Adaptational changes in the cone system of the isolated frog retina

Vision Res. Vol. 12, pp. 875-888. Pergamon Press 1972. Printed in Great Britain.

ADAPTATIONAL CHANGES OF THE ISOLATED

IN THE CONE SYSTEM FROG RETINA

DONALD C. Hook

Department of Psychology,ColumbiaUniversity,New York, New York 10027,U.S.A. (Received 10 September 1971)

INTRODUCTION LIGHT adaptation of the vertebrate visual system produces not merely a change in sensitivity but also a change in the system’s ability to resolve temporal variations in light intensity. GRANIT (1935) found in a number of vertebrates that the ability of the retina to resolve temporal variations in light strongly depended on the state of light adaptation. Granit’s measure of the retina’s temporal resolving power was the critical ticker frequency (cff) of the electroretinogram (ERG). He showed that when stimulating conditions favored the cones the cff of the ERG increased during light adaptation and decreased during dark adaptation. DODT and HECK (1954) observed like changes in cat ganglion cell firing. In fact, a similar phenomenon has been reported in the human psychophysical literature. LYTHGOE and TANSLEY (1929) observed that the cff for fovea1 stimulation, or for long-wave stimulation of the peripheral retina, decreased during dark adaptation and increased with light adaptation. The temporal resolving power of the frog retina, as measured by the ERG, also shows this strong dependence on the state of light adaptation. When an ERG is evoked by intermittent light (flicker ERG) the cff of this flicker ERG is far greater for the light adapted retina than it is for the dark adapted retina (GRANITand RIDDELL,1934; DODT and HECK, 1954). GORDON (1967) has demonstrated first that the frog’s ticker ERG is a cone response and second that the functional change of the retina with light adaptation can be shown by recording a flicker ERG to an intermittent light of a constant frequency and intensity. Figure 1 is essentially a replication of this second finding and shows sample flicker ERG responses to a lo-Hz white light following 35 min of dark adaptation. The response is very small initially but increases drastically over the 20-min period of stimulation. It is important to note that this increase in response amplitude is due to light adaptation by the intermittent stimulation. If an equivalent steady adapting light is substituted, but turned off for tests with the same lo-Hz stimulation, the same growth occurs. Why is the cone system’s cff lower, or the flicker amplitude to a constant intensity smaller, in the dark adapted eye when the intensity of stimulation is well above cone threshold? GRANIT(1938) proposed two possible mechanisms to account for these findings. One, which will be called here cone system adaptation, involves some sort of alteration, due to light adaptation, of the system producing the flicker. The second, which Granit called “rodcone inhibition”, involves the inhibition of the flicker producing receptors, cones, by the red rods. GRANIT(1935, 1936, 1938, 1947) consistently preferred the explanation based on rodcone inhibition. 875

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200 msec FIG. 1. Flicker ERG records from a live curarized frog in response to white 10 Hz stimulation as a function of time after onset of stimulation. The 2Omin period of 10 Hz stimulation was preceded by 25 min of dark adaptation. The bars to the right of these records indicate 100 pV. Thus the five lower responses so represented are half as large as their appearance would suggest when viewed in the same display as the rest of the responses. The latencies of the responses in this figure and succeeding figures can not be compared since all traces were initiated manually.

A test for cone system adaptation could be made if the red rods could be eliminated from the frog’s retina. Although this removal of the rods is physically impossible, they can be functionally removed by using the isolated retina preparation. The term “isolated retina” is used to designate a retina freed from the pigment epithelium and removed from the eye. The frog isolated retina can be maintained for over 1 hr and responds to photic stimulation with a normal ERG, low thresholds, and spike activity at the ganglion cell level (SICKEL,1961; BAUMANN, 1964; BAUMANNand SCHEIBNER,1968). The most important structural difference between the intact and the isolated retinas is the absence in the latter of the pigment epithelium. Because the pigment epithelium is absent, little or no rhodopsin (502 pigment) regenerates in the isolated retina (KUHNE, 1879; ZEWI, 1939; BAUMANN, 1965; GOLDSTEIN,1967). However, GOLDSTEIN(1967, 1970)

