodopsin concentration (dark adapted concentration = 100) during adaptation to red light, as determined by pigment extraction. FIG. I.
In another set of experiments, the period of adaptation was extended to eight minutes. Test flashes of 420, 490, 548, 590 and 630 nm which generated 30-60 ttV responses were presented to a dark adapted retina. The retina was then exposed to the red adapting light. Beginning at 2 min after the initiation of adaptation, test flashes were presented every 30 set until one test flash at each wavelength had been presented. This series of test flashes was then repeated at six minutes after the start of adaptation. In half of the experiments test flashes were presented in ascending order (420-630 nm), and in the other half in descending order (630-420 nm). Figure 2 shows the results of these experiments. The percentages of the dark adapted response, 2-4 and 6-8 min after the beginning of red-adaptation, are plotted against the wavelength of the test flash, The amplitude decreases most during the first minute of red-adaptation and only slightly further after 4 min. At 630 nm the amplitude decreases to almost zero, whereas at 420 nm the amplitude decreases only to 78 percent of its dark adapted value after 2-4 min of adaptation, and 64 percent after 6-8 min. The results presented in Figs. 1 and 2 indicate that more than one mechanism is responsible for generating the ERP in the isolated frog retina. The long-wavelength mechanism 2 If the pigment density were high, self-screening by the pigment (cf. BRDIDLY,1960;GOLKSTEINand WILLIAMS,1966)
would cause the course of ERP adaptation to depend somewhat on the wavelength of the test flash. However, the effect of self-screening is small in the present experiments since the maximum absorbance of the Iong-wavelength cone pigment is only about 020 (LJEEMAN, personal comrmmication).
956
E. BRUCE GOLDSTEIN
is almost completely eliminated by adaptation with red light, whereas the shorterwavelength mechanism is only slightly affected by red adaptation. This short-wavelength mechanism, however, is eliminated by irradiation with white light. Thus it too is photosensitive. I
I
w ‘oo-
(: 2
w” 50Lz
w” k
so-
a
2 g
.o-
d
!s w to: 0
420
460
200
140
520
620
WAVELENGTH-nm Fro. 2. ERP amplitude
Measured
at 2-4
min
I
during red adaptation as a function of wavelength of test flash. of adaptation (@); measured at 6-8 min (0). Averages of five experiments.
’
z 2.05 I= cn $
l.5-
Y 5
l.O-
ii
a
(3 o.ss L
J
I
420
460
500
540
S60
WAVELENGTH
- nm
620
660
FIG. 3. Action spectra for generating the ERP of the frog retina under various conditions. Sensitivity is the reciprocal of numbers of photons per test flash that excite a constant amplitude of ERP. Dark adapted (e); Bleached and recovered (0); Red adapted (&; Red adapted, from equation 1: 2-4 mitt of adaptation (o), 6-g min adaptation (+). The points calculated by equation 1 are positioned on the ordinate to reflect the actual differences in sensitivity between the dark adapted and red-adapted retinas. The sensitivity of the recovered and dark adapted retinas have been equated at 630 nm. Relative rhodopsin concentrations: dark adapted = 100; red adapted = 100; recovered = 5. The red adapted sensitivity (& is the result of experiments on nine retinas. The other functions were determined on 4-5 retinas.
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
957
Actron spectra. The action spectrum of the ERP remaining after red adaptation was measured by determining the energy necessary to elicit a criterion response of about 20 PV at 420,460,490,54& 590 and 630 nm during red adaptation3. The action spectrum is indicated by the filled triangles in Fig. 3, which represent the averages of sensitivity measurements on nine retinas. The sensitivity could not be measured accurately at 630 nm since adaptation caused the response to decrease nearly to the noise level of the preparation. The sensitivity of the red adapted retina can also be determined from the data in Fig. 2 since the relation between ERP amplitude and intensity of test flash (A-I function) in the red adapted retina (Fig. 4), and the sensitivity of the dark adapted retina are known. The dark-adapted sensitivity has been determined by GOLDSTEIN(1967b) and was redetermined in the present experiments (cf. Fig. 3). The action spectrum of the red-adapted retina was determined from the following formula (cf. Appendix):
log %l
= log s,, -
log 100 - log(%DA) C m
1
(1)
where S,, = sensitivity of the red adapted retina at a given wavelength. S = sensitivity of the dark adapted retina at the same wavelength. O/,& = percent dark adapted response remaining after adaptation with the red adapting light. m = slope of the log amplitude vs. log intensity function for the red-adapted retina. The slope is 0.80 in the present experiments (cf. Fig. 4). The action spectrum determined by equation (1) is indicated by the open circles and S’s in Fig. 3. The circles and +‘s represent, respectively, the sensitivities calculated from the data obtained after 24 and 6-g min of adaptation. The ERP action spectrum of the red adapted retina presents a broad maximum from about 420 to 500 nm, decreasing at longer wavelengths. It seems clear that the mechanism that generates the ERP in the red-adapted retina is much more blue-sensitive than the mechanism that dominates response in the dark adapted retina. The flatness of the curve suggests that more than one visual pigment may be responsible for the short-wavelength sensitivity. Rhodopsin (A,,, = 502 nm) and the visual pigment of the green rods (&lax = 433 nm; DARTNALL, 1967) may contribute to the generation of the short-wave response. The spectral sensitivity of the ERP of the dark adapted retina was determined earlier at 5 wavelengths (GOLDSTEIN,1967a,b), and here has been re.-measured at 11 wavelengths. Since the presentation of 11 test flashes to a retina would bleach appreciable amounts of pigment, the action spectrum was determined in two separate experiments. In one set of retinas the relative sensitivity was determined at 420, 490, 548, 590 and 630 nm, and in the other set, at 420, 460, 522, 560, 600, 620 and 660 nm. The two functions were then combined by equating the relative sensitivities at 420 nm, the common wavelength in each series. The resulting curve, shown in Fig. 3, indicates that the maximum sensitivity is at about 580 nm. -\Theaction spectrum was measured during adaptation because the redgmpitive pignent -tea in the dark (Go-, 1967a,b). This pigment also regenuntea continuously in tbe light, as ewknced by the fact that the amplitude of the 630 nm test flash decrrpses to a steady state level wblcb is maintained as long as the light is on (GOLD-, unpublished observations). Increa&g the adapting izttansity decreases the level of the steady state. In the present experiments a steady state kvel close to zero is reached after about 1 min. of light adaptation.
958
E.
