Dark-adaptation processes in the rhodopsin rods of the frog's retina

Vision Rrs. Vol. 7, pp. 17-41.

Pergamon Press1967. Printed inGreatBritain.

DARK-ADAPTATION RHODOPSIN

RODS

PROCESSES OF THE

FROG’S

IN THE RETINA

K. 0. DONNER and TOM REUTER Department

of Zoology, University of Helsinki (Received 20 June 1966) INTRODUCTION

RECENTLY, we (DONNER and REUTER, 1965) analysed the dark-adaptation of the excised and opened frog’s eye by recording the impulse activity of isolated ganglion cells in the retina. The sensitivity of these units was measured in terms of the light intensity necessary to produce a threshold response. We found that if the stimulus was small (a circular spot less than 0*3-0*4 mm in diameter centered on the receptive field) no changes in the nervous organization of the field could be detected, spatial summation being constant over the whole course of dark-adaptation. Measured in this way the sensitivity increase observed during dark-adaptation would therefore be caused by changes in the properties of the receptors, their light-catching properties, etc. Possible changes in temporal summation have not been studied, but they do not, as judged by the latency of the discharge at threshold, seem to be of any considerable magnitude. With test fields <0*3-0*4 mm diameter and light of wave-length 500 nm, dark-adaptation curves of the type shown in Fig. 1 (uppermost curve) were obtained when the preceding light-adaptation had been strong and of fairly long duration, thus leaving only small amounts of rhodopsin in the rods (REIJTER, 1964, 1966). To begin with the sensitivity is determined by the cones, as can be shown by measurements of the spectral sensitivity at this stage, and by the presence of a Stiles-Crawford effect (DONNERand RUSHTON, 1959b). This cone branch lasts for 10-15 min under the conditions used (Donner and REU-ER, 1965). Then follows the rod branch of the curve, during which, at 500 run, the rhodopsin rods determine the sensitivity, with possibly a small contribution from the cones in the very beginning of the curve. Adaptation is complete after about 180 min, which coincides in time with the full regeneration of rhodopsin at the temperature of the experiment (13-14”). If the rod branch of dark-adaptation curves, such as that in Fig. 1 (uppermost curve) is analysed, omitting the rapid increase of sensitivity between the end of the cone branch and about 40 min of dark-adaptation, we found that the increase in sensitivity can be accounted for by the following processes: (i) the withdrawal of the black melanin pigment from the receptor interspaces. The functionally significant part of this process occurs 6&80 min after the beginning of darkadaptation under the conditions used here (BRICK, DONNER and REUTER, 1965); (ii) the increase in the density of rhodopsin in the rods, which will increase the rate of quanta caught at a given intensity of light; (iii) a third process, which we concluded depends on the rate of regeneration. Since 17

B

18

K. 0.

DONNER

and

TOM

bJTER

the effects of (i) and (ii) can be accounted for quantitatively, it is possible to calculate the relative rate of quanta absorbed (AZ,) at threshold at any instant of the dark-adaptation process. This gives a curve of the shape shown in Fig.1 (middle curve, broken line).

FIG. 1. Dark-adaptation curves for single units in different states of light adaptation. Thresholds measured at 503 nm. Upper curve (circles): test field @ 19 mm in dia., the eye strongly light-adapted before beginning of dark-adaptation. Middle curve (dots, broken line): Relative values for the number of quanta absorbed at threshold in the experiment described by the upper curve (see DINNER and RIWTER, l%S). Lowest curve (circles): test field 0.24 mm in diameter, the eye originally fully dark-adapted, thresholds after 60 set of 462 nm exposure which bleaches about 6 per cent of the rhodopsin. Temperature in both experiments 13”.

Expressed in this way we found that AI, (after more than 40 min of dark adaptation) showed the same relation to the rate of regeneration of rhodopsin as the increment threshold of the rhodopsin rods to the intensity of a steady background field (DONNER,1959). The simplest interpretation of this finding seems to be that equivalent sensitivity-reducing effects were produced by the processes initiated by the isomerization of rhodopsin by light and by the synthesis of new molecules of rhodopsin in the rods. We calculate that the reduction of sensitivity produced by the isomerization of 1 molecule of rhodopsin per rod per set would be the same as that caused by a regeneration rate of 4.7 x 10s molecules per rod per sec. This interpretation leads to the conclusion that the sensitivity of the rhodopsin rods, expressed in terms of the number of quanta caught at threshold, is independent of the amount of rhodopsin present. Obviously, however, there is an indirect connection because the rate of regeneration during the later stages of dark-adaptation appears to depend on the amount of unregenerated rhodopsin in a simple way.

Dark-Adaptation

Processes in the Rhodopsin

Rods of the Frog’s Retina

19

This explanation regarding the later part of the rod dark-adaptation process in the frog (DONNER and REUTER, 1965) appears to be the simplest and most economical. Considering, however, the results obtained and the conclusions drawn regarding roa dark-adaptation in the rat and in man (DOWLING, 1963; RUSHTON, 1961a, b, 1965) it appeared desirable to gain additional evidence about the significance of the rate of regeneration. In the frog this process is highly temperature-dependent, with a Q10 of about 4 (ZEWI, 1939 : Rana esculentu). This gives an opportunity of altering the rate of regeneration by changing the temperature of the excised eye. Moreover, this makes it possible to devise an experiment where, for instance, a sudden reduction of the temperature during darkadaptation should give rise to entirely different effects depending on whether the rate of regeneration or the amount of rhodopsin is the significant factor. In the former case an immediate sensitivity increase should be observed, in the latter case again a retardation of the rate of dark-adaptation. The outcome of such experiments is reported in the first part of the present paper. They fully support the conclusion previously reached (DONNER and REUTER, 1965) that it is the rate of regeneration that is significant. It is evident however, that this explanation cannot apply to the initial part of the rod adaptation curve. As seen in Fig. 1, there is a rapid increase in sensitivity from the conerod transition to about 40 min of dark-adaptation. This occurs during a period (15-40 min) when the rate of regeneration is practically constant (REUTER, 1966). The process that causes this rapid sensitivity increase is analysed in the second part of this paper. A rapid adaptation phase of this kind was earlier described and studied in the frog by GRANIT et al., (1938), who found it to be present even after very weak preceding light-adaptation. Its presence can be demonstrated with our conditions of recording and stimulation also, where neural reorganization of the receptive field is excluded. This is shown in Fig. 1 (lowest curve), which gives the course of dark-adaptation of a single unit after the rapid bleaching of approximately 6 per cent of the rhodopsin in an initially fully dark-adapted eye. The curve can be seen to follow approximately the same course as the uppermost curve in Fig. 1, which refers to an eye where strong preceding light-adaptation has removed more than 95 per cent of the rhodopsin. This suggests that the initial rapid adaptation of the rods is caused by the same process in both cases and is unrelated to the regeneration of rhodopsin or to the rhodopsin content. Though such rapid adaptation processes have been described for other eyes, including those of man (e.g. RUSHTONand COHEN, 1954; DOWLING, 1963), and have frequently been attributed to neural factors, it appears that this explanation is less likely for our experiments. It is then necessary to examine the possibility that some chemical event or state in the thermal decomposition of the rhodopsin molecule after the intial isomerization by light is the cause of this rapid adaptation phase. MATERIAL AND METHODS Material and preparation Common frogs (Rana temporaria) from South Finland wem used. They were caught in the autumn and stored at 2-6” in a refrigerator. Before use the animals were kept for about 12 hr at 15-20” in darkness. If light-adapted eyes were to be used the frogs were given 1 hr in light before the eyes were excised and opened, and were then further light-adapted under the mi croscope lamp of the diion microscope. The procedure was in all its details identical with that described by DONNW and REWIZR (1%2,1965) and I&KITER(1966). At the beginning of dark-adaptation these eyes contained 1.5-2 per cent of the full amount of rhodopsin (REUTER, 1966). In the case of initially dark-adapted eyes the animals were treated in the same way, except that there was no preliminary light-adaptation. The eyes were excised and opened under a deep red light. Rhodopsin and metarhodopsin measurements These were all carried out according to the description given by REUTER (1966). Rhodopsin was