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has shown by recording early receptor potentials (ERP) that the cone pigments are regenerated in the frog isolated retina even after extensive bleaching. The important implication for this study is that the frog’s cone system can be functionally isolated by using an isolated retina in which light has inactivated the red rods by bleaching away their 502 pigment. The evidence for functional isolation of the cone system is quite convincing. The effectiveness of quanta stimulating the isolated retina of a dark adapted frog matches the absorption spectrum of rhodopsin (BAUMANN, 1967a). This finding, based on records of the b-wave of the ERG, is in complete agreement with results similarly obtained from the whole frog. However, after extensive light adaptation and subsequent dark adaptation, the spectral sensitivity curve for the isolated retina ‘preparation has a h,, between 560 nm and 580 nm, as shown by b-wave amplitudes (BAUMANN, 1967a, b). Under similar experimental conditions, the intact frog eye would regenerate rhodopsin and therefore show the sensitivity of the 502 pigment. BAUMANN and SCHEIBNER (1968) have shown similar functional isolation of the cone system by recording from ganglion cells of the frog isolated retina. As long as less than 40 per cent of the 502 pigment has been bleached, the influence of the red rods on the spectral sensitivity of the retina can be predicted from studies of the intact frog eye. However, when more of the 502 pigment has been bleached, the spectral sensitivities based on records of

ganglion cell responses are those of a light adapted (photopic) intact eye even when the isolated retina is dark adapted. These data agree with BAUMANN’S(1967b) ERG recordings in which 45 per cent of the 502 pigment had to be bleached before the sensitivity of the dark adapted isolated retina became photopic. Evidence of CRESCITELLI and SICKEL (1968) suggests that red rods may still be active in these bleached isolated retinas, but are inhibited by the cones. Whether or not this is true, the conclusion is that a dark adapted, isolated retina with less than 60 per cent of its 502 pigment functions as a cone retina. In the experiments to be reported, over 80 per cent of the 502 pigment was removed by extensive light adaptation (bleaching) of the isolated retina. Since the cone system was thus isolated, we can ask whether the increase in flicker ERG in response to 10 Hz stimulation is due to a change in the cone system.

METHODS Stimulation

Stimulation of the isolated retina was controlled by a projection system that flooded the entire retina with light. The light source for this system was a 45 W quartz-iodine tungsten filament lamp operated at 6.3 amps. The spectral composition of the light from channel I was measured by placing a spectral radiometer in the plane of the retina. This spectral composition was best fitted by a black body curve for 2400°K. This output from channel I will be referred to below as white light. The wavelength of the stimulation for Experiment 2 was controlled by two Baird-Atomic interference filters. These filtkrs were calibrated with a spdctrophotometer and were shown to have peak transmissions at 636 nm and 549 nm and half-band widths of 7 nm. Full intensity 636 nm stimulation resulted in 0.50 log unit greater quanta1 flux at the retina than 549 mn stimulation. SedHooD (1971) for radiometric calibration procedures. Using a MacBeth Illuminometer, the maximum illuminance on the retina from channel I was determined to be 56-2 ft-c for white light and 2.73 ft-c for 549 M-Ilight. Since 1 ft-c is equivalent to 1.076 x lo-” lm/mm* these values correspond to 6.07 x lo-“ lm/mm’ and 2.97 x 10m5Im/mm2, respectively. For the 549 nm light this represented 1.18 x 10” quanta/set/mm’. The intermittent stimulation used in these experiments was controlled by a disk that was rotated by a synchronous motor. The flash rate, calibrated by a stroboscope, was 10.1 Hz with equal dark and light periods. Rise and decay times of each light pulse were less than 1 msec.