BRUCE GOLDSTEIN
Rhodopsin and the frog ERP
Five experiments were performed to investigate further the observation that there is little correlation between the ERP and rhodopsin in the isolated frog retina (GOLDSTEIN, 1967a,b). In the first experiment, rhodopsin concentration was measured during 30 set of adaptation with red light. The results of this experiment, shown in Fig. 1, indicate that though this adaptation reduces the response to a 630 nm test flash almost to zero, it bleaches little rhodopsin. In the other four experiments, the action spectra, effect of red adaptation, A-I functions, and waveforms were measured in retinas from which most of the rhodopsin had been removed by bleaching. The results of these experiments are compared with the results of analogous experiments on dark adapted retinas. Action spectrum. Retinas were irradiated with white light for 2 min, followed by a 2-min bleach with orange light. The retinas were then kept in darkness for 30 min to allow the ERP to recover. Isolated retinas that had been bleached and then dark adapted are referred to as “recovered” retinas 4. Direct extraction of the rhodopsin 30 min after light adaptation indicated that these retinas contained less than five percent of their dark adapted concentration of rhodopsin. The sensitivity of the recovered retina is indicated by the open squares in Fig. 3. The points at 420 and 490 nm fall only slightly below the dark adapted action spectrum. Thus, decreasing the rhodopsin concentration has little effect on the spectral sensitivity. This result is in agreement with GOLDSTEIN’S(1967b) finding that the action spectra of dark adapted retinas and retinas that contain only 25 percent of their dark/adapted concentration of rhodopsin are identical. Red adaptation. If recovered retinas that contain only five percent of their rhodopsin are irradiated with the red adapting light, the ERP amplitude measured with the 630 nm test flash decreases more than the ERP amplitude measured with the 490 nm test flash. After 2 min of adaptation, the response generated by the 630 run flash decreases almost to zero. The 490 response, however, decreases only to 50 percent of its amplitude prior to adaptation. This finding is in agreement with the results shown in Fig. 2 for the dark adapted retina. Thus, the short-wavelength response of the red-adapted retina does not depend upon rhodopsin.5 Amplitude vs. intensity. Amplitude vs. intensity (A-I) functions for dark adapted (A), recovered (B), and red-adapted (C) retinas are plotted in Fig. 4. The red-adapted function represents the response of the pigments remaining after bleaching the long-wavelength cone pigment; the recovered function represents the response of a retina containing regenerated cone and possibly green-rod pigments and ten percent of the retina’s dark adapted concentration of rhodopsin. 6 A comparison of the dark adapted and red-adapted 4 The procedure for bleaching and dark adaptation was not the same in all experiments. The procedures used for preparing recovered retinas in each set of experiments were as follows: Acrion spectrrtnt and red udaptarion: 2-min white bleach followed by 2-min orange bleach; 30-min dark adaptation. Amplirrrde vs. intensity: 3-min white bleach; 20-min dark adaptation. Waveform: 1 msec flash bleach (log relative intensity = 0); 3%min dark adaptation. 5 This experiment does not rule out, however, the possibility rhodopsin is contributing to the short-wavelength response.
that the small amount
of residual
6 Rhodopsin concentrations were measured by extraction immediately after the three-minute white bleach, 20 min after bleaching, and after a flash of log I = 0 at 23 min after bleaching. The concentration was ten percent of the dark adapted concentration under all conditions, indicating that no regeneration of rhodopsin occurred in the dark and that there was no net production of rhodopsin due to photoregeneration by the high intensity flash presented at t = 23 min.
Visual Piefnents and the Early Receptor Potential of the Isolated Frog Retina
959
functions indicates that red adaptation reduces the response amplitude by about 060 log unit but causes little change in the slope of the function. The slope of the recovered A-l function is smaller than the slope of the dark-adapted function. r-
to-
., -
o-
.5-
6 .Oi
I
-3
-2
LOG
RELATIVE
-I
0
INTENSITY
FJG. 4. Relation between log amplitude and log intensity of test flash in: (A) dark adapted retinas (relative rhodopsin concentration = 100); (B) recovered retinas (R = 10); (C) redadapted retinas (R = 100). Curves B and C have been shifted down @15 log unit on the ordinate. The actual difference between curves A and B at log f = -3.02 is Q07 log unit. For the dark adapted and recovered retinas four white ffashes were presented to each retina, three of log I = -3.02, -272, -248, and one of higher intensity. The same procedure was used for the red-adapted retina, but only two flashes were presented to each retina, one of low intensity (log 2 = -2.3) and one of high intensity. By using this procedure it was possible to combine the results from many retinas which differed in responsiveness. In order to compensate for differences in the level of responding in different retinas, alI responses for a given retina were multiplied by a constant in order to minimixe variability when the data for different retinas were combined. The huge points on curve C are for monochromatic test fhshes of 490 (0) and 548 (0) nm. Bach point is baaed on eight experiments. The 490 and 548 nm functions have been positioned on the ordinate to correspond to the actual response amplitude generated by the gashes but have been arbitrarily positioned on the abscissa. It was not possible to determine the A-I functions at other wavelengths due to lack of flash energy. All measurements on red-adapted retinas were begun after two minutes of adaptation.
The slope of the dark adapted A-I function in Fig. 4 is about 0.80 at low intensities; another independent set of experiments yielded a slope of 0.85. These slopes are smaller than CONE’S (1964) value of l-0 for the slope of the albino rat A-I function7 but compare favorably with the slope of the ground squirrel ERP A-I function (PAK and EBREY, 1966). The A-I function of the albino rat determined in the present experiments using the same apparatus and procedure as was used for the frog, has a slope of about 0.86. Thus, in the present experiments the slope of the rat A-I function is slightly lower than that determined by Cone, but the difference between the frog and rat functions is not significant. 7 However, a line with slope less than 1.0 fits Cone’s data well if his low intensity data are weighted more heavily.
960
E.BRWCEGOLDSTEIN
Waveform. Differences between dark adapted and recovered retinas are also reflected by differences in the Rl/R2 ratio of the ERP. In Table 1 RI/R2 is tabulated for dark adapted and recovered retinas. The rhodopsin concentration in these recovered retinas is 20 percent of the dark adapted concentration. TABLE 1
Log relative intensity
N
RI/R2 Dark adapted
RI/R2 Recovered
DA-Recovered
-2.3 0.0
8 20
0.36 1.47
0.37 2.00
-0*01* -0*53t
N = nnmber of retinas. * DiRerenw not statisticallysignificant. t Differencesignificantat
Bleaching causes little change in Rl/R2 at low intensities but causes a large change at high intensities. A comparison of the ERP amplitudes of dark adapted and recovered retinas indicates an analogous relationship. The amplitudes generated by low intensity flashes are almost identical but the responses generated by high intensity flashes are greater in the dark adapted retina (cf. Fig. 4). Thus, there is little Merence between the ~phtudes or waveforms of the responses of dark adapted and recovered retinas at low intensities. There is, however, a large difference at high intensities. Perhaps the ERP generated by rhodopsin becomes measurable only at high intensities. If this is the case, rhodopsin would have only a small effect on the spectral sensitivity functions determined at relatively low intensities. On the other hand, the differences between the high intensity responses in the dark adapted and recovered retinas may be caused by unknown factors, i.e. other than by differences in rhodopsin concentration. Factors such as changes in the regenerated cone pigments, responses generated by photoproducts of bleaching, or ageing of the retina could, in fact, be causing the differences between dark adapted and recovered retinas. DISCUSSlON These experiments confirm the earlier observation that the ERP of the dark adapted isolated frog retina is dominated by a cone pigment that absorbs maximally at about 570-580 nm. Although this pigment accounts for only about one percent of the total visual pigment of the retina (LIEBMANand ENTINE, 1968)s it dominates the ERP. One possible explanation of the cone domination of the frog ERP is based on the morphological differences between the rods and cones of the frog retina. It has frequently been suggested that a necessary condition for recording ERP’s may be that the visual pigment molecules be contained in membranes that are in contact with the extracellular space (cf. LJZTTVIN. 1965). The visual pigments in the frog cones satisfy this condition since the lamellae of the cone outer segments, which contain the cone pigments, are infoldings of the plasma membrane. The rod pigments, on the other hand, are contained in lamellae that float in an intracellular matrix inside the plasma membrane of the rod outer segment, and consequently make no contact with the extracellular space (MOODY and ROBERTSON,1960; COHEN,1963; NILSSON,1965). The only rod pigment possibly in contact with the external * Seenote added in proof.