20

K. 0. DONNERand TOMREUTER

measured in digitonin extracts of the retinae, with the addition of hydroxylamine and with special precautions to stop the regeneration process when the retina was removed from the eye. The decomposition of metarhodopsin was measured on whole mounts of the retina in a Beckman Model B spectrophotometer. Fit the retina was exposed for 30 set to a blue-green light (495 nm interference filter), which bleached more than 90 per cent of the rhodopsin. Following this the density changes in the retina were measured, for instance at 480,420 and 390 run, with 600 mn (for measurements at 480 nm) or 640 nm as a reference. This was carried out in a temperature-controlled room and with a thermocouple attached to the cell holder of the spectrophotometer for direct readings of the temperature of the preparation. Electrophysiologicalexperiments Single-unit activity was recorded with the aid of 3 M-NaCl-filled glass micropipettes from nerve fibres or ganglion cells close to the retinal surface. The apparatus for recording and display as well as the optical system have been described by DONNERand REUTER(1965). The lights used were measured in terms of quanta with the aid of a calibrated vacuum thermocouple and a sensitive galvanometer. As a modifkation of the previous technique provision was made for varying the temperature of the excised and opened eye during the course of the dark-adaptation process; that is, during the recording

0

5cm

I

I

FIG. 2. Arrangement used for the recording. A, opened eye; B, piece of chalk supported by Perspex holder; C, test tube with Ringer’s solution; D. thermocouple; E, coil of copper tube with rurming water. from a single unit (Fig. 2). The opened eye (A) rested on a piece of chalk supported by a Perspex holder (B) so that most of the chalk was submerged in Ringer’s solution contained in a small test tube (C). This was surrounded by a coil of copper tube (E) in close contact with the glass. Water of the required temperature could be run through the copper tube. The temperature of the test tube and of the preparation could thus be kept at any desired value, or the temperature could be rapidly altered by changes in the temperature of the water running through. A change of 5” in either direction in 5 min was a fairly normal value in the region 5-18”. The copper coil and the test tube were imbedded in a block of foam plastic. The temperature inside the test tube, close to the preparation, was measured by a thermocouple (D) connected to a spot galvanometer outside the black box, which contained this arrangement as well as the micromanipulator and the final lenses of the optical system. Thus the temperature of the opened eye could be read continuously during the experiments.

Dark-Adaptation

21

Processes in the Rhodopsin Rods of the Frog’s Retina RESULTS

1. The effect of temperature on rod sensitivity during dark-adaptation

As stated in the introduction, changes in temperature should affect the sensitivity during the rod adaptation phase, because of its effect on the rate of regeneration. Further, if the Qis of the regeneration process is known, the sensitivity changes should be quantitatively predictable. If, on the other hand, it is the amount of rhodopsin in the rods that is significant, no sudden sensitivity changes should be observed when the temperature is altered, but only a change in the slope of the dark-adaptation curve corresponding to the changed rate of increase of rhodopsin. A typical experiment showing the effect of temperature changes is illustrated in Fig. 3A, which gives the dark-adaptation curve for the off-effect of an on/off-unit, starting from

%--J 1

25

50

75

I

100 lime,

_

I u

1 --

25

P-

I

5

min

FIG. 3. A. Dark-adaptation of fully light-adapted on/off-unit, off-thresholds (upper curve). 503 nm, field Q24 mm in dia. Lower curve gives the temperature (ordinates on the right). Time given from the beghming of dark-adaptation. B. On-threshold of fully dark-adapted on/off-unit during a temperature change. 503 mn, D24 mm field. Lower curve gives the temperature. Time from the instant when the -unit was found.

the completely light-adapted condition (see Methods). The lower curve gives the temperature at each instant. It is seen that a lowering of the temperature causes a rapid rise in sensitivity, the opposite being true of a rise in temperature. Because these experiments refer to the recording of impulses from single ganglion cells or optic nerve fibres, however, it is clear that the excitatory effects from the rods have passed at least two synapses between the receptors and the site of recording. It follows that before effects such as those in Fig. 3A can be accepted as evidence for any changes

22

K.O.

DONNER

and TOMREUTER

or events in the rods it is necessary to show that the transmission channel from the rods is, as such, unaffected by changes in the temperature. That this is the case is shown in Fig. 3B, which gives the result of measurements of the sensitivity of a fully dark-adapted on/off-unit (on-effect). Here where there is no regeneration of rhodopsin going on, a rise in temperature of about 5” does not give rise to any effect at all, except for a small transient sensitivity reduction during the initial phase of the temperature change. This is the only effect on the sensitivity that has been observed in several such experiments, a reduction of temperature sometimes giving the same transient effect. In either case sensitivity quickly returns to the original value. We conclude, therefore, that the effects observed during dark-adaptation (Fig. 3A) are actually caused by thermal effects on the mechanism of adaptation in the receptors. Figure 4 shows the result of an experiment (on/off-unit, average of on and off thresholds) where a higher temperature has been applied twice, once during the fast phase of regeneration and ‘once during the end of the dark-adaptation process. The effect of the second rise i

25

50

15

100 Time, min

125

150

175

FIG. 4. Dark-adaptation of fully light-adapted on/off-unit. Average of on- and off-thresholds (upper curve). 503 nm, @24 mm field. Lower curve gives temperature. Time from the beginning of dark-adaptation.

in temperature (from 155 min ownards) is clearly smaller than the first, as would be expected near the absolute threshold because of the curved shape in this region of the increment threshold curve (DONNER, 1959). These experiments thus qualitatively co&m that the rate of regeneration and not the amount of rhodopsin (or free opsin) is important during dark-adaptation. In order to make quantitative comparisons possible it is necessary, however, to measure the actual rates of rhodopsin synthesis under the same experimental conditions as during the electrophysiological measurements with rapid temperature changes of the eye. ZEWI (1939) obtained a Qrs for the regeneration process of about 4. However, his data refer to the species Rana esculenta and to a comparison between frogs that were kept at the same

Dark-Adaptation Processesin the Rhodopsin Rods of the Frog’s Retina

23

temperature for the whole period of dark-adaptation. His data do not show whether a change in temperature during regeneration would cause an immediate decrease or increase in the rate of synthesis. For these reasons rhodopsin measurements were carried out according to the following procedure, using the regeneration curve of RJXITER(1964, 1966) at 13-14” as a basis. Both light-adapted, excised and opened eyes from the same frog were left to dark-adapt at 135” for 48 min, which corresponds to the regeneration of 50 per cent of the rhodopsin. Then one of the eyes was taken for extraction and the other eye allowed to dark adapt for a further 20 min at 8-Y or 13.5” or for 15 min at 18-O”,and was subsequently extracted. At 18” only 15 min was necessary because of the rapid regeneration at this temperature. while a 20-min period would bring the rhodopsin to such a concentration that the regeneration curve no longer follows a linear course. The density loss at 500 nm after complete bleaching of the extract was used as a measure of the amounts of rhodopsin present. By comparison of the amount of rhodopsin in one 48-min eye with the amount in the 68-min (respectively 63-min) eye from the same frog, values for the rate of regeneration were obtained for limited periods at 8-5 or 18” as well as when the temperature had remained unchanged at 13.5”. This method uses the fact that although there is a fairly large variation in the rhodopsin content of eyes from different frogs in the same state of adaptation, both eyes from the same frog generally give rhodopsin values not differing by more than 5 per cent from each other (ZEWI, 1939). TABLE1 Rate of