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Preparation Frogs (Ranupipiens)from 1.5 to 3 in. in body length were housed in a refrigerator maintained at approximately 6°C. Twenty-four hr or more before they were used, they were removed from the refrigerator and placed in an aquarium that was well lighted. Thus, a11frogs were light adapted and warmed to room temperature prior to experimentation. Room temperature, considering seasonal variation, ranged from 20°C to 26°C. After at least 24 hr of light adaptation, frogs were dark adapted for a minimum of 24 hr. A dark adapted frog was decapitated and then an eye enucleated. The anterior portion of the eye was removed by cutting below the ora serrata with iridectomy scissors. The retina was carefully teased away from the pigment epithelium and dropped into a dish of Ringer’s solution. The retina was then floated, receptors up or down, onto a cotton pad soaked in Ringer’s soWion. This entire procedure was performed under long-wave light ~ratten filter No. 29). The cotton pad with the isolated retina was placed in a plastic chamber. This chamber, similar in principle to that of MACNICHOL and SVAETICHM (1958), had a moat filled with Ringer’s solution surrounding, but not touching, the retina. A mixture of 95 % Oa and 5 % CO, flowed slowly into the chamber, creating a moist, oxygenated atmosphere. Isolated retinas prepared as described survived up to 2.5 hr in this chamber. Recordingsystem The Xmger-soaked cotton wickelectrodes in contact with Ag-AgCl wire were connected to the differential input leads of a low level preamplifier (Tektronix type 122). The output of the proposer was connected to an oscilloscope (Tektronix type 502A). The low frequency cutoff of the preamplifier was set at O-2Hz and the high frequency cutoff at 250 Hz. The wick of one electrode, the reference, was placed under the cotton pad and the wickof the other, the

recording electrode, was allowedto just touch the surface of the retina.

PROCEDURE Isolation

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The frog’s cone system was isolated by bleaching the isolated retina with 1.5min of full intensity light at a flash rate of 10 Hz. Extractions indicated this procedure bleached more than 80 per cent of the rod pigment. According to BAUMANN (1967a, b) this procedure should isolate the frog’s cone system. In fact, after the isolated retina was allowed to dark adapt for 25 min the ERG had a spectral sensitivity that agreed closely with Baumann’s sensitivity curves. The intact eye would regenerate 502 pigment during the dark interval and the spectral sensitivity would be heavily influenced by the red rods (see DODT and JESSEN, 1961). Every preparation in the experiments below was tested for isolation of the cone system. This was accomplished by presen~ng 549 nm and 636 nm single flashes that were adjusted to be equally effective stimuli for the cone system. This adjustment was made based on energies giving equal flicker ERG responses in the intact frog. This functional matching of the 549 nm and 636 nm stimuli resulted in a 636-nm stimulus that had 0.50 log unit more quanta than had the 549-nm stimulus. The 636-nm stimulus was too intense by approximately 0.10 log unit based on Baumann’s isolated retina curve. Experiment 1. Light adaptation of the cone system

The purpose of Experiment 1 was to test for an increase in flicker ERG amplitude when only the cone system is functioning. Preparation I. After the retina was isolated it was placed in the apparatus, ganglion cells toward the light. The retina was then dark adapted for 12 min. At this time, a single flash of 636 nm light attenuated by 4.14 log units and of 0.5 set duration was presented. One and one-half min later the photopically equivalent 549 nm stimulus, also attenuated by 4.14 log units, was presented for 0.5 sec. The responses to these stimuli are shown by the top two records of Fig. 2A. The response to the 549-nm stimulus is considerably larger than the

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response to the 636~nm stimulus since most of the 502 pigment is still present, and consequently the red rods are still active. After l-5 min, the original 636 nm stimulus was repeated the response to this stimulus is the third trace in Fig. 2A. After 15 min of dark adaptation the full intensity 10 Hz white light illuminated the retina for 15 min and ERG responses were photographed. Samples of the responses to this 10 Hz stimulus are presented in Fig. 2B. The response to this 10 Hz stimulus increased markedly over the 15 min of stimulation. The time course of this increase is quite similar to that seen in Fig. 1. After the 15-min period of 10 Hz stimulation, the retina was dark adapted for 20 min. This is the procedure shown above to isolate the cone system. At this time, O-5 set 636 nm and 549 nm stimuli were presented, separated by l-5 min. Since both these stimuli were A 636nm

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FIO. 2. ERG responses from an isolated retina to single flashes of 549 nm and 636 run light and to intermittent white light stimulation. See text for explanation of sequence of conditions. Amplitude and time calibration lines shown at the bottom of the figure are for the flicker responses. The calibration lines for each set of single flash responses appear to the right of these responses.