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
961
medium is that contained in the few basal discs of the rod outer segments, which are infoldings of the plasma membrane. If these notions are relevant, it may be that all of the cone pigments can contribute to the ERP, whereas only the small fraction of the rod pigments in the open basal lamellae can generate this response. This might explain why the ERP of the frog retina is dominated by the cones. The chromatic adaptation experiments indicate that, besides the long-wav&mgth cone pigment, visual pigments that absorb at shorter wavelengths also generate an ERP. These short wavelength pigments account for about 20-25 percent of the ERP, as indicated by the fact that the maximum sensitivity of the red-adapted retina is only about one-fifth of the maximum dark adapted sensitivity (Fig. 3). Also, red adaptation reduces the amplitude generated by a white flash (color temperature ~5800°K) to about 2&25 percent of its dark adapted value (Fig. 4). The broadness of the action spectrum of the red-adapted retina indicates that the short-wavelength component of the ERP may be generated by more than one visual pigment, perhaps rhodopsin (&, = 502 nm) and the visual pigment of the green rods intermediate of bleaching &IX+,= 433 nm). It is also possible that a shop-wavelen~ may generate a response similar to the ERP (cf. CONE, 1967; PAK and BOES, 1967; ALDEN, et al., 1966). However, changes in the response latency, such as are characteristic of the responses generated by photoproducts of rhodopsin, were not observed during redadaptation in the present experiments. Any photoproduct responses present would have to have latencies similar to the dark adapted ERP. Furthermore, the intermediates of bleaching of cone pigments appear to be more short-lived than the photoproducts of the bleaching of rhodopsin. LIEBMAN (personal communication), in his microspectrophotometric studies of frog cone pigments, has found no cone photoproducts absorbing above 420 mn shortly after bleaching, and P.K. BROWN(personal collation) has found no evidence of long-lived, ~sibly-absorb~g photoprodu~s in human and monkey cones. Nate udded in proof--In an article received after submission of this paper, Liebman and Entine (Visual pigments of frog and tadpole (R. pipiena), Vision Res. 8,761-775) report that, in addition to the 570-580 nm pigment of the frog’s “principal cones”, there is also a pigment spectrophotometrically indistinguishable from rhodopsin (hmax= 502 nm) contained in the frog’s “accessory cones”. This 502 nm “cone pigment” accounts for 05 percent of the visual pigment in the retina and could contribute to the short wavelength response observed in the p,resent experiments. thank Prof. GEORGEWALDand Dr. RX!IURD A. Corm for their helpful suggestions and criticism of the manuscript.
AcknowIedgmem-1
REFBRENcEs
ALDEN,G. B., IKRDA,H. and SIEOEL,I. M. (1966). New components of the mammalian receptor potential and their relation to visual photochemistry. Y&&XI Res. 6,373-384. BAECK,I., Do=, K. 0. and Rsurq T. (1965). The scmening &ect of the pigment epithelium on the retinal rods in the frog. P&x Res. 5, 101-I 11. Bamoas, C. D. B, (1957). Cationic extracting agents for rhodopsin and their mode of action. Brkkz. J. 66,372383. BIUNDLEY, G. S. (1960). Phys~I5~ of rhe Retina md the Visnai Pathway. Edward Amold, Lundon. BROWN,K. T. (1965). An early potential evoked by light from the pigment ep~~~id complex of the eye of the toad. Nature, Lmd. 2U7.124~1253. BROWN,IL T. and M URUCA~.~,M. (1964). A new receptor potential of the monkey retina with no detectable latency. Nuture, Land. 201, 626-628. COHEN,A. I. (1963). Vertebrate retinal cells and their organization. BioI. Rev. 38,427-459. CONE, R. A. (1964). Early receptor potential of the vertebrate retina. Nature, Lmd. 204, 736-739.
E.
962
BRUCE GOLDSTEIN
CONE,
R. A. (1965). Early receptor potential of the vertebrate eye. Co/d SprLrg Harbor Symp. qrram. Biol. 30, 483491. CONE, R. A. (1967). Early receptor potential: photoreversible charge displacement in rhodopsin. Science, N.Y. 155, 1128-1131. DARTNALL, H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. EBREY, T. G. and CONE, R. A. (1967). Melanin, a possible pigment for the photo-stable electrical responses of the eye. Nature, Lond. 213, 360-362. GOLDSTEIN, E. B. and WILLIAMS,T. P. (1966). Calculated effects of “screening pigments.” Ksiorr Res. 6, 39-50. GOLDSTEIN, E. B. (1967a). Source of the early receptor potential in the isolated frog retina. J. opt. Sot. Am. 57, 1425A. GOLDSIZIN, E. B. (1967b). Early receptor potential of the isolated frog (Rana pipiens) retina, Vision Res. 7, 837-84s. GOLDSTEIN, E. B. (1968). Early receptor potential of the isolated frog (Runa pipierrs) retina. Doctoral dissertation, Brown Univ., Prov., R.I. LETTVIN, J. Y. (1965). General discussion: early receptor potential. Cofd Spring Harbor Symp. quant. Biol. 30, 501-502. LIEBMAN, P. and ENTINE, G. (1967). Cyanopsin, a visual pigment of retinal origin. ?/urure, Lond. 216, 501-503. MOODY,M. F. and ROBERISON,J. D. (1960). The fme structure of some retinal photoreceptors. J. Biophys. Biochcm. Cytol. 7, 87-91. NILSSON,S. E. G. (1965). The ultrastructure of the receptor outer segments in the retina of the leopard frog (Rana pipiens). J. Ultrastruct. Res. 12. 207-231. PAK, W. L. and BOES, R. J. (1967). Rhodopsin: responses from transient intermediates formed during its bleaching. Science, N. Y. 155, 1131-l 133. PAK, W. L. and CONE, R. A. (1964). Isolation and identification of the initial peak of the early receptor potential. Nature, Lond. 204.436438. PAK, W. L. and EBREY, T. G. (1966). Early receptor potentials of rods and cones in rodents. J. gen. Physiol. 4, 1199-1208. SIEGEL, S. (1956). Nonpurametric Stutistics. McGraw-Hill, New York. ZEWI, M. (1939). On the regeneration of visual purple. Acta. Sot. Sci. Fenn. N.S. B, l-57. APPENDIX Calculation of the action spectrum of the,red-adapted retina At a given wavelength a flash of intensity 11 generates a response of amplitude A1 in the dark adapted retina. After adaptation with the red adapting light, a flash of intensity 11 generates a response A? where A2 < Al. The lower sensitivity of the red-adapted retina is reflected by the higher intensity 12 (where retina. The decrease in log 12 >, 4) necessary to generate a response of amplitude A1 in the red-adapted sensitivity due to adaptation equals log 12 - log 11, i.e. an increment of intensity A log I = (log 12 - log 11) must be added to the original flash of log intensity. log II, to bring the response .back to the original dark adapted level Al. Thus, the sensitivity of the red-adapted retina, log Srn, equals the sensitivity of the dark adapted retina, log SdO. minus A log I. In the red-adapted
retina
A log I = ( A log A)/m
where A log A = log Al - log A2 = -log
(AZ/AI)
m = slope of the A-I function Since the “percent dark adapted response”,