Temp. during Frog no. the later period of adaptation :

3 4

8.5 8.5 8.5

Density x 103 at 48 min

at 68 min

21.2 192

26.3 23.2

28.3 25.2

32.8 28.8

MWl

DiSerence re8cncration inpercent inpercentof of initial 48 min value value per min 208 24.1 15.9 14.3

18.8

5

13.5

21.8

33.2

52.3

76

13.5

18.5 148

24.2 21.6

46.0 u)8

Mean 8 9 10 11

43.0 18.0 18.0 18.0 18.0

20.1 26-O 18.8 27.3

at 63 min 299 42.4 33.8 40.2

M&Xl 8.5-13.5” 13~5-18*0” 8*5-18.0”

5.2 3.9 4.6

2.15

48.8 63.1 798 47.3 598

QIO

0.94

3.98

24

IL 0. DONNEX and TOMREUTER

The results are given in Table 1, expressed as density values obtained from extracts of single retinae and corrected for differences in retinal area (DOWER and REUTER,1962). From these values the rate of regeneration in per cent/mm has been calculated for the period of changed temperature, and finally, based on the averages obtained, the Qlo. The values so obtained show fair agreement with the value 4.1 given by ZEWI (1939) for the temperature interval 7*2-17.2”. It can further be calculated from his data that Qls between 7.2 and 12.2” is 4.9. It therefore seems justifiable to conclude that the changes in temperature applied during the present experiments result in an imme~ate change in the rate of regeneration, and, as judged by the agreement found with Zewi’s data, to such values as would be obtained in eyes kept at a constant temperature during the whole period of regeneration. Experiments such as those shown in Figs 3A and 4 allow the magnitude of the rise or drop in threshold caused by the temperature change to be evaluated with fair accuracy. This has been done, as indicated in Fig. 3A, by extrapolation of the course of the darkadaptation curve to its expected value in the absence of the tem~rature change and then reading off the difference in sensitivity. In Table 2 such values are given from 9 individual experiments. These data all refer to temperature changes applied between 40 and 100 TABLE 2 Change in temperature Af” -8.1 -10.0 -9.9 -8.0 -9.6 -1@8 2-10-S i-4.6 i4-7

Observed increase (+) or decrease (-) in fog intensity at threshold -0.7 -0.8 -07 -0.7 -0.6 -0.8 +0*23 -+0*6 +0*4

mitt of dark-adaptation and with initial temperatures between 10 and 15”. It appears that a temperature change of 10” causes a change in threshold of about O-8 log units and one of 5” of about 0*5-0~6 log units. On the other hand a temperature change of 10” alters the rate of regeneration by a factor of about 4.6 (Table 1). This then is the change in “equivalent background” that in our formulation (DONN~R and RJSITER,1965) acts in the same way as a real light. In the present experiments this background is then expected to change by 4.6 or, in log units, 0.66 per 10”. From the rod increment threshold curve for the frog it can be calculated that a change of this magnitude in log background should give a change in threshold of O-8 log units, which thus fully agrees with the results of the temperature experiments {Table 2). It should be noted here that the increment threshold curve of the frog rods does not follow an exactly 45” course in a log-log plot. These changes in threshold occur during such short time intervals (S-10 min) that the amount of rhodopsin in the rods can be considered practically constant, and does not, then, affect the results in an appreciabIe way. It should further be noted that the interval 6080 min has been avoided for measurements of this kind because of the occurrence at this stage of the significant part of the photomechanical movements. These experiments thus also quantitatively corroborate our former conclusion regarding the effect of the rate of regeneration upon rod sensitivity during dark-adaptation.

Dark-Adaptation Processes in the Rhodopsin Rods of the Frog’s Retina

25

2. The early, rapid phase of dark-adaptation in the rods

Referring to Fig. 1, it was stated in the introduction that the initial rapid adaptation phase observed from the cone-rod transition (10-15 min) to about 40 min of dark-adaptation cannot be caused by changes in the rate of regeneration. Moreover, a very similar and, under certain conditions, even identical rapid adaptation phase can be recorded and studied in isolation when the eyes are initially dark-adapted and then given a weak and short light-adaptation that bleaches only a minute fraction of the rhodopsin present (Fig. 1, lowest curve; see also GRANIT,et al., 1938). In such a case the regeneration process should be of no, or only very small, significance. As regards the nature of this rapid adaptation phase it may be remarked that it shows a very similar time-course to that of the conversion of metarhodopsin to retinal and opsin as can be seen if measurements of the fading of metarhodopsin (REUTER, 1966) in the excised retina are compared with the curves in Fig. 1. Similar measurements of the fading of metarhodopsin in situ have been reported by DENTON(1959), MATTHEWSet al. (1963) and BAUMANN(1965, 1966), and, indeed, this slow fading of a yellow compound in the retina after bleaching was even seen by K~&E (1879). In the introduction we referred to the similarity between the course of the two darkadaptation curves shown in Fig. 1 over the time-interval of 1540 min. Here the first (uppermost) curve refers to a strongly light-adapted eye initially containing only 15-2.0 per cent of the full amount of rhodopsin (actually a mixture of equal amounts of rhodopsin and isorhodopsin) and about 5-10 per cent metarhodopsin, as can be estimated from the data for this particular experiment. In the lowest curve of Fig. 1 about 6 per cent of the

1

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Dark-adaptation of initially fully dark-adapted on/off-unit after 30 set exposure to white light, bleaching about 55 per cent of the rhodopsin. Off-thresholds, 503 mn, 0.24 mm field. Time from the end of the exposure, which is indicated by the thick vertical line. The threshold before the illumination given to the left of this line. The cono-rod transition is indicated by an arrow. Temperature 13”.

26

K. 0. DONNER and TOMREV~ER

rhodopsin in the initially fully dark-adapted eye has been bleached, giving then 6 per cent metarhodopsin and 94 per cent rhodopsin at the beginning of dark-adaptation. In both cases then roughly equal amounts of metarhodopsin are present at 0 min, while the amounts of rhodopsin are vastly different. We have further observed that strikingly different dark-adaptation curves can be recorded after rapidly bleaching a large fraction of the rhodopsin instead of using long periods of light-adaptation. This is illustrated in Fig. 5, which shows the dark-adaptation of a single unit after the bleaching of about 55 per cent of the rhodopsin in 30 set in an initially dark-adapted eye. Here cones determine the sensitivity during the first 40 min, the cone-rod transition in Fig. 5 being indicated by an arrow. The rapid bleaching of rhodopsin then appears to cause a prolonged desensitization of the rods by comparison with the state when a long light-adaptation has given time for most of the metarhodopsin to decompose. This kind of dependence on the length of the period of preceding lightadaptation has been found in the human eye also (WALD and CLARK, 1937; CRAWFORD, 1946; MOTE and FORBES,1957), where if the product Ix? is kept constant, small values of t give initially higher rod thresholds. These qualitative observations suggest that the decomposition of the amount of metarhodopsin in the rods, or some process related to it, actually affects in a significant way the sensitivity of the rods. Quantitative experiments have been carried out in order to test this possibility more accurately. Before they are presented it is however necessary to give a more detailed account than before (REUTER,1966) of the decomposition of metarhodopsin in the excised retina as observed in spectrophotometric measurements on whole mounts of freshly excised, dark adapted retinae. The fading of metarhodopsin I and II in the isolated retina The work of MATTHEWSet al. (1963) established that metarhodopsin,