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attenuated by 3.54 log units, these 549 nm and 636 nm stimuli were O-60 log unit more intense than the earlier ones. As expected, the amplitudes of response to these 549 nm and 636 nm stimuli are quite similar as seen in Fig. 2C. Finally, after 25 min of dark adaptation, the IO-Hz white light was presented for 15 min. Again the response to the IO-Hz stimulus increased with light adaptation, even though only the cone system appeared to be functional. Figure 2D shows that the ERG response continued to increase during the first 5 min, then decreased slightly. (The smaller amplitude of flicker in the right hand column vs. that in the left hand column indicates that the retina was probably deteriorating. However, the responsiveness of this particular retina remained relatively stable for 70 min, although its maximum amplitude was smaller than that typically seen in other preparations.) Preparation 2. The retina was placed in the chamber, again with ganglion cells toward the light, and immediately subjected for 15 min to the lo-Hz full intensity light. Following this period of light adaptation, the retina was dark adapted for 20 min. After 18 min of dark adaptation had taken place, a I-set 549~nm stimulus was presented. After another 1.5 min a 636~nm 1-set stimulus was presented. These stimuli were adjusted as above to be equal for the cone system and were produced by attenuating channel I stimulation by 2.9 log units. The responses to the 549~nm and 636~mn stimuli were nearly identical. Prominent OM (b-wave) and o#(d-wave) components are seen in these records. After 25 min of dark adaptation the full intensity 10 Hz white light was presented for 20 min. The ERG flicker responses to this 10 Hz light are presented in Fig. 3. The amplitude of the ERG response increased during the first 3 min of light adaptation and then decreased again. This experiment again demonstrates that the ERG flicker amplitude increases during light adaptation even when only the cone system is functional. Though only two preparations are presented here this experiment was repeated on numerous isolated retinas. Three other common properties of the isolated retina preparation are illustrated in Fig. 3. First, the flicker response reached a maximum amplitude greater than 250 pV. Thus, the isolated retina is capable of responses as large as those seen in the whole frog. Second, the amplitude of the response to the IO-Hz stimulation shows a steady decline after the initial increase. This decline, often present in the isolated retina preparations, has also been observed in deteriorating whole frog and eyecup preparations. The decline, following the increased amplitude due to cone system adaptation, can sometimes be seen a second and third time if the retina is alternately light and dark adapted. Therefore, the phenomenon of the decreased response is not simply due to the aging of the preparation. Finally, the growth in flicker ERG amplitude of the isolated cone system was sometimes even smaller than seen in Fig. 3. This small growth in some preparations may have been partly due to the decline in response amplitude mentioned above. The conclusion from Experiment 1 is that when only the cone system is functioning the ERG flicker response increases in amplitude with light adaptation. Experiment 2. Is there another mechanism controlling adaptation ?

The purpose of Experiment 2 was to hold cone system adaptation constant while varying adaptation of any mechanism with a different sensitivity. Preparation I. The isolated retina was placed in the apparatus, again with the ganglion cells toward the light, and was light adapted with full intensity 10 Hz white light for 15 min. A lo-Hz 549~nm stimulus attenuated by l-07 log units was then presented for 20 min. Samples of the responses after 15 min and 20 min of 549 nm stimulation are shown in

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FIG. 3. ERG responses from an isolated retina to &@a flasks of 549 nm and 636 nrn light and to iWmittent white light stimulation. See text for explanation of sequence of conditioris. Amplitude and time calibration lines are shown to the right of the records for both single flash and ticker responses.

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Fig. 4A. After 20 min of 549 nm stimulation a photopically equal 636 nm 10 Hz stimulus was presented for 20 min and responses such as those in Fig. 4B were photographed. The amp~tude of these responses was quite constant during the 20-min period. The original IO-Hz 549-nm stimulus was then reinstated for 10 min. The responses to the 549-nm stimulus are shown by sample records in Fig. 4C. These responses also remained approximately constant for the IO-min period.

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FIG.4. ERG fiicker responses from an isolated retina to 10 Hz stimulation of 636 nm and 549 nm light. The order of presentation was 15 min adaptation with 10 Hz white light, 20 min stimulation with 10 Hz 549 nm light, 20 min stimulation with 10 Hz 636 mn light, and 10 min stimulation with 10 Hz 549 nm light. Amplitude and time calibration lines are shown in the lower right hand comer.

Pre~~uti~~ 2. The initial procedure for this preparation was-identical to that for preparation 1 (i.e. 15 min of light adaptation, 20 min of 10 Hz 549 nm stimuIation, 20 min of 10 Hz 636 nm stimulation, then 15 min more of 10 Hz 549 nm stimuIation). Figure 5 shows the same general results as Fig. 4.