%DA, equals lOOAp/A1
A log A = --log (%DA/lOO) = log(100) - log(%DA) Substituting
into (1) above
A log I =
log 100 - log (%DA) m
Thus
iOg s,,
=
log
sd, -
(log 100 - log %DA) f?,
(1)
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina Abstract-The early receptor potential (ERP) of the isolated frog retina is generated primarily by a cone pigment that absorbs maximally at ca. 570-580 MI. irradiation of the retina with red light, which selectively bleaches this cone pigment, indicates that, in addition to the 580 nm pigment, the ERP of the isolated frog retina is generated by a pigment or pigments absorbing at shorter wavelengths. Bleaching most of the rod pigment rhodopsin causes appreciable changes in the response waveform and slope of the amplitude vs. intensity function but causes only small changes in the ERP action spectrum. Resume-Le potentiel pr&oce de rkepteur (ERP) de la r&tine isok de grenouillc est engendre surtout par un pigment de cone qui pOss&de son absorption maximale vers 570-580 nm. Par irradiation de la r&tine avec une lumi&e rouge qui d&&ore s&Ctivement ce pigment de cone, on constate que, outre ce pigment 580 MI, I’ERP de la r&tine isoMe de grenouille est engendrl par un ou des pigments absorbant vers les longueurs d’onde plus courtes. La dtcoloration presque totale de la rhodopsine des biltonnets change nettement la fonne de I’onde de rtponse et la pente de la fonction amplitude et intensitd, mais settlement faiblement le spectre d’action de I’ERP. Zusammenfassnng-Das frtihe Rezeptorpotential (ERP) der isolierten Froschnetzhaut wird hauptsiichlich durch ein Zapfenpigment erzeugt, welches maximal bei cu. 570-580 nm absorbiert. Die Bestrahlung der Netzhaut mit rotem Licht, das dieses Zapfenpigment selektiv ausbleicht, weist darauf hin, daD zus&zlich zu dem 580 nm Pigment das ERP der isolierten Froschnetzhaut noch von einem Pigment oder Pigmenten etzeugt wird, die bei ktirzeren WellenBngen absorb&en. Durch das Ausbleichen des graDten Teils da Zapfenpigmentrhodopsins werden merkliche Verilnderungen in der Welknform der Antwort und der Amplitudenintensit&sfunktion hervorgerufen, wt[hrend im ERP-Aktionsspektrum nur geringe Ver&tderungen auftreten.
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963
Pergamon Press 1968. Printed in Great Britain.
VISUAL PIGMENTS AND THE EARLY RECEPTOR POTENTIAL OF THE ISOLATED FROG RETINA1 E. BRUCEGOLDSTEIN TheBiological Laboratories, Harvard University, Cambridge, Massachusetts 02138 (Received 18 March 1968)
THE EARLY receptor potential (ERP) has been identified as a response that is generated by the direct action of light on visual pigments in the outer segments of the photoreceptors I, 1964; CONE, 1964, 1965, 1967). Much evidence in support of (BROWN and MURAICAM this conclusion has come from work on the rod-dominated eye of the albino rat. CONE (1964) and PAK and CONE (1964) have shown that the action spectrum of the rat ERP corresponds to the absorption spectrum of rhodopsin. CONE (1964) found that the amplitude of the ERP is linearly related to the amount of rhodopsin bleached by a brief gash. He also found that, following a bleaching exposure to light, the amplitude of the rat ERP increases in the dark at a rate comparable with the rate of pigment regeneration. If, by irradiating the rat eye with a light that bleaches the rhodopsin, the ERP is eliminated, it returns when the rhodopsin is photoregenerated (from long-lived intermediates of bleaching) with a blue flash (ARDEN,etal., 1966; CONE, 1967). All these results indicate that in the albino rat the ERP is closely linked to rhodopsin, the dominant visual pigment of the rat retina. In the frog however, a different situation exists. Although at least ninety percent of the visual pigment in the frog retina is rhodopsin (LIEBMAN,personal communication), this pigment appears to play only a small role in generating the ERP. GOLDSTEIN(1967a,b) has shown that after a light adaptation that bleaches most of the rhodopsin and reduces the ERP amplitude, the ERP recovers in the dark long before there has been an appreciable regeneration of rhodopsin. Furthermore, the action spectrum of the ERP of the isolated frog retina is maximally sensitive at CQ.580 run, whereas a response mediated by rhodopsin should be maximally sensitive at about 502 nm. Thus, the frog ERP arises predominantly from a pigment whose spectrum lies far to the red from rhodopsin, presumably the iodopsin of the cones (GOLDSTEIN,1967a,b). LIEBMANand ENTINE (1967) have measured the absorption spectrum of frog iodopsin by microspectrophotometry of single cones and find that it absorbs maximally at about 570-580 nm. In the present experiments, the retina was irradiated with red light which selectively bleached the long-wavelength cone pigment. This adaptation shifted the maximum sensitivity of the ERP to shorter wavelengths, indicating that pigments other than the longwavelength pigment also generate an ERP. The relationship between the ERP and rhodopsin was also investigated by comparing the ERP of dark adapted retinas with that of retinas in which almost all the rhodopsin had been bleached. * This research was supported by a National Science Foundation grant to George Wald and a US Public Health Service Postdoctoral Research Fellowship to the author. 953
E. BRUCEGOLDSTEI~~
954
METHODS AND MATERIALS All animals were dark adapted for a minimum of 12 hr at room temperature prior to the cxperimcnts, and all dissections were performed under dim red light. The frog retina was dissected out by carefully teasing it away from the pigment epitheiium under Ringer’s solution. Retinas so isolated, rather than the whole eye or live animal, were used in all experiments in order to eliminate the electrical response generated by the pigment epithelium-choroid complex (cf. BROWN,1965; EBREYand CONE, 1967) and to block rho&p&t regeneration (cf. ZEWI, 1939; GOLDSTUN,1967b). The retina was placed receptor side down on a moist cotton pad. Eiectrical contact was through cotton wick Ag-AgCl electrodes, one under the cotton pad, and the other in contact with the vitreal side of the retma. During the ex~riments the cotton pad and retina were placed on an aluminum block kept at a constant temperature of 23°C by circulating water through it from a Haake constant-tem~rature bath. Temperature was measured with a thermister probe and a Yellow Springs Instruments telethermometer. The stimulating light was a Honeywell 65C electronic flash gun with flash duration 1.3 msec. The adapting light from a General Electric 1OOWmicroscope lamp passed through a Jena KG 3 heat filter. The stimulating and adapting beams were superimposed by means of a beamsplitter, and were focused on the retina by a single lens. Both the stimulating and the adapting lights illuminated the entire retina. Their intensities were controlled by Bausch and Lomb