Bleaches Are Substantial; Thus the Rapid Recovery Phenomenon Lies Outside the Conditions Here Examined

612 J. Phyaiol. (1965), 181, pp. a12-628 With 5 text-ftgure8 Printed in Great Britain

DARK ADAPTATION AND INCREMENT THRESHOLD IN A ROD MONOCHROMAT By C. B. BLAKEMORE AND W. A. H. RUSHTON From the Physiological Laboratory, University of Cambridge (Received 30 April 1965)

THE OBJECT The well-known fall in sensitivity of the eye when exposed to light and the subsequent rise in the dark has long been attributed to the bleaching and subsequent regeneration of rhodopsin and of cone visual pigments. This conjecture, held for thirty years without evidence, was shown to be on the right lines when Dowling (1960) demonstrated in rats that, after full bleaching, there was at each stage of regeneration a linear relation between the amount of rhodopsin in the retina and the log e.r.g. threshold at that moment. In man a quite similar relation was found between the log visual threshold measured in the usual way and the rhodopsin concentration measured directly upon the same area of retina by the technique of retinal densitometry. Though this result could be inferred with some confidence from work on normal eyes (Rushton, 1961a) it was displayed more clearly in the eye of a special subject (Rushton, 1961 b) who lacked nearly all cone vision but whose rhodopsin and rod vision appeared quite normal. If the threshold is expressed in units of the fully dark-adapted value, the relation found was that log threshold is proportional to the amount of rhodopsin bleached. But, as was pointed out in the brief Discussion of the previous paper (Rushton, 1961 b), there are two respects in which this simple relation fails. (a) If the brief adapting light bleaches only a small fraction of rhodopsin, visual sensitivity recovers rapidly, and all agree that the process is in some way different from that linked to the slow regeneration of rhodopsin after substantial bleaching. In the present paper, as in the former one, all bleaches are substantial; thus the rapid recovery phenomenon lies outside the conditions here examined. (b) The simple relation found that log threshold was proportional to the amount offree opsin could not be quite right. To be sure, something changes uniquely with the rhodopsin concentration and may affect the threshold in the way described, but it is not the threshold itself that stands in unique relation to pigment concentration. For several workers have shown that threshold depends also upon the kind of test flash used to measure it. In DARK ADAPTATION IN A ROD MONOCHROMAT 613 his last paper Lythgoe (1940) discussed dark adaptation with great insight and pointed out that something like a reorganization of nerve connexions must play an important part. Craik & Vernon (1941) measured the visual threshold during dark adaptation using either large or small test flashes and found quite different shapes for the corresponding dark adaptation curves, confirmed by Crawford (1947) and Arden & Weale (1954). Wolf & Zigler (1950) placed an acuity grating in the test flash and found that the shape of the resulting dark adaptation curve depended upon whether the threshold task was to see the flash or to resolve the grating. These results were confirmed and extended by Brown, Graham, Leibowitz & Ranken (1953). There would of course be little difficulty in reconiciling these obser- vations with the unique dependence of dark adaptation upon the regeneration of rhodopsin, if a change in the nature of the test flash did nothing more than shift the log threshold curve bodily up and down. But all investigations have shown that not only the position but the shape of the dark adaptation curve alters and that the range of the log threshold change for a given bleach depends upon the criterion by which the threshold is measured. It is plain that we should be faced with a heavy task in explaining the way in which adaptation depends upon rhodopsin bleaching if first we had to explain how threshold depends upon the varied criteria which may be used in its measurement. Fortunately as long as thirty years ago views were being advanced which suggested a great simplifica- tion of the problem. Holladay (1926, 1927), Stiles (1929, 1930) and others worked upon the effect of glare-the change in properties of one region of the retina when a distant region of small area is brightly illuminated. They found that the effect was as though a luminous veil was spread over the test field, and they measured this equivalent luminance by replacing the glare by the uniform veil that produced identical results. 