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FICL5, ERG Bicker responses from an isolated retina to 10 Hz stimulation of 636 m and 549 m light. The order of presentation was 15 min adaptation with X0Hz wbite light, 20 min striation with 10 Hz 549 mn light, 20 min stimulation with 10 Hz 636 mn light, and 10 min stimulation with 10 Hz 549 nm light. Amplitude and time calibration lines are shown in tbe lower right hand corner,

After 15 min of the second 549~nm period, a 636-nm stimulus 1.07 log units more intense than the previous 636 nm stimulus was presented. The responses to this 636 nm stimulus are illustrated in Fig. 6. These responses increase in amplitude with light adaptation and reach a maximum at 3-5 min. The growth in the ERG response to this 636 nm stimulus is another demonstration of the effects of cone system adaptation in the isolated retina. The conclusion from Experiment 2 is that only adaptation of the cone system determines flicker ERG amptitude. If adaptation of some other system were involved then prolonged 549 nm and 636 nm stim~ation should differentially affect the amplitude of the flicker ERG.

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FIG+.6. ERG flicker responses from an isolated retina to 10 Hz stimulation of 636 mn light. See text for precediig conditions. Amplitude and time calibration lines are shown in the lower right hand comer. DISCUSSION This study was concerned with the fact that after dark adaptation, prolonged exposure to intermittent light produces ERG responses of increasing amplitude. The main conclusion is that in the isolated retina, when only the cone system is functioning, the ERG ticker response increases in amplitude with light adaptation. The procedures employed in this study did indeed isolate the cone system as was expected from prior studies. In Experiment 1 it was shown that under conditions yielding a functional isolation of the cone system the flicker ERG increased in amplitude as it does in the live frog. At this point it could have been argued that the other receptors, e.g. red rods, of the isolated retina, although not contributing directly to the tlicker ERG, were somehow modulating its amplitude. However, Experiment 2 demonstrated that only adaptation of the cones affected the flicker ERG response in the bleached isolated retina. Although all four preparations in this study had their ganglion cells toward the light, the main conclusions were verified with isolated retinas with receptors toward the light. The effects of adaptation cannot be attributed to either the pigment epithelium or the iris since neither is present in the isolated retina. In addition, DITTLFIR(1907) observed that

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the cones of the frog’s isolated retina contract with light but do not elongate during darkness. Therefore, the cones are presumably contracted before the increase in flicker ERG due to light adaptation in Experiment 2. Consequently, the mechanism of this adaptation is more proximal than the cone myoids. It is interesting to speculate on the possible loci of these mechanisms of adaptation. The sites of adaptation cannot be more central in the retina than the site of the most proximal ERG generator, cells of the inner nuclear layer (INL). The probable loci of adaptation can be further delineated by examining the nature of the ilicker ERG. Figure 7 from GRANITand RIDDELL (1934) shows the frog ERG response under light adapted and dark adapted conditions. With light adaptation, the c-wave disappears, the b-wave decreases in size, and the d-wave markedly increases in amplitude and in rate of rise. The increased flicker amplitude in the light adapted eye can be understood on the basis of these changes, since the fast cone flicker is due to an interaction of a- and d-waves (GRANIT, 1947). Since the d-wave is an interaction of PI1 and PIII, its increase with light adaptation must reflect changes in these components. Indeed, during light adaptation PI11 shows a large increase in size and a faster return to baseline (GRANIT and RIDDELL, 1934; THERMAN,1938). The increased flicker amplitude appears to be largely due to these changes in PI11 (GIUNIT and RDDELL, 1934). PIII has been shown to consist of two processes: a distal process generated by the receptors, and a proximal process generated by cells in the INL (TOMJTA,1963, 1965; MURAKAMIand KANEKO,1966). Since a portion of PI11 is generated by the receptors, the increase in flicker ERG amplitude may be due to a change in the cone receptor potentials. The cone receptor potentials may increase with light adaptation or more likely change in form, e.g. a faster decay time. The S-potentials recorded from external horizontal cells in fish, which receive their input strictly from cones, indicate that rise and decay times decrease with light adaptation (MITARAI, SVAETICHIN,VALLECALLE,FATECHAND,VILLEGASand LAUFJB, 1961). Changes in rise and decay times could change flicker ERG amplitudes. + I-2r

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7. Componentanalysisof the frog ERG elicitedby a singleflash of 2 set duration. Upper: dark adapted. Lower: light adapted (modified from GRANIT and RIDDELL, 1934).