neutral filters, and their wavelengths by BairdAtomic interference filters having half-bandwidths of 6-15 nm (stimulating light) and by a Wratten #92 filter (adapting light). Calibration of the interference filters with a Cary 11 recording spectrophotometer indicated that the rn~rn~ transmission at wavelengths outside the main bandpass was O-05 percent. The temperature rise at the retina caused by the adapting light was, at most, 0*5”C. The energy output of the flash gun at the wavelengths of peak transmission by the interference filters was measured with a calibrated Type 3 Weston Photronic Cell. Photovoltaic and electrical artifacts were eliminated by appropriate shielding. The electrical responses were amplified either by a Tektronix type FM 122 low-level preamplifier or by a Grass P5 AC preamplifier. The preamplifier bandpass was 0*2-10,000 Hz. Responses were displayed on a Tektronix 502 oscilloscope. Rhodopsin concentrations were determined by extracting the pigment with hexadecyltrimethylammonium bromide (cf. BIUDOES, 1957), and measuring the change in absorbance at 502 nm upon bleaching in a Cary 11 recording spectrophotometer. The absorbance change of the rhodopsin extracted from the experimental retina was compared with that of the rhodopsin extracted from the unbleached retina of the same frog’s other eye. The rhodopsin concentration of the experimental retina was expressed as a percentage of the concent~tion in the unbieach~ eye. BAECK,et uf. (196Jf, GOLDSTEIN (1968) and ZEWI (1939) have found that the two eyes of a frog yield equal amounts of rhodopsin within 5 percent.
RESULTS Chromatic adap’tation of the ERP Isolation of a short-wavelength component of the ERP. To determine whether the ERP of the isolated frog retina is generated by pigments other than the long-wavelength cone pigment (h,,, = 570-580 nm), the retina was irradiated with red light (X> 630 nm) which bleaches this pigment while sparing rhodopsin and pigments that absorb at shorter wavelengths. The intensity of a monochromatic test flash was first adjusted to produce a 30-60 PV response in the dark adapted retina. A single test flash of this intensity causes only a small decrease in the amplitude of the ERP generated by subsequent flashes. ERP amplitudes were measured from the peak of the cornea-positive phase of the ERP, Rl, to the peak of the negative phase, R2. To determine the course of adaptation to red Iight, two or three monochromatic test flashes were presented to the retina at various intervals after the start of the ~l~u~nati~n. Such measurements, made at 420, 490, 548, 590 and 630 nm, are shown in Fig. 1. The ERP amplitudes, expressed as percent of the dark adapted amplitude, are plotted vs. time. If only one pigment were responsible for the ERP, the course of adaptation plotted as percentage of the dark adapted response at each wavelength should be independent of
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
955
the wavelength of the test flash. 2 The results in Fig. 1, however, indicate that the extent of adaptation increases markedly with the wavelength. This suggests that some pigment in addition to the long-wavelength cone pigment must generate an ERP. The dashed line in Fig, 1 indicates that the adapting light bleaches little rhodopsin.
ADAPTATION TIME - MINUTES Chromatic adaptation of the ERP. ERP amplitude as a function of adaptation time for monochromatic test flashes presented during adaptation with red light. The number beside each curve indicates the wavelength of the test flash. All amplitudes are expressed relative to the ampiitude generated by the test Dash in the dark adapted retina. Each curve is the average of two experiments. The dashed line represents the reiative rhodopsin concentration (dark adapted concentration = 100) during adaptation to red light, as determined by pigment extraction. FIG. I.
In another set of experiments, the period of adaptation was extended to eight minutes. Test flashes of 420, 490, 548, 590 and 630 nm which generated 30-60 ttV responses were presented to a dark adapted retina. The retina was then exposed to the red adapting light. Beginning at 2 min after the initiation of adaptation, test flashes were presented every 30 set until one test flash at each wavelength had been presented. This series of test flashes was then repeated at six minutes after the start of adaptation. In half of the experiments test flashes were presented in ascending order (420-630 nm), and in the other half in descending order (630-420 nm). Figure 2 shows the results of these experiments. The percentages of the dark adapted response, 2-4 and 6-8 min after the beginning of red-adaptation, are plotted against the wavelength of the test flash, The amplitude decreases most during the first minute of red-adaptation and only slightly further after 4 min. At 630 nm the amplitude decreases to almost zero, whereas at 420 nm the amplitude decreases only to 78 percent of its dark adapted value after 2-4 min of adaptation, and 64 percent after 6-8 min. The results presented in Figs. 1 and 2 indicate that more than one mechanism is responsible for generating the ERP in the isolated frog retina. The long-wavelength mechanism 2 If the pigment density were high, self-screening by the pigment (cf. BRDIDLY,1960;GOLKSTEINand WILLIAMS,1966)
would cause the course of ERP adaptation to depend somewhat on the wavelength of the test flash. However, the effect of self-screening is small in the present experiments since the maximum absorbance of the Iong-wavelength cone pigment is only about 020 (LJEEMAN, personal comrmmication).
956
E. BRUCE GOLDSTEIN
is almost completely eliminated by adaptation with red light, whereas the shorterwavelength mechanism is only slightly affected by red adaptation. This short-wavelength mechanism, however, is eliminated by irradiation with white light. Thus it too is photosensitive. I
I
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200
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WAVELENGTH-nm Fro. 2. ERP amplitude
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FIG. 3. Action spectra for generating the ERP of the frog retina under various conditions. Sensitivity is the reciprocal of numbers of photons per test flash that excite a constant amplitude of ERP. Dark adapted (e); Bleached and recovered (0); Red adapted (&; Red adapted, from equation 1: 2-4 mitt of adaptation (o), 6-g min adaptation (+). The points calculated by equation 1 are positioned on the ordinate to reflect the actual differences in sensitivity between the dark adapted and red-adapted retinas. The sensitivity of the recovered and dark adapted retinas have been equated at 630 nm. Relative rhodopsin concentrations: dark adapted = 100; red adapted = 100; recovered = 5. The red adapted sensitivity (& is the result of experiments on nine retinas. The other functions were determined on 4-5 retinas.