'There is actually such a veil due to the diffusion of light within the eye, but it explains only a part of the phenomenon and the remainder is a fictitious luminance which represents [retinal] inhibition' (Le Grand, 1957). The tentative conclusion reached at that time was that there is a 'state of adaptation' of the retina which could result from various conditions (e.g. either from a uniform veil or from a distant localized glare), and the properties of the retina appeared to depend upon its state of adaptation but not upon the particular conditions which produced it. Lythgoe & Tansley (1929) (see also Lythgoe, 1940, fig. 2) investigated the flicker fusion frequency using a small flickering patch of fixed intensity with a luminous surround. They either varied the intensity of the surround or they light-adapted the eye and followed the critical fusion frequency (CFF) throughout the subsequent dark adaptation. In a general way it appeared that, for each intensity of flickering light, the effect upon CFF 614 C. B. BLAKEMORE AND W. A. H. RUSHTON of light adaptation was the same as the effect of some bright surround, and, as dark adaptation proceeded, the CFF results were as though the surround was progressively dimmed. If these thirty-year-old ideas are true we need not bother about the complex way in which threshold depends upon the parameters of the test flash, in our attempt to relate bleaching to adaptation. It is only necessary to find the luminance that is equivalent by one criterion of threshold, and it will be equivalent by all others. 'Threshold' drops out of the picture and the equivalent luminance that remains is the retinal state that should stand in unique relation to bleaching. But how true are those ideas, and in particular how well is dark adaptation represented at each stage by some equivalent luminance whose value is independent of the criterion by which it is measured? The most complete and accurate comparison is that ofCrawford (1947), where the increment threshold of a flash upon various intensities of background is compared with the threshold in dark adaptation at various times following an exposure which he has told us amounted to 20,000 troland seconds of retinal illumination. The area of test flash was the same in each pair of experiments, and in different pairs it varied between 0.180 and 5.70 in angular subtense. What Crawford found was that, if dark adaptation is plotted not as log threshold against time but as log equivalent luminance against time, the curve is very nearly the same, independent of the area of test flash used. The experiments, however, were undertaken for certain practical ends and have three imperfections from the present academic view-point. First; there was no fixation point provided, so the subject used whatever part of the retina suited him best at the moment, and no doubt this lay closer to the central fovea the more the eye was light adapted and the smaller the test flash. Second, no attempt was made to separate rod thresholds from cone thresholds, and, since in general the equivalent luminance of dark adaptation cannot be the same for rod and for cone vision, the exact relation was somewhat obscured. Third, the bleaching was certainly not substantial; it was only about 0*2 % of the total amount of rhodopsin present. If rhodopsin is bleached by an exposure (It) troland sec applied for less than 1 min (so that regeneration is very small; Campbell & Rushton, 1955), then the fraction p of the initial pigment that remains unbleached is given (to good approxi- mation) by loglologlo(I/p) = log1o (It)-7.3. (1) This formula is derived from the assumption that the rate of bleaching (-dp/dt) is always proportional to the rate at which quanta are being absorbed (kpI), and it has been shown to hold for rods (Rushton, 1956) DARK ADAPTATION IN A ROD MONOCHROMAT 615 and for cones (Rushton, 1958, 1963a, 1965a). The figure 7*3, which applies to rods only, is derived from measurements of the rate of bleaching of rhodopsin in normal eyes (Rushton, 1956). Crawford's log (It) was 4 3, thus log I/p = 0.001 and the fraction bleached was 0-0023. The minute extent of the bleaching in Crawford's experiments does not detract from their importance in demonstrating that the subsequent stages of recovery may each be represented by a background whose equivalence is valid no matter what size of test flash is used. But without further investigation we may not conclude that this principle of 'equivalent luminance' applies to the quite different kind of adaptation that is linked to rhodopsin; for that requires the substantial bleaching of rhodopsin that Crawford did not employ. In the present paper, therefore, we have repeated some of Crawford's experiments, using large bleaches, and following the subsequent adaptation over a millionfold range of thresholds. This is not possible with normal subjects but one of us (Rushton, 1961b) has shown in experiments upon a subject with very little cone function that rod dark adaptation and increment threshold can be measured over a range of about 6 log units, using large test areas. The other of us (C.B.B.) appears to have no cone vision whatever and is experienced both as investigator and as subject in psycho- physical measurement. Between us we have been able to extend the principle of equivalent luminance to the conditions following massive bleaching.