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However, two facts must be kept in mind. First, PI11 increases in amplitude with light adaptation, and second, the major portion of PIII, proximal PIII, is generated by structures of the INL, not from the receptors (TOMITA,1963; MURAKAMIand KANEKO,1966). Some older ERG studies can be used to argue that it is the proximal PI11 that is undergoing change. BERNHARDand SK~GLUND(1941) have shown that alcohol changes the responses of the light adapted frog eye to responses similar in form to those of the dark adapted eye. That is, the a- and d-waves decrease and the b-wave increases. Since the distal PI11 appears to be the receptor potential, it can be assumed that it is still present, since PI1 and ganglion cell responses are present. Therefore, it is likely that the proximal PI11 is eliminated or greatly decreased by alcohol and that the proximal PI11 reflects some changes in organization during light adaptation. The change in organization during light adaptation manifested by the increased PI11 undoubtedly reflects increased inhibition. GRANIT(1933, 1947) demonstrated that the PI11 was concerned with inhibition. Indeed, GRANITand THERMAN(1934, 1935) showed that an u-wave falling on a d-wave, due to reillumination of the retina, produced an inhibition of the ganglion cell firing. In addition, BERNHARD and SKOGLUND(1941) demonstrated that if the PI11 was decreased, so were the effects of inhibition as reflected in the response to a single flash or the flicker response to intermittent stimulation. This led Granit to conclude that, with prolonged stimulation and light adaptation, PI11 and inhibition increased. In short, the cone adaptation mechanism involves an organizational change of the retina probably mediated by cells generating proximal PIII, possibly the horizontal cells. This change in organization is reflected in the ERG by an increased PI11 component and may involve inhibitory connections that change the entire function of the retina. The change in function is reflected in the increased cff and amplitude of the flicker ERG. To conclude the flicker ERG increases in amplitude with light adaptation when the cone system alone is functional. Consequently, one mechanism responsible for this increase involves a change within the cone system, not an inhibition from the rods. The next paper (Hc~~D, 1972) shows an additional mechanism is involved in the intact retina. Acknowledgement-This work formed part of a dissertation submitted to the Graduate School of Brown University in partial fulfillment of the requirements for the Ph.D. degree. I wish to express my gratitude to LORRIN A. RIGGS for his support of thii research and for his helpful comments on this manuscript. This research was supported by a research contract between Brown University and the Office of Naval Research, and by a predoctoral fellowship awarded to the author by the National Institute of Mental Health. Preparation of the manuscript was aided by a grant from the Institutional Scientific Research Pool of Columbia University.

REFERENCES BAIJMANN,CH. (1964). Die absolute Schwelle der isolierten Froschnetzhaut. Ppiigers Arch. ges. Physiol. 280, 81-88. BAUMANN,CH. (1965). Die Photosensitivitat des Sehpurpurs in der isolierten Netzhaut. Vision Res. 5, 425-434. BAUMANN,Cu. (1967a). Sehpurpurbleichung und Stiibchenfunktion in der isolierten Froschnetzhaut. II. Die Begrenzung der Stibchenfunktion durch Helladaptation. P&en Arch. ges. Physiol. 298, 61-69. BAUMANN,CH. (1967b). Schpurpurbleichung und St%chenfunktion in der isolierten Froschnetzhaut. III. Die Dunkeladaptation des skotopischen Systems nach partieller Sehpurpur bleichung. Pj%gers Arch. ges. Physiol. 298,70-81. BAUMANN,CH. and SCHEIBNER,H. (1968). The dark adaptation of single units in the isolated frog retina following partial bleaching of rhodopsin. Vision Res. 8, 1127-l 138. BERNHARD,C. G. and SKOGLUND,C. R. (1941). Selective suppression with ethylalcohol of inhibition in the optic nerve and of the negative component PI11 of the electroretinogram. Acta physiol. stand. 2, 10-21.