the first more stable photoproduct of rhodopsin, exists in two tautomeric forms, metarhodopsin I and at about 480 and 380 nm respectively. The equilibrium between them can be II, with AmaX. affected in various ways; thus the proportion of metarhodopsin II is favoured by higher temperature or acidity, neutral salts, glycerol and some specific reagents, which have this effect even at low concentrations. Using solutions of cattle rhodopsin MATTHEWSef al. (1963) further established that the half-return time of the reaction metarhodopsin 1-t II is about 1 min at 1” (QlO~lOO) and that the equilibrium is very rapidly reached at biological temperatures, metarhodopsin I being the substance formed from lumirhodopsin. By comparison metarhodopsin II is relatively slowly transformed to the hydrolyzed and reduced product retinol+opsin (MATTHEWSet al., 1963). The hydrolysis of metarhodopsin II thus determines the rate at which the amounts of metarhodopsin I and II in equilibrium decrease. Measurements on the isolated frog retina of the fading of metarhodopsin I and II have shown that there is an exponentially decreasing density, irrespective of whether a green measuring light (BRICKet a/., 1965; BAUMANN,1965, 1966), which is absorbed by metarhodopsin I, or an ultraviolet light, absorbed by metarhodopsin II (MATTHEWSet al., 1963), is used for measurement. The measurements of REUTER(1966) of the decomposition of metarhodopsin I and II at 12-15” deviate in two respects from this simple scheme. First, as is seen also in Fig. 6 he established that the decomposition curves were not exponential when measured at 390 or 417 nm. Secondly, Reuter observed that during the first three minutes after the cessation of the bleaching light the density at 390 nm rapidly decreased, with a corres-

Dark-Adaptation

I

Processes in the Rhodopsm

I

I

27

Rods of the Frog’s Retina

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f

SO-

.LO-4

.35 -

*30-

z 3 : 0

.25-

.204 j

.15 -

-10 -

.05-

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FIG. 6. Density changes (“dark reactions”) in fresh isolated retinae after 30 set 495 nm bleaches at various temperatures and wav&ngths. Bleach represented by hatched area, and the symbols on the left side of it the density immediately before the bleach. The time is given from the end of the bleach. Filled symbols: single experiment with measurements at 390,420 and 480 run (15”). Open triangles and squares: single experiment with measurements at 390 and 420 nm at 15”. Open circles: three separate experiments with measurements at 480 nm at 6.8, 15 and 25.5”. The density scale (ordinate) gives the actual density difirences measured in the experiment with 6lled symbols, but the curves are arbitrarily displaced vertically on the scale. The measurements indicated by the open symbols are scaled to facilitate comparison with the values given by the filled symbols. The broken lines indicated in the 480 nm curves at 6.8, 15 and 25.5” describe, together with the later part of the curves in full, exponentially decreasing densities with 9, 4.5 and 1.75 min half-return times respectively. The final level at 6.8” is indicated by an arrow.

28

K.O.

DONNER

and TOM FWJTER

ponding increase in the density at 480 nm, suggesting a transformation of metarhodopsin II into a coloured compound (probably metarhodopsin I). At 417 nm, which is the isosbestic point for cattle metarhodopsin I and II (MaTTHEws etal., 1963), a slowly decreasing density was observed. The curves in Fig. 6 show these features, established in some new experiments. Measurements at a number of wave-lengths in the same experiment have confirmed that the orange compound formed during the first minutes in the dark is actually metarhodopsin I, as judged by the agreement obtained with the spectra published for cattle metarhodopsin I (MATTHEWS et al., 1963), frog metarhodopsin I in glycerol at - 65” (HUBBARD et al., 1959) and metarhodopsin I in situ in the frog retina (BAUMANN, 1966).

20

min

FIG. 7. Density changes in an isolated retina after two subsequent bleaches. Dots: measurements at 480 run. Triangles: measurements at 390 run. The density values at 480 nm are all in a correct relationship to each other, as are those at 390 run, but the 480 and 390 nm values have been vertically displaced in relation to each other. Temperature 15”. Bleaches and initial density levels indicated as in Fig. 6. First exposure: 558 nm for 50 set, bleaching about 27 per cent of the rhodopsin. The curves given (left) are those from Fig. 6 for 480 and 390 nm at 15”, both scaled in the same way to give the best fit. Second exposure: standard 495 nm bleach, curves (right) drawn to fit the points. Note that the normal equilibrium between metarhodopsin I and II is not restored after the second exposure. The 480 run points, immediately after the first exposure, suggest that a moderate bleach of this kind does not displace the metarhodopsin equilibrium towards metarhodopsin II to the same extent as the stronger, standard bleach (curve).

Dark-Adaptation Processesin the Rhodopsin Rods of the Frog’s Retina

29

The rapid conversion of metarhodopsin II into metarhodopsin I during the first minutes after the cessation of the bleaching light indicates that strong illumination shifts the equilibrium towards metarhodopsin II and that the opposite shift occurs afterwards in the dark. This observation is in apparent disagreement with the statement by MATTHEWS et al. (1963) that the equilibrium is not promoted in either direction by light. However it can be shown using weak bleaching lights that the effect is not caused by the absorption of light by metarhodopsin I. In Fig. 7 (left part) an experiment is shown where a 50-set bleach at 558 nm has bleached 27 per cent of the rhodopsin. Immediately after the bleach the amount of metarhodopsin I is found to be about 25 per cent of the total metarhodopsin formed, rising after a few minutes to 50-60 per cent. Thus a radical displacement of the metarhodopsin equilibrium has occurred, although it can be calculated that only 8 per cent of the metarhodopsin molecules would have absorbed a light quantum even if they all had been in the metarhodopsin I-form during the bleaching (calculated from the per cent of rhodopsin bleached, from the assumption that the quantum efficiency of the bleaching process is 1, and from the relative extinctions of rhodopsin and metarhodopsin I respectively at 558 nm). These observations suggest then that the bleaching of rhodopsin in situ is accompanied by the liberation of a metabolite or an inorganic ion (possibly H+ ions), which displaces the equilibrium towards metarhodopsin II and which in the dark is afterwards consumed if the retina is still sufficiently fresh. As can be seen in Fig. 7 (right) the capacity to restore the equilibrium has been considerably weakened during 30 min in the measuring apparatus. Although the fading of metarhodopsin I, when the normal equilibrium has been reached, can be described by a first-order reaction, it is evident (Fig. 6) that the measurements at 390 and 420 nm do not give an exponential decrease. This is probably caused by the formation and subsequent decomposition of an unknown intermediate between metarhodopsin II and retinol+opsin. When measurements are carried out at 390 nm at 15” with the retina suspended in an alkaline buffer (Fig. 8), the accumulation of this compound appears as a secondary maximum or as a plateau on the decomposition curve 8-20 min after the end of the bleaching. The extinction of this unknown intermediate seems to be < 50 per cent at 420 nm of its extinction at 390 nm, with no appreciable extinction at 480 nm. It is then unlikely that it is identical with free retinal+opsin, which would be the logical intermediate between metarhodopsin II and retinol+opsin. The measurements at 480 nm in Fig. 6 show that metarhodopsin I, when the equilibrium between I and II is reached, fades exponentially with the following half-return times: 6.8” 9-O min 15.0” 4.5 min 25.5” 1.75 min. These values give a Qis=2*4 for the range 6.8-25*5”, which is in good agreement with the value 2-3 given by MATTHEWS et al. (1963) for metarhodopsin in solution. It appears likely that these Qto-values reflect the summed effect of two different actions of temperature. First, a higher temperature increases the probability of hydrolysis of a metarhodopsin II molecule and, second, a rise in temperature increases the proportion of metarhodopsin II. Our calculations on the effect of temperature on the metarhodopsin equilibrium (see below) suggest that the Qio-value 2.4 can be divided into a hydrolysisfactor 1.54 and an equilibrium-factor 1.56.