THE SUBJECT C.B.B. has been examined and briefly described by Pickford (1957). He has no known relatives with a similar condition, and his brother has been an air pilot. He was 28 years of age at the time of these experiments (in 1961) and the nystagmus of his boyhood was no longer noticeable. He avoids strong light because it dazzles, utterly 'fogs' the visual scene and is psychologically unpleasant, though not painful. He was able, however, to tolerate the very strong bleaching lights used in these experiments. There was no lasting visual impairment such as that described by Walls & Heath (1954). Visual acuity by test type under standard conditions is 6/36, and he can read ordinary books, preferring dim light andholding the print rather close, and in fact he reads a great deal. He fixates upon a retinal region some 100 from the fovea rather close to a large blood vessel, and he was asked to fixate in this way upon the text flash in the experiments of this paper. C. B. B. shows no evidence ofany active cones. He has never experienced the sense of colour and has only a theoretical idea of what colour means. A blue and a yellow light which match at normal scotopic levels he finds indistinguishable in all conditions investigated (except occasionally owing 616 C. B. BLAKEMORE AND W. A. H. RUSHTON to their difference in refraction). The curves of dark adaptation, increment threshold and fficker fusion, however, have all shown breaks at high intensity levels. This cone-like segment has already been described by Lewis & Mandelbaum (1943), Hecht, Shlaer, Smith, Haig & Peskin (1948), Sloan (1954, 1958), and Alpern, Falls & Lee (1960). We confirm that it exhibits the spectral sensitivity of rhodopsin and we find no Stiles- Crawford effect. It is therefore due to rods. We have analysed this high- level mechanism and hope to describe its nature in a future publication. It is sufficient here to say that in omitting further reference to it we do not affect any of the considerations of the present paper.

METHOD Increment thre8hold The apparatus is shown diagrammatically in Fig. 1. The light source A was a 6 V, 3 A motor head-lamp with a straight vertical filament which was focused upon the subject's pupil P by the lens L after reflexion or transmission in the beam-splitting mirror M. Fine adjustments upon M brought the two images into coincidence upon the pupil. Stops S1, S2 in the focal plane of L were seen sharply. The stop S2 which limited the background field subtended usually 120 at the eye. The superposed flash S1 was of various sizes, and this beam

rd W A° f " I S2~~~~~~~F

wX1 j R F1 Fig. 1. Diagram of the apparatus for measuring increment thresholds. For description see text. was continually interrupted by rotating sectors at R, 1 sec on, 1 sec off. The intensities of each beam were varied by filters F1, F2 and wedges W1, W,. The subject maintained his position by biting upon a dental impression and by steadying his head with a brow rest. The two beams were adjusted to pass through the centre ofthe pupil and, since the coincident images of the filament were very small, they easily passed through the pupil even when it was constricted by bright fields. Many of the results were also confirmed with the pupil dilated by homatropine. Light adaptation At first some difficulty was experienced in bleaching the required retinal region. The dazzling bleaching light entirely incapacitated the cone-free eye as regards fixation and it DARK ADAPTATION IN A ROD MONOCHROMAT 617 induced unconscious (and hence uncontrolled) rolling movements of avoidance. Thus when the bright exposure was finished the after-image was often seen not to lie over the fixation point. A satisfactory procedure was to place the eye to be bleached 7 cm from a 75 W pearl glass lamp (with heat absorber interposed), and to place in front of the other eye a mirror in which a distant light point could be seen. A 2-0 density filter was placed in front of the first eye so that the light bulb could be seen dimly, and the mirror was adjusted so that the light point seen in the other eye appeared to be in the middle of the bulb. When the 2-0 filter was removed suddenly, the subject could hold the dazzled eye steady and in the correct position by fixating with concentration upon the light point with the other eye. The subsequent appearance of a large after-image centred correctly confirmed the success of the method. The luminance of the pearl glass was 10,000 cd/M2, the natural pupil during the bleach about 3 mm in diameter, and thus the retinal illumination was about 4.9 log td. The duration of exposure was 2 min and it is likely that about 50% of rhodopsin was bleached. We did not measure this on the densitometer since C. B. B.'s unsteadiness of fixation made measurements too unreliable. Dark adaptation This was simply done with the apparatus of Fig. 1. Usually a screen was placed at F2 to block out all background, and C. B. B. moved the wedge W1 until he was satisfied that the flahing light was just at threshold intensity. He then signalled, the time was read, and then the corresponding wedge setting recorded. Sometimes a fixation point was given to help locate the test flash. When the flash area was large a bright unflashing point was placed at its centre. When the flash was small a faint circular line was placed around it. Fixation The pure rod monochromat (as distinct from the cone-poor subject of the former paper (Rushton, 1961 b)) naturally does not fixate with the fovea, since that would bring the image to be examined on to a retinal region probably devoid of active receptors. In all the experiments of this paper C. B. B. fixated upon the test flash, and at the time we supposed that this ensured that the test always fell upon the same retinal region. This belief, however, is probably wrong, as will be mentioned in the Discussion.