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
957
Actron spectra. The action spectrum of the ERP remaining after red adaptation was measured by determining the energy necessary to elicit a criterion response of about 20 PV at 420,460,490,54& 590 and 630 nm during red adaptation3. The action spectrum is indicated by the filled triangles in Fig. 3, which represent the averages of sensitivity measurements on nine retinas. The sensitivity could not be measured accurately at 630 nm since adaptation caused the response to decrease nearly to the noise level of the preparation. The sensitivity of the red adapted retina can also be determined from the data in Fig. 2 since the relation between ERP amplitude and intensity of test flash (A-I function) in the red adapted retina (Fig. 4), and the sensitivity of the dark adapted retina are known. The dark-adapted sensitivity has been determined by GOLDSTEIN(1967b) and was redetermined in the present experiments (cf. Fig. 3). The action spectrum of the red-adapted retina was determined from the following formula (cf. Appendix):
log %l
= log s,, -
log 100 - log(%DA) C m
1
(1)
where S,, = sensitivity of the red adapted retina at a given wavelength. S = sensitivity of the dark adapted retina at the same wavelength. O/,& = percent dark adapted response remaining after adaptation with the red adapting light. m = slope of the log amplitude vs. log intensity function for the red-adapted retina. The slope is 0.80 in the present experiments (cf. Fig. 4). The action spectrum determined by equation (1) is indicated by the open circles and S’s in Fig. 3. The circles and +‘s represent, respectively, the sensitivities calculated from the data obtained after 24 and 6-g min of adaptation. The ERP action spectrum of the red adapted retina presents a broad maximum from about 420 to 500 nm, decreasing at longer wavelengths. It seems clear that the mechanism that generates the ERP in the red-adapted retina is much more blue-sensitive than the mechanism that dominates response in the dark adapted retina. The flatness of the curve suggests that more than one visual pigment may be responsible for the short-wavelength sensitivity. Rhodopsin (A,,, = 502 nm) and the visual pigment of the green rods (&lax = 433 nm; DARTNALL, 1967) may contribute to the generation of the short-wave response. The spectral sensitivity of the ERP of the dark adapted retina was determined earlier at 5 wavelengths (GOLDSTEIN,1967a,b), and here has been re.-measured at 11 wavelengths. Since the presentation of 11 test flashes to a retina would bleach appreciable amounts of pigment, the action spectrum was determined in two separate experiments. In one set of retinas the relative sensitivity was determined at 420, 490, 548, 590 and 630 nm, and in the other set, at 420, 460, 522, 560, 600, 620 and 660 nm. The two functions were then combined by equating the relative sensitivities at 420 nm, the common wavelength in each series. The resulting curve, shown in Fig. 3, indicates that the maximum sensitivity is at about 580 nm. -\Theaction spectrum was measured during adaptation because the redgmpitive pignent -tea in the dark (Go-, 1967a,b). This pigment also regenuntea continuously in tbe light, as ewknced by the fact that the amplitude of the 630 nm test flash decrrpses to a steady state level wblcb is maintained as long as the light is on (GOLD-, unpublished observations). Increa&g the adapting izttansity decreases the level of the steady state. In the present experiments a steady state kvel close to zero is reached after about 1 min. of light adaptation.
958
E.
BRUCE GOLDSTEIN
Rhodopsin and the frog ERP
Five experiments were performed to investigate further the observation that there is little correlation between the ERP and rhodopsin in the isolated frog retina (GOLDSTEIN, 1967a,b). In the first experiment, rhodopsin concentration was measured during 30 set of adaptation with red light. The results of this experiment, shown in Fig. 1, indicate that though this adaptation reduces the response to a 630 nm test flash almost to zero, it bleaches little rhodopsin. In the other four experiments, the action spectra, effect of red adaptation, A-I functions, and waveforms were measured in retinas from which most of the rhodopsin had been removed by bleaching. The results of these experiments are compared with the results of analogous experiments on dark adapted retinas. Action spectrum. Retinas were irradiated with white light for 2 min, followed by a 2-min bleach with orange light. The retinas were then kept in darkness for 30 min to allow the ERP to recover. Isolated retinas that had been bleached and then dark adapted are referred to as “recovered” retinas 4. Direct extraction of the rhodopsin 30 min after light adaptation indicated that these retinas contained less than five percent of their dark adapted concentration of rhodopsin. The sensitivity of the recovered retina is indicated by the open squares in Fig. 3. The points at 420 and 490 nm fall only slightly below the dark adapted action spectrum. Thus, decreasing the rhodopsin concentration has little effect on the spectral sensitivity. This result is in agreement with GOLDSTEIN’S(1967b) finding that the action spectra of dark adapted retinas and retinas that contain only 25 percent of their dark/adapted concentration of rhodopsin are identical. Red adaptation. If recovered retinas that contain only five percent of their rhodopsin are irradiated with the red adapting light, the ERP amplitude measured with the 630 nm test flash decreases more than the ERP amplitude measured with the 490 nm test flash. After 2 min of adaptation, the response generated by the 630 run flash decreases almost to zero. The 490 response, however, decreases only to 50 percent of its amplitude prior to adaptation. This finding is in agreement with the results shown in Fig. 2 for the dark adapted retina. Thus, the short-wavelength response of the red-adapted retina does not depend upon rhodopsin.5 Amplitude vs. intensity. Amplitude vs. intensity (A-I) functions for dark adapted (A), recovered (B), and red-adapted (C) retinas are plotted in Fig. 4. The red-adapted function represents the response of the pigments remaining after bleaching the long-wavelength cone pigment; the recovered function represents the response of a retina containing regenerated cone and possibly green-rod pigments and ten percent of the retina’s dark adapted concentration of rhodopsin. 6 A comparison of the dark adapted and red-adapted 4 The procedure for bleaching and dark adaptation was not the same in all experiments. The procedures used for preparing recovered retinas in each set of experiments were as follows: Acrion spectrrtnt and red udaptarion: 2-min white bleach followed by 2-min orange bleach; 30-min dark adaptation. Amplirrrde vs. intensity: 3-min white bleach; 20-min dark adaptation. Waveform: 1 msec flash bleach (log relative intensity = 0); 3%min dark adaptation. 5 This experiment does not rule out, however, the possibility rhodopsin is contributing to the short-wavelength response.
that the small amount
of residual
6 Rhodopsin concentrations were measured by extraction immediately after the three-minute white bleach, 20 min after bleaching, and after a flash of log I = 0 at 23 min after bleaching. The concentration was ten percent of the dark adapted concentration under all conditions, indicating that no regeneration of rhodopsin occurred in the dark and that there was no net production of rhodopsin due to photoregeneration by the high intensity flash presented at t = 23 min.