30

K.O. DONNERXI~TOM

REIJTER

In order to determine the different metarhodopsin equilibria at different temperatures and pH-values from the curves measured at 390 nm and 480 nm, the following method has been used. The method is based on the following assumptions: (i) the end product of the metarhodopsin decomposition, retinol+opsin, has no appreciable absorption at 390 and 480 nm; (ii) at 480 nm metarhodopsin II has no extinction while metarhodopsin I has its highest extinction. At 390 nm the extinction of metarhodopsin II is nearly maximal (about 97 per cent of the maximum) and of metarhodopsin I 17.5 per cent of the maximum (calculated from data for cattle metarhodopsin, MATTHEWSet al., 1963, Figs. 2 and 3); (iii) the molar extinctions of metarhodopsin I and II at their absorption maxima show the relation 45(I) to 42(11). It can be questioned whether (iii) applies to the situation in situ, because the relative extinctions in that case depends on the orientation of the pigment molecules in the rod outer segments (SCHMIDT, 1938; DENTON, 1959; LIEBMAN,1962; WALD et al., 1962). It is possible however to check whether metarhodopsin I and II are oriented in the same way as rhodopsin. First, the density of an amount of metarhodopsin II produced by the bleaching of a known amount of rhodopsin can be established, in conditions that favour metarhodopsin II. Figure 8 (pH 6) and the right-hand side of Fig. 7 may serve as illustrations of how this is carried out. The 390 nm curve is extrapolated to a point representing the middle of the bleaching period. The density difference between that point and the final density level gives the total density of metarhodopsin II (the small amounts of metarhodopsin I present at the the end of the bleaching can be “translated” to metarhodopsin II and added to this value). On the other hand the density difference at 480 nm between the value before bleaching and the final value represent the density of bleached rhodopsin. (This value has to be corrected to account for the fact that 600 nm has been used as the reference wavelength, where the extinction of rhodopsin is 4.5 per cent of the maximum.) Further, we must account for the fact that difference spectra of isolated retinae are often slightly displaced towards longer wavelengths as compared with difference spectra of rhodopsin in solutions containing NHzOH. We have found that the density decrease at 480 nm on bleaching rhodopsin has to be multiplied by l-19 to give the value at 500-505 nm, whereas the corresponding factor for a difference spectrum of frog rhodopsin solutions in the presence of NH20H is l-12. This discrepancy is probably caused by an increase in the light-scattering properties of the retina induced by the bleaching and is especially marked at shorter wavelengths. This effect probably also explains the fact that the density measured at 390 nm does not, after bleaching, return to its initial level after complete fading of the metarhodopsin (Figs. 6-8, and Fig. 11 in MATTHEWSet al., 1963). In the present measurements at 390 nm this effect can be neglected because the density of metarhodopsin II is evaluated from the difference between two levels, both measured after bleaching. Accounting for these complications we find in the preparations used, where it has been established by microscopic inspection that the measuring light enters parallel to the rod axes (DONNERand REUTER,1965), that the density of a certain number of rhodopsin molecules is nearly the same as the density of the same number of metarhodopsin II molecules at their absorption maxima respectively. The right-hand side of Fig. 7 gives the value O-228 for rhodopsin and O-239 for metarhodopsin II. Figure 6 (at 15”) gives O-371 and 0.383 and Fig. 8 O-302 and O-318 respectively. Because the molar extinctions of rhodopsin and metarhodopsin II in solution at their maxima are the same (MATTHEWSet al., 1963)

Dark-Adaptation Processes in the Rhodopsin Rods of the Frog’s Retina

31

it is justifiable to conclude that the metarhodopsin II molecules are oriented in the same way as the rhodopsin molecules; that is, perpendicular to the long axis of the rods (see also DENTON, 1959). WALD er al. (1962) have shown that the orientation is reversed when the bleaching products are converted to retinal oxime+opsin. The extinction of this compound in situ is at its lowest when the extinction of rhodopsin is at its highest; i.e., when the measuring light enters parallel to the rod axes. We have confirmed this result using Ringer’s solution containing NHzOH. It also appears that metarhodopsin I is orientated in the same way as metarhodopsin II. which is borne out by an analysis of the fast metarhodopsin II +I transition immediately after the bleach. These assumptions and arguments allow calculation of the relative amounts of metarhodopsin I and II. As an illustration of the procedure the curves in Fig. 6 measured at 480 and 390 nm and 15” may be used. Here the normal equilibrium between metarhodopsin I and II is apparently Ieached 8 min after the bleach, at the point where the broken line meets the continuous one. At this point, however, the measurements at 390 nm are clearly influenced by the unknown third intermediate mentioned earlier. For this reason we have started from the density values at a point intermediate between the density maximum at 480 nm and the point where the equilibrium has been reached, and corrected for the deviations from the final equilibrium. With this method we find that in a freshly excised retina in Ringer’s solution metarhodopsin I constitutes the following percentages of the total metarhodopsin present: Temperature 6.8” 15.0 25-5

Per cent metarhodopsin

I

68 50 36

These values give a good fit with the slope of the straight line relating temperature and per cent cattle metarhodopsin I in neutral 33 per cent glycerol solution (MATTHEWSet al. 1963). In the same way we have determined the different metarhodopsin equilibria which appear at 15” when the retina is placed in different buffer solutions. It has been shown by BRIDGES (1962) that the rod outer segments are readily permeable to hydrogen ions. In our experiments both KHzPOa-NaOH-buffers and Mcllvaine’s buffers approximately isotonic with Ringer’s solution have given the same results. With McIlvaine’s buffers the following metarhodopsin I percentages were obtained (for pH 6.0 and 8.0 see Fig. 8):

PH 5.6 6.0 7.0 8.0

Per cent metarhodopsin 23 25 60 79

I

K. 0. DONNER and TOM I~UTER

32

*70P

a-

300 nm

*so 0 A A c) ._

o-o_-0

-

L20 nm

0

480 nm -Q--o.o

: *40al P pH

6.0

$30-

0 .20 -

: .lO-

480 nm --o--

*OOI

0

10

20

mln

30

t

0 1

40

50

FIG. 8. Density changes in two fresh isolated retinae gently washed in MCILVAINE’Sbuffers at pH 6.0 and 8-O respectively and measured in the same buffers at the wave-lengths indicated. Curves displaced vertically in relation to each other for clarity. Temperature 15”. Note difference in metarhodopsin equilibrium as clearly seen from the 480 and 390 nm curves. These values lie close to the curve given by MATTHEWS et al. (1963) for cattle metarhodopsin in solution at 3.2”. It is evident that although the buffer solutions do not seem to affect the metarhodopsin equilibrium during illumination, the effect of pH on the equilibrium in the rods when in darkness is the same as that in solution. One would expect that metarhodopsin I and II would show a faster rate of decomposition at a lower pH-value when the proportion of metarhodopsin II is higher. This is

Dark-Adaptation

Processes in the Rhodopsin

Rods of the Frog’s Retina

33

apparently the case as judged from the curves measured at 480 nm, the 390 nm curves being too much influenced by the unknown third intermediate to be used for this comparison. However, the rate of decomposition of metarhodopsin is possibly not exactly proportional to the amount of metarhodopsin II. Figure 8 shows that the half-return times at pH 8-O and 6.0 are approximately 65 and 2.5 min respectively, while the corresponding percentages of metarhodopsin II are 21 and 75. It appears as if the increased fraction of metarhodopsin II would to a certain extent be balanced by a higher stability of the metarhodopsin II-molecule. The same stabilization, but more pronounced, can be observed during illumination that also favours metarhodopsin II. For instance, REUTER (1966) showed that there were surprisingly large quantities of metarhodopsin in retinae after 5-min bleaches, quite inconsistent with the assumption of a high rate of decomposition during and immediately after illumination. A comparable delaying effect of illumination on the rate of rhodopsin synthesis from retinal and opsin has been observed by REUTER(1966). Quantitative correlations between the rapid adaptation process and the fading of metarhodopsin

We have studied the rapid adaptation process using bleaches that remove small fractions of the rhodopsin present. In Fig. 9 curves are given for two experiments, one carried out I . 0..0 i-

.