RESUILTS Size of test flash The results of one experiment are shown in Fig. 2a and b, where the curves on the left plot dark adaptation, those on the right the log increment threshold against various log intensities of background. Immediately following the 2 min bleaching exposure, the log threshold for the flashing light was determined by presenting alternately a 60 test patch and a 5' patch in succession throughout 45 min of dark adaptation. Then the increment thresholds were measured for each size of test patch against a background which started at zero and was gradually raised to 1000 td. The two dark adaptation curves ofFig. 2a show the change ofshape with change in area of test flash that was noted by Craik & Vernon (1941), Crawford (1947), Arden & Weale (1954) and others, and show it over a far greater rod range in the absence of any cone-rod break. 618 C. B. BLAKEMORE AND W. A. H. RUSHTON The increment threshold curve for 60 test patch (Fig. 2b) follows closely the shape already found in a similar cone-weak subject (Fuortes, Gunkel & Rushton, 1961) and previously by Aguilar & Stiles (1954) in the normal subject using a special technique. If the line rose at 450 it would correspond exactly to the Weber-Fechner relation: actually it is slightly less steep. The line for the 5' test patch, on the other hand, is very much less steep as Barlow (1957) has found, though it does not drop to a gradient of & as would be required for a constant signal/noise ratio. Barlow found that it needed a brief flash of only a few msec to achieve this, and, in the 1 sec of exposure which we used, eye movements may have spread the small image over a larger area.

a b d

6 ~~~~~~~~~~~ 6 5~~~~~~'flash' 6 I6I1b .t,I 0

4m 4 4 6la flash bo 3 . .. 3 . .~ . - 0-A 2 2 0 120 30 0 4 -32 -

0 0 10 20 30 40 -4 - 3 -2 -1 0 1 2 3 Minutes log ID Background in log trolands Fig. 2. a, Left half, dark adaptation curve, log threshold plotted against time in the dark; b, right half, increment threshold curve, log threshold plotted against log background field. Threshold flash subtending 60, open circles; subtending 5', filled circles. At higher background fields the curves rise sharply and 'saturate' at a field intensity of about 1000 td as Aguilar & Stiles (1954) have shown. The fact that the curve with a small test patch rises in the same way and saturates at about the same background level has not previously been shown. Equivalent luminance. The object of the experiment of Fig. 2 was to find how exact is the idea that at each moment of dark adaptation the threshold corresponded to some equivalent background whose value was independent of the size of the test patch. Figure 3 shows dark adaptation expressed as DARK ADAPTATION IN A ROD MONOCHROMAT 619 equivalent log background plotted against time, open circles from measurements with the 60 test patch, filled circles from the 5' test. It is simple to obtain most of this curve from those of Fig. 2. At each duration of dark adaptation (e.g. at 14 min) we erect an ordinate to give the log threshold of dark adaptation as measured (say) by the 60 test. Now we carry this ordinate across to the log increment threshold curve (also 60 test) and find to what log background this size of ordinate corresponds. If it is 05 log td then in Fig. 3 against the duration of 14 min a white circle is plotted with ordinate 05 log td. The same thing is done where the ordinate at 14 min