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CR~SCITELLX, F. and SICKEL,E. (1968). Delayed off-responses recorded from the isolated frog retina. Vision Res. 8, 801-816. DITTLER,R. (1907). uber die Zapfenkontraktion an der isolierten Forschnetzhaut. Arch.f. d. gesum. Physiol., 117,295-328. DODT, E. and HIXK, J. (1954). Einfllisse des Adaptationszustandes auf die Rezeption intermittierender Lichtreize. Pflcgers Arch. ges. Physiol. 259, 212-225. DODT, E. and J~SSEN,K. H. (1960). Depression of cone sensitivity during dark-adaptation. Experienriu. 16, 205-206. GOLDSTEIN,E. B. (1967). Early receptor potential of the isolated frog (Runa Pipiens) retina. Vision Res. 7, 837-845. GOLDSTEIN,E. B. (1970). Cone pigment regeneration in the isolated frog retina. Vision Res. 10, 1065-1068. GORDON,J. (1967). ERG spectral sensitivity of the frog, Runa pipiens. Master’s thesis, Brown Univ., Prov., R.I. GRANIT, R. (1933). The components of the retinal action potential in mammals and their relation to the discharge in the optic nerve. J. Physiol., Land. 11,207-239. GRANIT,R. (1935). Two types of retinas and their electrical responses to intermittent stimuli in light and dark adaptation. J. Physiol., Lond. 85,421-439. GRANIT,R. (1936). Die Elektrophysiologie der Netzhaut und des Sehnerven mit besonderer Berucksichtigung der theoretischen Begriindung der Flimmer-methode. Acru ophthaf. 14,1-98. GRANIT, R. (1938). Processes of adaptation in the vertebrate retina in the light of recent photochemical and electrophysiological research. Documenta Ophth. 1, 7-77. GRANIT, R. (1947). Sensory mechanism ofthe retina. Oxford University Press, New York. GRANIT, R. and RIDDELL,H. A. (1934). The electrical responses of light- and dark-adapted frogs’ eyes to rhythmic and continuous stimuli. J. Physiol., Land. 81, l-28. GRANIT, R. and THERMAN,P. 0. (1934). Inhibition of the off-effect in the optic nerve and its relation to the equivalent phase of the retinal response. J. Physiol., Land. 81,47 P. GRANIT, R. and THJXRMAN, P. 0. (1935). Excitation and inhibition in the retina and in the optic nerve. J. Physiol., Land. 83, 359-381. Hook, D. C. (1972). Suppression of the frog’s cone system in the dark. Vision Res. 12, 889-907. K~HNE, W. (1879). Chemische Vorg%nge in der Netzhaut. In Hands. Physiol. (edited by HERMANN),3, 235-342. LYTHGOE,R. J. and TANSLEY,K. (1929). The relation of the critical frequency of flicker to the adaptation of the eye. Proc. R. Sot. B. 105,60-92. MACNICHOL, E. F., JR. and SVAETICHIN,G. (1958). Electric responses from the isolated retinas of fishes. Am. J. Physiol. 46, 26-4Q. MITARAI,G., SVAETICHIN,G., VALLECALLE, E., FATECHAND,R., VILLEGAS,J. and LAUFER,M. (1961). Glianeuron interactions and adaptational mechanisms of the retina. In The visual system: neurophysiology andpsychophysics (edited by JUNO and KORNHUBER), pp. 463-481. Springer-Verlag, Berlin. MURAKAMI,M. and KANEKO,A. (1966). Differentiation of PI11 subcomponents in cold-blooded vertebrate retinas. Vision Res. 6, 627-636. SICKEL,W. (1961). Stoffarechsel und Funktion der isolierten Netzhaut. In Neurophysiologie undPsychophysik der Visuellen System, pp. 80-94. Springer-Verlag, Berlin. THERMAN,P. 0. (1938). The neurophysiology of the retina in the light of chemical methods of modifying its excitability. Actu Sot. Sci. fenn. 2, l-74. TOMITA,T. (1963). Electrical activity in the vertebrate retina. J. opt. Sot. Am. 53,49-57. TOMITA,T. (1965). Electrophysiological study of the mechanisms subserving color coding in the fish retina. Cold Spring Harb. Symp. quant. Biol. 30,559-566. ZEWI, M. (1939). On the regeneration of visual purple. Actu sot. Sci. fenn. N.S.B. 2, l-57. Abstract-An intermittent light presented to a dark adapted frog eye produces a small “flicker ERG” (response to the individual light pulses). After light adaptation the flicker ERG increases drastically in amplitude. This study showed that a qualitatively similar increase is seen in an isolated retina in which the rods were inactivated by bleaching 80 per cent of their pigment, rhodopsin. This bleach does not permanently affect the cones since, unlike the rods, their pigment regenerates in the isolated retina. Consequently, in the isolated retina the increase involves a change within the cone system, not an inhibition from the rods. The next paper shows an additional mechanism is involved in the intact retina. R&u&--Dans l’oeil de grenouille adapt& & l’obscuritt, une lumibre intermittente produit un petit “flicker ERG” (rkponse aux pulsations individuelles de lumikre). Apr&s adaptation & la lumitre, le flicker ERG augmente considerablement en amplitude. On montre dans le