7-c 0

i-

I

0

I

10

20

.

,

30

Timt~ min

50

60

70

FIG.9. Dark-adaptation

of two single units, one at 7” and the other at 15” after 558 exposure of the fully dark-adapted eye for 60 set (circles) and 30 set (dots), bleaching about 6 and 3 per cent of the full amount of rhodopsin respectively. 503 nm, 0+24 mm field. At both temperatures off-thresholds of on/off-units. The broken line indicates the level of the absolute threshold before the bleach. The curves are exponentials on a linear scale of ordinates with half-return times 9 min (7”) and 4.5 min (15’). The ratio between the ordinates of the curves at each temperature is 2 : 1. In this Fig. and in Fig. 10, threshold measurements with 30 set intervals. Note different time-scale as compared to Figs. l-5. C

34

K. 0. DONNER and TOM&UTER

at 7” (upper curves) and the other at 15” (lower curves). In both cases we have started from a completely dark-adapted eye. Then the recovery of sensitivity has been studied after 558 nm bleaches of 60 and 30 set duration, calibrated to bleach approximately 6 and 3 per cent of the total amount of rhodopsin. Beginning from the end of each bleach the thresholds are here given as circles (6 per cent bleach) and dots (3 per cent). The curves drawn are exponentials, with half-return times 9 min (7”) and 4.5 min (IS’) and thus corresponding to the values obtained for the decomposition of metarhodopsin (p. 29). Further, in each case the lower curve (dots) is a curve where the ordinates are l/2 of those of the higher curve on a linear scale starting from the level of the absolute threshold, indicated by the broken line (measured before the application of the bleach). It can be seen that both sets of points at each temperature start from the same sensitivity level, which is determined by the cones as judged by the fact that at this stage spectral sensitivity is maximal at 560 nm. After this short cone phase that lasts only 2-3 min at 15” and 4-6 min at 7” comes a period of rapid sensitivity increase of the rods and the darkadaptation process begins to follow an exponential course. As shown in Fig. 9 this happens after about 6-7 min at 15” and 9-11 min at 7”. This coincides approximately in time with the moment when the metarhodopsin equilibrium is reached as indicated by the spectrophotometric measurements (Fig. 6). The rest of the adaptation process follows the exponential curves drawn, with the exception of a deviation frequently observed at 15” with stronger bleaches from about 15 min onwards. Here there is a slightly lower sensitivity than would be expected, probably caused by an onset of regeneration, as judged by the fact that the deviation is greater with strong bleaches and much less marked at low temperatures (Fig. 9, upper curves). Thus two facts in Fig. 9 suggest a quantitative relationship to the fading of metarhodopsin: (i) log sensitivity during the main part of the rod adaptation process can be described by an exponential on a linear scale of ordinates, with the same half-return time as that of metarhodopsin at the corresponding temperature (see p. 29); (ii) when the amount of rhodopsin is reduced to l/2 (Fig. 9, dots) and hence the amount of metarhodopsin produced is halved, the ordinates of the exponential curve that fits the points are correspondingly reduced. This implies a linear relation between log sensitivity and amount of metarhodopsin. Thus logAZ/A

= kM

(1)

where A Z is the threshold intensity, A is the absolute threshold, M is the amount of metarhodopsin in the rods at the corresponding instant and k is a constant. This formulation is equivalent to the relation given by RUSHTON(1965) between rhodopsin bleached and threshold in the human eye if the amount of metarhodopsin is substituted by the fraction of rhodopsin bleached. When curves such as those in Fig. 9 are examined in detail it is found that when the same bleach has been given at a certain temperature in different experiments, the absolute desensitization produced is not always the same; in other words, there is some variation in the constant k in (1) from one eye to another. This may be caused by individual differences between the eyes, but the possible influence of slight variations in the bleaching capacity of the light due to small differences in the angle of incidence on the retina cannot be excluded. This simple proportionality between the amount of metarhodopsin and log threshold raises the question “What is the actual factor that exerts the effect observed?” Both the

35

in the Rhodopsin Rods of the Frog’s Retina

Dark-Adaptation Pr-

total metarhodopsin, as well as the amounts of metarhodopsin I and II decrease exponentially with the same time-constants. Further, the rate of hydrolysis of metarhodopsin II will also show this property, as well as the net change metarhodopsin I+II. This latter process is, however, the result of a dynamic equilibrium, where the actual change in both directions is larger and has, moreover, a very large temperature coefficient (MATTHEWS el al., 1963). The fact that the equilibrium between metarhodopsin I and II is affected by temperature can be utilized to decide, at least tentatively, between the different alternatives. At a lower temperature the fraction of metarhodopsin II is smaller (p. 31). It follows that an analysis of the sensitivities in experiments such as are shown in Fig. 9 could provide an answer, were it not for the fact that there is a variation in the constant k from (1) between different experiments. This can be avoided, however, by determination of the adaptation curves on a single unit with the same amount of rhodopsin bleached but at two different temperatures. Even this procedure, however, involves the assumption that the constant k is independent of temperature. Figure 10 shows the result of an experiment (on-effect, on/off-unit) with the same bleach applied twice but at two different temperatures, 15 and 7”. The recovery of sensitivity is given by circles (15’) and dots (7”). Between the bleaches the sensitivity returned to the /---SC

il

0

/

10

20

30

LO Time,

50

50

70

min

FIG. 10. Dark-adaptation of a single on-unit at two different temperatures after 60 set 558 nm exposure of the fully dark-adapted eye, bleaching about 6 per cent of the rhodopsin. 503 nm, tield 024 mm in dia. Open circles, 15“; dots, 7”. The curves are exponentials with half-return times 4.5 min (15’) and 9 min (7”) placed so that theii ordinates at 0 min on a linear scale from the level of the absolute threshold gives the ratio 1.56, which is the ratio between the amounts of metarhodopsin II at the two temperatures. Inset: relation between the number of quanta absorbed per rod/sez at threshold and the number of metarhodopsin II molecules per rod. Full explanation in the text.