o 3

0 1

0 0b

Ca > 2

10 20 30 40 Minutes Fig. 3. Replot of Fig. 2 to eliminate 'threshold'. A horizontal line drawn across Fig. 2 at any level of log threshold cuts the left curve at a 'time in the dark' and the right curve at a 'background lu-rminance'. These two quantities are plotted against each other in Fig. 3; open circles when the test flash subtended 60, filled circles when it subtended only 5'. cuts the 5' curve of Fig. 2a. The ordinate is carried across to meet the 5' curve of Fig. 2b, and the log background is measured. It is nearly the same as for the 6° test; thus in Fig. 3 at 14 min white and black circles would nearly coincide. Though most of the curve may be plotted in this way, the method becomes useless at the very end of dark adaptation, since here the equivalent log background assumes some indefinite large negative value, and the exact way to obtain the (dotted) tail of the curve will be described later. It is plain from Fig. 3 that the open and filled circles lie upon the same curve; thus the principle of equivalent luminance that Crawford showed valid during recovery from weak light exposures applies equally to the recovery from substantial bleachings. We may therefore conclude that the 620 C. B. BLAKEMORE AND W. A. H. RUSHTON essential change in adaptation that is uniquely correlated with bleaching and regeneration of rhodopsin is the equivalent luminance, for that is independent of the area of the test flash used to measure it. Grating test It might be argued from the foregoing results that, though 'equivalent background luminance' certainly is an invariant with respect to the test area used, it is a clumsy and possibly a misleading way of describing the facts. If in Fig. 2 we slide the 5' curves (filled circles) down so that the absolute dark threshold coincided with that of the open circles, it is clear that both the 5' and 60 curves would differ simply by the scales of their ordinates. The same is seen in Crawford's (1947) similar but larger family of curves all of which when so displaced appear to differ simply in their ordinate scales. It might be argued that the simplest formal description of the situation is that the state of adaptation is measured by log threshold multiplied by a constant factor depending upon the area of test flash used. According to this, the equivalent background is but a roundabout way of incorporating ordinate scaling without mentioning that simplifying concept. The use of grating acuity as a threshold test shows clearly the superiority of the concept of equivalent background, for, despite the fact that thresholds no longer change by simple scaling, the principle of equivalence still holds. Several investigators have measured dark adaptation using as criterion of threshold the resolution of an acuity grating (Wolf & Zigler, 1950; Brown et al. 1953; Brown, 1954). They found with coarse gratings that the dark adaptation curve in the normal subject is composed of a cone and a rod branch as usual; with a somewhat finer grating the rod branch appears later and adapts less; finally with a grating below the resolution of the rods there is naturally no rod branch left. We have repeated upon C. B. B. the experiments of the first part of this paper, using the resolution of various gratings as the threshold criterion both in dark adaptation and as increment threshold against various backgrounds. Figure 4 shows the result of one experiment plotted as in Fig. 2. The open circles give the result when threshold was the resolution of a grating whose bright bars and dark bars each subtended 11'. In full dark adaptation the threshold for resolution was about 2 log units above that for the detection of a flash of light through the grating. The filled circles represent the result when the grating bars each subtended 7'. The dark threshold is seen to be about 1x5 log units higher than with the 11' grating. Triangles show the curves when threshold was the detection of light from a small patch subtending 10', as in the former experiments. DARK ADAPTATION IN A ROD MONOCHROMAT 621

4 It0 . , 3

bO2 19

0 10 20 301 -2 -1 0 1 2 3 Min Log ID Log (ID +I) in trolands Fig. 4. Dark adaptation (a) and increment threshold curves (b) plotted as in Fig. 2 with two modifications. Triangles show results when the test was the detection of a 10' patch; open circles when it was the resolution of a grating each bar of which subtended 11'; filled circles when the grating bar subtended 7'. The abscissa in the right half of Fig. 4 plots not the log of I, the background luminance, but the log of (I+ ID) the total background where Eigengrau, ID = 0-0015 td; see Discussion.

aC) 3

IC)-~ 0

e. o. -1

-2

0 10 30 40 Minutes Fig. 5. Replot of Fig. 4 to eliminate 'threshold' in the same way as in Fig. 3. Triangles, open and filled circles have the same significance as in Fig. 4. The dotted part of the curve in Figs. 3 and 5 show where the addition of the Eigengrau ID to the applied field make any difference. 622 C. B. BLAKEMORE AND W. A. H. RUSHTON It will be observed from the increment threshold curves of Fig. 4b that at absolute threshold it requires nearly the same intensity of flash to detect the circular patch 10' in diameter as to resolve a grating of 11' bar width (i.e. the luminance of the little circle is nearly the same as that of the bright bars). However, against a bright background, the luminance required to detect the little circle is nearly sufficient to resolve a 7' grating. Similarly with the dark adaptation curves of Fig. 4a. In the dark the triangles lie close to the open circles; early in dark adaptation, they approach the filled circles. Obviously no simple scaling will make these three curves coincide. But Fig. 5 shows that, when dark adaptation is plotted as log equivalent luminance against time, the three criteria of threshold give results which lie upon the same curve. It follows that the condition which stands in unique relation to the amount of bleaching and regeneration is not log threshold nor any power of the threshold, but log equivalent luminance.