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prksenttravailque l'on constate un accroissement qualitativement semblable dans une &tine isolQ oti on inactive les bltonnets en d&olorant 80 pour cent de leur pigment, la rhodopsine. Cette d&coloration n’affecte pas les c&es en permanence puisque, contrairement aux batonnets, leur pigment se rbg&re dans la r&ine isolr5e. Dans la r6tine isol&, l’accroissement suppose done un changement dans le systkme des c&es et non une inhibition venant des b&tonne&. L’article suivant montre que dans la &tine intacte il existe un m&a&me supplbmentaire.

Zusammenfassung-Beim dunkeladaptierten Froschauge erzeugt ein Flimmerlicht ein kleines Flimmer-ERG (Antwort auf die individuellen Lichtpulse). Nach Helladaptation nimmt die Amplitude des Flimmer-ERG stark zu. Diese Arbeit zeigt, da13eine qualitativ lihnliche Zunahme in isolierten Netztiuten beobachtet werden kann, in denen die %&hen durch Ausbleichen von etwa 80 Prozent ihres Pigmentes, Rhodopsin, inaktiviert wurden. Diese Bleichung beeinflul3t die Zapfen im Gegensatz zu den %&hen nicht dauernd; ihr Pigment regeneriert in der isolierten Netzhaut. Deshalb bedeutet die Zuanhme in der isolierten Netzhaut eine hderung im Zapfensystem, nicht eine Hemmwirkung von den StPbchen. Die ngchste Arbeit zeigt, dal.3 bei der lebenden Netzhaut ein zusgtzlicher Mechanismus mitspielt. Pe3EOMe--l-lpepbIBHCTbrk CBeTBbI3bIBileTBTeMHO-aAalYITEpOBaHEIOMrJIa3y JI5IrjTKJXEHE3KyIO no abfn.miTyfle%xenbKa~oruyro 3PT” (Pm Ha 0TAeJIbnbre sihmynbcbI cBeTa). Ihxe cseToBo& aaanraquw Menbxammaa 3PT pesro BospacTaeT no ahmnm yne. B AaHHOM ECCJIe,QOBaFIHHlIOKa3aHO,'ITOKaWCTBeHHO CXOAHOe YBelDi'IeHEeHa6JImAaeTcn B H30JIEpOBaHHOB CeTsaTKe, UaJIoSKH KOTOpOi ElIiaKTEBEpOBaHbI 06eCUe¶EBaEfieM 80% Kx WrMeHTa, pO,I(OIICIiHa. 3TO 06eCmwniBaHIie He OKa3bIBaeT JJJIEiTeJIbHOrO BO3AeztcrGEUlHa KOJI~OW~, llOCKOJIbK~,B OTJlEWie OT IIalIO'IeK,HXWTMeHT pereHepIipyeT BE30JlEpOBaHHOk CeTYaTKe. BHyTpH KOJI6O'iKOBOfi 3~0 ysememie MenbKaromeP 3Pr onpeAeer0-i rf3MeHew CECTeMbI,a He TOpMOX(eHHeM CO CTOpOHbI IYIaJIO'ieK. CJIegyKJmaX pa6ora IIOKa3bIBaeT,'ITO 6 HHTaKTHOzt CeT’IaTKe AefiCicrByeT eme AO6aBO'iHbti MeXaHH3M.