Visual Piefnents and the Early Receptor Potential of the Isolated Frog Retina
959
functions indicates that red adaptation reduces the response amplitude by about 060 log unit but causes little change in the slope of the function. The slope of the recovered A-l function is smaller than the slope of the dark-adapted function. r-
to-
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6 .Oi
I
-3
-2
LOG
RELATIVE
-I
0
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FJG. 4. Relation between log amplitude and log intensity of test flash in: (A) dark adapted retinas (relative rhodopsin concentration = 100); (B) recovered retinas (R = 10); (C) redadapted retinas (R = 100). Curves B and C have been shifted down @15 log unit on the ordinate. The actual difference between curves A and B at log f = -3.02 is Q07 log unit. For the dark adapted and recovered retinas four white ffashes were presented to each retina, three of log I = -3.02, -272, -248, and one of higher intensity. The same procedure was used for the red-adapted retina, but only two flashes were presented to each retina, one of low intensity (log 2 = -2.3) and one of high intensity. By using this procedure it was possible to combine the results from many retinas which differed in responsiveness. In order to compensate for differences in the level of responding in different retinas, alI responses for a given retina were multiplied by a constant in order to minimixe variability when the data for different retinas were combined. The huge points on curve C are for monochromatic test fhshes of 490 (0) and 548 (0) nm. Bach point is baaed on eight experiments. The 490 and 548 nm functions have been positioned on the ordinate to correspond to the actual response amplitude generated by the gashes but have been arbitrarily positioned on the abscissa. It was not possible to determine the A-I functions at other wavelengths due to lack of flash energy. All measurements on red-adapted retinas were begun after two minutes of adaptation.
The slope of the dark adapted A-I function in Fig. 4 is about 0.80 at low intensities; another independent set of experiments yielded a slope of 0.85. These slopes are smaller than CONE’S (1964) value of l-0 for the slope of the albino rat A-I function7 but compare favorably with the slope of the ground squirrel ERP A-I function (PAK and EBREY, 1966). The A-I function of the albino rat determined in the present experiments using the same apparatus and procedure as was used for the frog, has a slope of about 0.86. Thus, in the present experiments the slope of the rat A-I function is slightly lower than that determined by Cone, but the difference between the frog and rat functions is not significant. 7 However, a line with slope less than 1.0 fits Cone’s data well if his low intensity data are weighted more heavily.
960
E.BRWCEGOLDSTEIN
Waveform. Differences between dark adapted and recovered retinas are also reflected by differences in the Rl/R2 ratio of the ERP. In Table 1 RI/R2 is tabulated for dark adapted and recovered retinas. The rhodopsin concentration in these recovered retinas is 20 percent of the dark adapted concentration. TABLE 1
Log relative intensity
N
RI/R2 Dark adapted
RI/R2 Recovered
DA-Recovered
-2.3 0.0
8 20
0.36 1.47
0.37 2.00
-0*01* -0*53t
N = nnmber of retinas. * DiRerenw not statisticallysignificant. t Differencesignificantat
Bleaching causes little change in Rl/R2 at low intensities but causes a large change at high intensities. A comparison of the ERP amplitudes of dark adapted and recovered retinas indicates an analogous relationship. The amplitudes generated by low intensity flashes are almost identical but the responses generated by high intensity flashes are greater in the dark adapted retina (cf. Fig. 4). Thus, there is little Merence between the ~phtudes or waveforms of the responses of dark adapted and recovered retinas at low intensities. There is, however, a large difference at high intensities. Perhaps the ERP generated by rhodopsin becomes measurable only at high intensities. If this is the case, rhodopsin would have only a small effect on the spectral sensitivity functions determined at relatively low intensities. On the other hand, the differences between the high intensity responses in the dark adapted and recovered retinas may be caused by unknown factors, i.e. other than by differences in rhodopsin concentration. Factors such as changes in the regenerated cone pigments, responses generated by photoproducts of bleaching, or ageing of the retina could, in fact, be causing the differences between dark adapted and recovered retinas. DISCUSSlON These experiments confirm the earlier observation that the ERP of the dark adapted isolated frog retina is dominated by a cone pigment that absorbs maximally at about 570-580 nm. Although this pigment accounts for only about one percent of the total visual pigment of the retina (LIEBMANand ENTINE, 1968)s it dominates the ERP. One possible explanation of the cone domination of the frog ERP is based on the morphological differences between the rods and cones of the frog retina. It has frequently been suggested that a necessary condition for recording ERP’s may be that the visual pigment molecules be contained in membranes that are in contact with the extracellular space (cf. LJZTTVIN. 1965). The visual pigments in the frog cones satisfy this condition since the lamellae of the cone outer segments, which contain the cone pigments, are infoldings of the plasma membrane. The rod pigments, on the other hand, are contained in lamellae that float in an intracellular matrix inside the plasma membrane of the rod outer segment, and consequently make no contact with the extracellular space (MOODY and ROBERTSON,1960; COHEN,1963; NILSSON,1965). The only rod pigment possibly in contact with the external * Seenote added in proof.
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina
961
medium is that contained in the few basal discs of the rod outer segments, which are infoldings of the plasma membrane. If these notions are relevant, it may be that all of the cone pigments can contribute to the ERP, whereas only the small fraction of the rod pigments in the open basal lamellae can generate this response. This might explain why the ERP of the frog retina is dominated by the cones. The chromatic adaptation experiments indicate that, besides the long-wav&mgth cone pigment, visual pigments that absorb at shorter wavelengths also generate an ERP. These short wavelength pigments account for about 20-25 percent of the ERP, as indicated by the fact that the maximum sensitivity of the red-adapted retina is only about one-fifth of the maximum dark adapted sensitivity (Fig. 3). Also, red adaptation reduces the amplitude generated by a white flash (color temperature ~5800°K) to about 2&25 percent of its dark adapted value (Fig. 4). The broadness of the action spectrum of the red-adapted retina indicates that the short-wavelength component of the ERP may be generated by more than one visual pigment, perhaps rhodopsin (&, = 502 nm) and the visual pigment of the green rods intermediate of bleaching &IX+,= 433 nm). It is also possible that a shop-wavelen~ may generate a response similar to the ERP (cf. CONE, 1967; PAK and BOES, 1967; ALDEN, et al., 1966). However, changes in the response latency, such as are characteristic of the responses generated by photoproducts of rhodopsin, were not observed during redadaptation in the present experiments. Any photoproduct responses present would have to have latencies similar to the dark adapted ERP. Furthermore, the intermediates of bleaching of cone pigments appear to be more short-lived than the photoproducts of the bleaching of rhodopsin. LIEBMAN (personal communication), in his microspectrophotometric studies of frog cone pigments, has found no cone photoproducts absorbing above 420 mn shortly after bleaching, and P.K. BROWN(personal collation) has found no evidence of long-lived, ~sibly-absorb~g photoprodu~s in human and monkey cones. Nate udded in proof--In an article received after submission of this paper, Liebman and Entine (Visual pigments of frog and tadpole (R. pipiena), Vision Res. 8,761-775) report that, in addition to the 570-580 nm pigment of the frog’s “principal cones”, there is also a pigment spectrophotometrically indistinguishable from rhodopsin (hmax= 502 nm) contained in the frog’s “accessory cones”. This 502 nm “cone pigment” accounts for 05 percent of the visual pigment in the retina and could contribute to the short wavelength response observed in the p,resent experiments. thank Prof. GEORGEWALDand Dr. RX!IURD A. Corm for their helpful suggestions and criticism of the manuscript.