DISCUSSION Total background A century ago Fechner (1860) pointed out that the increment threshold AI of a flash superimposed upon a background I obeyed the relation AI = k(I+ID), where ID and k are constants. The absolute threshold is the value of AI when I = 0, and hence is kID. Fechner suggested that, even in complete external darkness, there is some intrinsic light of the eye (Eigengrau), and ID is the background of Eigengrau that must be added to the external background I. Barlow (1957) has worked out some implications of this in the modern concept of receptor noise, with its implications in the theory of information. Into this we need not go here; it suffices to take up two points. (i) If we are given the experimental curves of Fig. 2b both of which (below the onset of saturation) fit the formula log AI = a log (I+ID)+b (2) with different values ofa and b, how are we to extract the value of ID? The absolute threshold Alo is when I = 0; thus from eqn. (2) log AIo = a log (ID) + b. (3) When I is so large that ID is negligible log AI = a log I + b. (4) DARK ADAPTATION IN A ROD MONOCHROMAT 623 This is the linear part of the curve and a is its slope. If this line is pro- longed back until it reaches the level of the absolute threshold log AI, the value of I from eqn. (4) is given by log AIo = a log I+b, but from eqn. (3) it appears that this value of I is in fact ID. Thus by making this construction in Fig. 2b we find that the sloping line produced backwards meets the horizontal through the absolute threshold at ID = - 2'8 log td, a retinal illumination of Eigengrau very close to that found by Barlow (1957). So the rod monochromat is normal in receptor noise as in all other rod properties. It is seen in Fig. 2b that log ID is the same for the 60 and the 5' curves. This was to be expected if ID measures the level of dark retinal activity since that should not depend upon size of test flash used to measure it. (ii) If in Fig. 2b we add ID (= 00016 td) to each external background before taking the logarithm we shall plot log AI against log (I + D), the total background. Naturally this will make no difference to the curve except at the lowest backgrounds, where the last two points become displaced to the right and are plotted as 'diamonds'. The new curve is seen to run as a (dotted) straight line right up to the point where it meets the absolute threshold level at a background of ID. The new curve plotted against log total background (internal + external) avoids the practical difficulty of assigning an equivalent log background corresponding to thresholds only just above the absolute value. The old curve lay indefinitely to the left; the new curve runs to a clean finish. The dotted tail of Fig. 3 is plotted from the new (dotted) curves of Fig. 2 b. Since the log increment threshold is a straight line function of the log total background from ID right up to the beginning of saturation it follows that the dark adaptation curve as usually determined plots the log equivalent total background scaled by a factor equal to the slope of the Fechner line appropriate to the threshold flash employed. But the neighbourhood of saturation needs special consideration.