eo

I

36

K. 0. DONNERand TOMREUTER

original absolute threshold. The curves drawn are exponentials with 4.5 (15’) and 9 min (7”) half-return times, which apply for the fading of metarhodopsin at these temperatures. It is obvious that if the total amount of metarhodopsin were effective the two dark adaptation curves ought to be described by a pair of exponential curves with the halfreturn times mentioned, but originating in the same point at 0 min. This is certainly not consistent with the experimental facts. If, on the other hand, only the amount of metarhodopsin II is considered, the situation is different. We have found (see p. 31) that at 15” 50 per cent of the metarhodopsin is in the II form and at 7” 32 per cent. The total metarhodopsin produced by the bleach being the same at both temperatures, it is evident that on a linear scale the relation between the two ordinates at 0 min should be 50/32 = l-56. This ratio has been used in Fig. 10, and it is seen that the curves fit the points in a fairly satisfactory way. It is clear that application of the same argument on metarhodopsin I could not give a result that agrees with the experiments. Finally, the rate of hydrolysis of metarhodopsin II would at 0 min require a ratio of 1 : 2 of the ordinates, which is not the case either. The experiment then appears consistent only with the amount of metarhodopsin II being the critical factor. The fact that in Fig. 10 as well as in Fig. 9 the points for 15” do not follow the curve drawn does not constitute a serious discrepancy, because, as pointed out above, this is most likely due to an onset of regeneration, which is absent if the bleaches are small enough (Fig. 9, dots) and at lower temperatures. The argument presented above in connection with the experiment in Fig. 10, leading to the conclusion that the quantity of metarhodopsin II is the significant factor, contains an assumption that the fading of metarhodopsin is exponential from the beginning; alternatively, that the quantities of metarhodopsin in relation to each other behave as this were the case. It has, however, been stated above (p. 33) that the decomposition of metarhodopsin is slower immediately after the bleach than would be expected. Calculation of the amounts of metarhodopsin II from the spectrophotometric measurements at some later instant shows that this retardation of the metarhcdopsin decomposition at 15 and 7” respectively is such that the amounts of metarhodopsin II still behave in the manner predicted. For instance, it can be calculated that at 15” there is, after 8 min, 20 per cent remaining of the whole amount of metarhodopsin II formed by the bleach and, at 7”, 13 per cent after 16 min. And in Fig. 10 the ordinates at 8 min (15”) and 16 min (7”) almost exactly show the ratio 20/l 3. The finding that metarhodopsin II appears to be the desensitizing agent in the rods is supported by the fact (Fig. 10, dots) that it is often observed immediately after the cone-rod transition in the dark-adaptation curve that the rods are less sensitive than would be expected. This may be connected with the disturbance of the metarhodopsin equilibrium observed immediately after the bleach in the spectrophotometric measurements, so that more metarhodopsin II is present than during the normal equilibrium in the dark. This observation, refers, however, to conditions where relatively great amounts of rhodopsin have been bleached (for instance Fig. 7: 27 per cent in 50 set) and during fairly unphysiological conditions of the retina. It is, then, not known whether the same disturbance of the equilibrium occurs with 3-6 per cent bleaches. So far the amounts of rhodopsin bleached and the amounts of metarhodopsin have been given only as percentages of the full amount of rhodopsin in the rods. From the data given by WOLKEN (1961) we were able to estimate (DONNER and REUTER,1965) that

Dark-Adaptation Processesin the Rhodopsin Rods of the Frog’s Retina

37

each rod in Finnish Runa temporariu contains about 3 x 109 molecules of rhodopsin in the fully dark-adapted state. Bleaching 6 per cent, as has been done in the experiment shown in Fig. 10, means the formation of l-8 x 10s molecules of metarhodopsin per rod. It was stated above that after 8 min at 15” 20 per cent of this quantity remained; i.e., 3.6 x 107 molecules per rod. From this it is easily calculated that after 35 min (15’), when the threshold is practically the same as the absolute threshold 5.6 x 10s molecules of metarhodopsin II still remain in the rod, or in other words about 2 molecules per 10,000 molecules of rhodopsin. At 10 min, when the threshold is 1 log unit higher, there are 2.7 x 107 molecules or about 1 molecule of metarhodopsin II per 100 molecules of rhodopsin. This fairly minute quantity thus has a strong effect on the excitatory processes triggered by the relatively few quanta caught at threshold. Generally the intensity at the absolute threshold according to our measurements corresponds to the absorption of 80-160 quanta per set within the circular 04453 mm2 field used in the experiments. Measurements on freshly excised retinae have shown that there are approximately 850 rhodopsin rods within this area. This gives an absorption of about l-2 quanta per 10 rods per set, a value not far from those given by DONNERand RUSHTON(1959a) and BAIJMANN(1964). If metarhodopsin II, instead of the total amount of metarhodopsin, is significant it is clear that (1) should be written log A I/A = k Ml1

or

A I/A = ld( M1I

(2)

where the symbols are otherwise the same as in (1) but MII denotes the amount of metarhodopsin II at a given instant. It was stated above that for a single rod 2.7 x 107 molecules of metarhodopsin II raise the threshold 1 log unit. In that case A I/A = 10 and thus, referring to (2), k MII = 1. Since in this situation MI1 is known k can be calculated, and is found to be O-37 x 10-7 for the particular experiment shown in Fig. 10. In the inset the corresponding line has been drawn that then describes the relation observed. DISCUSSION Omitting effects on the sensitivity produced by changes in the organization of the receptive fields, the photomechanical movements (BXCK et al., 1965) and the increase in the quantum-catching properties of the rods caused by an increased density of the photopigment, it appears that the dark-adaptation of the rhodopsin rods in the frog can be described as being determined by two effects: (i) the effect of bleaching, which is caused by the presence of metarhodopsin II for a comparatively long period after the bleach. ; (ii) the effect of the synthesis of rhodopsin that depends quantitatively upon the rate of regeneration. The decomposition of metarhodopsin is the more rapid process, and hence the presence of metarhodopsin II, mainly affects the dark-adaptation curve at the beginning of its course, while the sensitivity during the later part is governed by the synthesis of new rhodopsin. The result is that the dark-adaptation curve for frog rods shows a division into two phases. Which of these phases is more prominent depends on the strength and duration of the previous light-adaptation. With very weak and short exposures the effect of (i) is predominant, while with the bleaching of large fractions of the rhodopsin (ii) becomes the more important. A corresponding property of the dark-adaptation of the cones in the frog is suggested by the shape of the initial cone branch in Fig. 5. It is also evident that the cone adaptation is more rapid in those experiments where only small