AcknowIedgmem-1
REFBRENcEs
ALDEN,G. B., IKRDA,H. and SIEOEL,I. M. (1966). New components of the mammalian receptor potential and their relation to visual photochemistry. Y&&XI Res. 6,373-384. BAECK,I., Do=, K. 0. and Rsurq T. (1965). The scmening &ect of the pigment epithelium on the retinal rods in the frog. P&x Res. 5, 101-I 11. Bamoas, C. D. B, (1957). Cationic extracting agents for rhodopsin and their mode of action. Brkkz. J. 66,372383. BIUNDLEY, G. S. (1960). Phys~I5~ of rhe Retina md the Visnai Pathway. Edward Amold, Lundon. BROWN,K. T. (1965). An early potential evoked by light from the pigment ep~~~id complex of the eye of the toad. Nature, Lmd. 2U7.124~1253. BROWN,IL T. and M URUCA~.~,M. (1964). A new receptor potential of the monkey retina with no detectable latency. Nuture, Land. 201, 626-628. COHEN,A. I. (1963). Vertebrate retinal cells and their organization. BioI. Rev. 38,427-459. CONE, R. A. (1964). Early receptor potential of the vertebrate retina. Nature, Lmd. 204, 736-739.
E.
962
BRUCE GOLDSTEIN
CONE,
R. A. (1965). Early receptor potential of the vertebrate eye. Co/d SprLrg Harbor Symp. qrram. Biol. 30, 483491. CONE, R. A. (1967). Early receptor potential: photoreversible charge displacement in rhodopsin. Science, N.Y. 155, 1128-1131. DARTNALL, H. J. A. (1967). The visual pigment of the green rods. Vision Res. 7, l-16. EBREY, T. G. and CONE, R. A. (1967). Melanin, a possible pigment for the photo-stable electrical responses of the eye. Nature, Lond. 213, 360-362. GOLDSTEIN, E. B. and WILLIAMS,T. P. (1966). Calculated effects of “screening pigments.” Ksiorr Res. 6, 39-50. GOLDSTEIN, E. B. (1967a). Source of the early receptor potential in the isolated frog retina. J. opt. Sot. Am. 57, 1425A. GOLDSIZIN, E. B. (1967b). Early receptor potential of the isolated frog (Rana pipiens) retina, Vision Res. 7, 837-84s. GOLDSTEIN, E. B. (1968). Early receptor potential of the isolated frog (Runa pipierrs) retina. Doctoral dissertation, Brown Univ., Prov., R.I. LETTVIN, J. Y. (1965). General discussion: early receptor potential. Cofd Spring Harbor Symp. quant. Biol. 30, 501-502. LIEBMAN, P. and ENTINE, G. (1967). Cyanopsin, a visual pigment of retinal origin. ?/urure, Lond. 216, 501-503. MOODY,M. F. and ROBERISON,J. D. (1960). The fme structure of some retinal photoreceptors. J. Biophys. Biochcm. Cytol. 7, 87-91. NILSSON,S. E. G. (1965). The ultrastructure of the receptor outer segments in the retina of the leopard frog (Rana pipiens). J. Ultrastruct. Res. 12. 207-231. PAK, W. L. and BOES, R. J. (1967). Rhodopsin: responses from transient intermediates formed during its bleaching. Science, N. Y. 155, 1131-l 133. PAK, W. L. and CONE, R. A. (1964). Isolation and identification of the initial peak of the early receptor potential. Nature, Lond. 204.436438. PAK, W. L. and EBREY, T. G. (1966). Early receptor potentials of rods and cones in rodents. J. gen. Physiol. 4, 1199-1208. SIEGEL, S. (1956). Nonpurametric Stutistics. McGraw-Hill, New York. ZEWI, M. (1939). On the regeneration of visual purple. Acta. Sot. Sci. Fenn. N.S. B, l-57. APPENDIX Calculation of the action spectrum of the,red-adapted retina At a given wavelength a flash of intensity 11 generates a response of amplitude A1 in the dark adapted retina. After adaptation with the red adapting light, a flash of intensity 11 generates a response A? where A2 < Al. The lower sensitivity of the red-adapted retina is reflected by the higher intensity 12 (where retina. The decrease in log 12 >, 4) necessary to generate a response of amplitude A1 in the red-adapted sensitivity due to adaptation equals log 12 - log 11, i.e. an increment of intensity A log I = (log 12 - log 11) must be added to the original flash of log intensity. log II, to bring the response .back to the original dark adapted level Al. Thus, the sensitivity of the red-adapted retina, log Srn, equals the sensitivity of the dark adapted retina, log SdO. minus A log I. In the red-adapted
retina
A log I = ( A log A)/m
where A log A = log Al - log A2 = -log
(AZ/AI)
m = slope of the A-I function Since the “percent dark adapted response”,
%DA, equals lOOAp/A1
A log A = --log (%DA/lOO) = log(100) - log(%DA) Substituting
into (1) above
A log I =
log 100 - log (%DA) m
Thus
iOg s,,
=
log
sd, -
(log 100 - log %DA) f?,
(1)
Visual Pigments and the Early Receptor Potential of the Isolated Frog Retina Abstract-The early receptor potential (ERP) of the isolated frog retina is generated primarily by a cone pigment that absorbs maximally at ca. 570-580 MI. irradiation of the retina with red light, which selectively bleaches this cone pigment, indicates that, in addition to the 580 nm pigment, the ERP of the isolated frog retina is generated by a pigment or pigments absorbing at shorter wavelengths. Bleaching most of the rod pigment rhodopsin causes appreciable changes in the response waveform and slope of the amplitude vs. intensity function but causes only small changes in the ERP action spectrum. Resume-Le potentiel pr&oce de rkepteur (ERP) de la r&tine isok de grenouillc est engendre surtout par un pigment de cone qui pOss&de son absorption maximale vers 570-580 nm. Par irradiation de la r&tine avec une lumi&e rouge qui d&&ore s&Ctivement ce pigment de cone, on constate que, outre ce pigment 580 MI, I’ERP de la r&tine isoMe de grenouille est engendrl par un ou des pigments absorbant vers les longueurs d’onde plus courtes. La dtcoloration presque totale de la rhodopsine des biltonnets change nettement la fonne de I’onde de rtponse et la pente de la fonction amplitude et intensitd, mais settlement faiblement le spectre d’action de I’ERP. Zusammenfassnng-Das frtihe Rezeptorpotential (ERP) der isolierten Froschnetzhaut wird hauptsiichlich durch ein Zapfenpigment erzeugt, welches maximal bei cu. 570-580 nm absorbiert. Die Bestrahlung der Netzhaut mit rotem Licht, das dieses Zapfenpigment selektiv ausbleicht, weist darauf hin, daD zus&zlich zu dem 580 nm Pigment das ERP der isolierten Froschnetzhaut noch von einem Pigment oder Pigmenten etzeugt wird, die bei ktirzeren WellenBngen absorb&en. Durch das Ausbleichen des graDten Teils da Zapfenpigmentrhodopsins werden merkliche Verilnderungen in der Welknform der Antwort und der Amplitudenintensit&sfunktion hervorgerufen, wt[hrend im ERP-Aktionsspektrum nur geringe Ver&tderungen auftreten.
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963