38

K. 0. Do-

and

TOMREUIZR

bleaches have been used (Fig. 1, lowest curve and Fig. 10) as compared with experiments with prolonged and strong light-adaptation (Fig. 1, upper curve). It is possible that this interpretation of the dark-adaptation process is applicable to other photoreceptors also. In the human eye, for instance, both rods (RUSHTONand COHEN, 1954; RUSHTON, 1965) and cones (RUSHTON and BAKER, 1963), under certain conditions that would seem favourable for bringing forward an effect of metarhodopsin and corresponding products of cone photopigments, show a rapid adaptation phase readily distinguishabIe from a later and slower phase. This is the case in measurements on the rat (DOWLING,1963) also, and on single photoreceptors in the Limulus eye (HARTLINEand MCDONALD,1947). In our results metarhodopsin II shows the relation to log threshold that would be be expected if this substance would enter the feed-back of a parametric feed-back model as proposed by FUORTESand HODGKIN(1964) in their analysis of the potentials recorded from the ommatidia of Limulus (see RU~HTON,1965). Thus metarhodopsin II will affect the gain of the ~p~cation mechanism triggered by the absorption of quanta in the rods. In this context it is interesting to note that it is possible to alter the amount of metarhodopsin II by changes in the internal concentration of some metabolite (e.g. H+-ions) as well as by the bleaching of rhodopsin and the subsequent slow decay of metarhodopsin. The existence of physiological processes of this kind is indicated by the observations of the changes in the metarhodopsin equilibrium during and after illumination in the s~trophotomet~c m~~ernen~, though admittedly during fairly unphysiolo~c~ conditions. It has been shown by SICKEL(1958, 1961), however, that a lowering of the pH-value causes a reversible reduction of sensitivity in the isolated frog retina, with the opposite effect of an increased pH-value. Contrary to this, the rate of regeneration does not act in this way, but appears to produce the same but weaker effects as the complete excitatory process initiated by the action of light. Possibly the synthesis of new molecules (see below) must run through the stages represented by the metarhodopsin II state and the change induced by the isomerization of the rhodopsin chromophore by light. At any rate it can be said that in terms of the feed-back model of FUORTF,Sand HODGICIN(1964) metarhodopsin II enters the feed-back but the regeneration process enters the input of the model. It was noted above that both me~h~opsin I and II are oriented in the rods in the same way as rhodopsin; i.e., transverseiy to the long axis of the rods. MA-S et al. (1963) assume that the conversion of metarhodopsin I to the II-form involves a rather large change in the steric relationships between the opsin molecule and the retinaldehyde chromophore. This reorganization of molecular cotiguration, which does not change the orientation of the chromophore, suggests a considerable change in the interna structure and position of the opsin. Possibly this opsin co~guration, specific for the metarhodopsin II state, may be causing the change in the sensitivity of the rod. It is clear, however, that the problem is far from solved on the molecular level. One may, for instance, ask whether the first rhodopsin intermediates, prelumirhodopsin and lumirhodopsin (WALD et al., 1950; GREILMANN et al., 1962; YOSHIZAWAand WALD, 1963), have any significanct effect. Their rapid conversion to me~hodopsin makes it, d&ult to establish whether such effects occur. A preliminary account of the present results was given to the XII Scandinavian Physiological Congress 22-25.8.1966 (DONNERand REUTER, 1966).

Dark-Adaptation Processes in the Rhodopsin Rods of the Frog’s Retina

39

Acknowle&ement-This work has been supported by grants from the National Research Council for Sciences in Finland to K.O.D. and from “Svenska Vetenskapliga Centralradet” and “Oskar &hmds Stiftelse” to T.R.

REFERENCES BA~~~ANN,01. (1964). Die absolute Schwelle der isolierten Froschnetzhaut.

P@g. Arch. ges. Physiof.

280, 81-88. BAUMANN, CH.

(1965). Die Photosensititvitat des Sehpurpurs in der isolierten Netzhaut.

Vision Res. 5,

425434.

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Abstract-In the excised and opened frog’s eye two phases of rod dark-adaptation can be distinguished. The slower phase, previously studied by us, depends on the rate of regeneration of rhodopsin. Supplementary evidence is here given based on measurements involving temperature changes of the eye during adaptation. The rate of regeneration, which has a Qre of about 4.6, is thereby rapidly altered. This brings about quantitatively corresponding changes in the sensitivity of the single units studied. The initial, rapid phase of rod adaptation is found to depend on the decomposition of metarhodopsin in the rods, which shows the same time-course at dit%.rent temperatures as the adaptation process. Here log threshold is found to be proportional to the amount of metarhodopsin. The evidence obtained in experiments at different temperatures further suggests that it is metarhodopsin II, which exists in a tautomeric equilibrium with metarhodopsin I, that causes this desensitization of the rods.

R&sum&On peut distinguer deux phases de l’adaptation a l’obscurid des batonnets dans l’oeil excise et ouvert de la grenouille. La phase lente, pr&demment ttudi&s par nous. depend du taux de r&gUration de la rhodopsine. On en donne ici une preuve supplementaire par des mesures oh la temperature de l’oeil change pendant l’adaptation. On ah&e ainsi rapidement le taux de r6gentration qui pos.&de un Qlc voisin de 4,6. Cela produit dans les unites iso1ee-s qu’on &die des changements de sensibilit6 en correspondance quantitative. On trouve que la phase initiale rapide de l’adaptation des batonnets depend de la d&cornposition de la metarhodopsine dans les batonnets, laquelle varie en fonction du temps independamment de la temperature pendant l’adaptation. On trouve ici que le log du seuil est proportionnel a la quantite de metarhodopsine. Les result&s obtenus a dif%rentes temperatures suggerent en outre que la desensibilisation des batonnets est cat&e. par la metarhodopsine II qui existe en equilibre tautom&ique avec la metarhodopsine I.

Zusamme&ssung-An herausgeschnittenen und geiiffneten Froschaugen konnen zwei Phasen der Dunkeladaptation der St%chen unterschieden werden. Die langsamere, bereits fruher von uns untersuchte Phase hangt von der Regenerationsgeschwindigkeit des Rhodopsins ab. Aufgnmd von Messungen bei Temperaturiinderungen des Auges w&end der Adaptation wird zus&zliches Beweismaterial dazu gebracht. Die Regenerationgeschwindigkeit die ein Qla von etwa 4,6 hat, lndert sich dabei stark. Das bringt quantitativ entsprechende Anderungen in der Emptindlichkeit der untersuchten einzelnen Einheiten mit sich. Die anfiingliche schnelle Phase der Stabchenadaptation hiingt von der Zersetzung des Metarhodopsins in den Stlbchen ab, die bei verschiedenen Temperaturen den gleichen Zeitverlauf zeigt, wie der Adaptationsprozess. Der Logarithmus der Schwelle ist proportional zum Metarhodopsingehalt. Das aus Experimenten mit verschiedenen Temperaturen erhaltene Beweismaterial weist femer darauf hin, dass die Desensibilisierung der Stilbchen durch Metarhodopsin II verursacht wird, das in einem tautomeren Gleichgewicht mit Metarhodopsin I steht.

Dark-Adaptation Processes in the Rhodopsin Rods of the Frog’s Retina Pesmie - I? 3HyKne~poaamoM H mcpbrro~ rna3y mxrynm MOXHO6mo pa3nmaTb jme ~a3brnano~o3o~~e~o~o#aAarrrarSrra.ljoAee~eaneHHaa~a3a, pamemysexxiaa mm, 3amcm OT cxopomi pertsepanmi p0Aoncma. 3Aecb A~z~TCX ~onomm~HOB~~~~XR~H~M~~~HHR~,B~~~~~H~M~B~T~~IexoX~aTe~, HHR TemepaTypM mAa38 ~0 ~perds aAamwmf. CxopOcTb pemiepauarr, KOTOP meer Qlo oxono 4,6, zi3-3a 3101’0 6wrpo ~3r.fe~lle~cs. 310 npmoAm x R~H~JIH~EXTeJIbFfO KOJUFIeCTBeHHO COOTBeTCTByrOII@iM E3hfeHeHWIM B WOCTR u3ylraehmx e;akniwrmx 3AeMemoB. Ha@xeHo, PTO Ha%-inbHan, 61~~pan Qliua xa~~o~o~o# &vvmmmi 3a~mm of paCnaAZI M~apOAOIICHBaB~~OPE~,XOTO~~ 06~py%3JBaeTTaKoeHceB~MeEHOe Tesemexpx ~Te~epa~,~~~~~~~~. ~pE3TOMRZ%&IeEO, 9~0 log nopora ~po~op~oE~eH ~0~1~1ecray M~apoAo~~a. CgaarM IfoJwiemrbxe B sxcuepmexrrax np~ pa3zroii nmepaType 3ac~am~ ~am5.e npe2monaram, ~0 3Ty AeW@KXi6XiJIE3aIGiW IIaJfOYeKBbI3JdBaeT MeTapOAOIICISlI 11, XOTOpd EaXOAETCH BTa~OMe~EDIeCKOMpaBEIOBeCE.ECMeTapOAOIICHHOMI.

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