The start of the log equivalent luminance plot in dark adaptation It is difficult to believe that the slow initial fall in the curves of Figs. 3 and 5 is a proper representation of what is going on. At a time when ob- jective measurements ofrhodopsin show it to be regenerating most rapidly, and when the log threshold curve is falling most steeply, it is obviously misleading to represent the condition by the equivalent background curve of Fig. 3. A glance at Fig. 2 reveals the source of the trouble. The Fechner line continues straight up to a background of about 2 log td, so that log threshold is linear with log total background (I + ID) up to this 624 C. B. BLAKEMORE AND W. A. H. RUSHTON point. But at higher backgrounds the log threshold rises steeply, or, put otherwise, log background does not increase linearly with the threshold. Thus the equivalent background of Fig. 3 flattens out at high thresholds and can never rise much above 3 log td since at this level the threshold approaches infinity. Aguilar & Stiles (1954) were the first to observe this rod 'saturation' as they called it, and pointed out that it occurred at a level where each rod was absorbing quanta at a rate of about 1000/sec. They suggested that signals could not be generated and transmitted steadily at so high a frequency, and consequently a large increment in light would produce little increase in signal. The transformation of Fig. 3 is thus rather similar to recording through an amplifier of limited grid swing. For moderate inputs the output is linear, but for large inputs we exceed the Fechner linear range and overloading occurs. And indeed Fig. 3 instead of displaying the familiar exponential dark adaptation curve looks very much like the record of a condenser discharge distorted by overloading the amplifier that records it. Until we have fuller understanding of the nature of rod saturation we shall not be able to speak with confidence about the highest levels of adaptation. It seems plain, however, that, up to the level where saturation sets in, three quantities run together in fixed proportions, namely log threshold, log total background and free opsin. And, even in the range of saturation, log threshold and free opsin still run together though here real backgrounds overload the transmission channels. It therefore appears reasonable to consider that log threshold still measures correctly the state of adaptation and to distrust the equivalent background in this region as we should distrust the record from an overloaded amplifier. The problem offixation The cone-poor subject of a previous investigation (Rushton, 1961 b) had enough foveal vision to be able to fixate there reliably. She was steady enough for densitometry measurements to be made with good rhodopsin density at 150 away from her point of fixation and no rhodopsin density upon the fixation point itself. And her log threshold during dark adaptation fell over 6 log units upon an exponential time curve of the same time constant as the exponential curve of rhodopsin regeneration. By contrast C. B. B. appears to have no cone function whatever, does not fixate with the fovea but somewhere close to a large retinal vein, and cannot hold any fixation point steadily enough to permit retinal densitometry. When asked to observe the test flash in threshold measurements he chose a retinal position that suited him. He was quite clear as to what region did and what did not suit him for detection of the flashing light, and DARK ADAPTATION IN A ROD MONOCHROMAT 625 when after faulty bleaching fixation he saw the after-image in the wrong place he said at once 'This won't do. The wrong region is bleached.' This led us to suppose that the 'right' region was a fixed place. There are no grounds for believing this and rather strong grounds for disbelieving it. We attempted to overcome some of the difficulties of fixation during the 2 min of bleaching by using an electronic bleaching flash presented in Maxwellian view. The Maxwellian lens was illuminated in addition by weak light and C. B. B. was asked to give his opinion upon some detail in the middle of it. At that instant the electronic flash was delivered as a sur- prise when his fixation was exactly centred upon the lens (as he admitted) yet he at once protested that the sharp after-image of the lens was well away from the flashing test light upon which he was now fixating. The only way of reconciling C. B. B.'s two statements is to conclude that the retinal region suitable for fixation in the dim light before the flash was quite different from that in the strong 'equivalent luminance of bleaching' immediately afterwards. Presumably in the subsequent course of dark adaptation the fixation point gradually shifted back to the first region again. We have not investigated the relation of fixation locus to luminance, but it is plausible to suppose that during the course of dark adaptation C. B. B. gradually shifted his gaze so as to bring in larger summating rod pools. Thus some of the fall in threshold of the 60 curve of Fig. 2 and some of its divergence from the 5' curve are probably due to this cause. In view of this it is remarkable that in Figs. 3 and 5 the open and full circles and triangles lie so exactly upon the same curve. For, if threshold depends in part upon the point of fixation, thresholds can only correspond as well as they do in Figs. 3 and 5 if the fixation points also correspond. We conclude that equivalent luminance not only determines threshold and the lateral summation and inhibition upon which the relative shapes of the curves of Fig. 2 depend; it must also determine the point offixation. The equivalent luminance We have discussed a few small difficulties that needed to be cleared up or at least aired, but they hardly affect the main purpose and proof of this paper. We set out to learn whether the principle of equivalent luminance would relate increment thresholds and the dark adaptation following a 50 % rhodopsin bleach in the manner that Crawford had shown after a 0-2 % bleach. And we have made use of the special faculty of the rod monochromat to give a great range of thresholds free from any contamina- tion by cones. Though some questions of interpretation remain open, it can hardly be doubted that, below the level where saturation sets in, the principle of equivalent luminance holds over the whole range of bleaching and re- 40 Physiol. 181 626 C. B. BLAKEMORE AND W. A. H. RUSHTON generation studied. Various test criteria were used and indeed the tests were more varied than those here described but they never showed an exception to the principle of equivalence. This enables us at last to express properly the relation between the amount of rhodopsin bleached and its effect upon visual performance, though the expression is not new, for Rushton (1963b) and Barlow (1964) have already proposed it-taking into consideration, however, the results of this paper which were known to us four years ago. The amount of free opsin is linear-not with log threshold, for that depends upon the kind of test used-but with log total equivalent luminance, for that is unaffected by the nature of the test. In symbols, if B is the fraction of rhodopsin in the bleached state, k is a constant, IB is the total equivalent luminance and ID the Eigengrau, then B = k log (IB/ID). Though this expression is analytically simple it introduces a retinal organization of some complexity. In a recent paper (Rushton, 1965b) it was shown that background luminance did not raise the increment threshold by making the rods themselves less sensitive; the characteristics of the 'adaptation pool' were changed so that more rod signals were needed for the detection of the test flash. In this paper we have shown that bleached rhodopsin affects visual performance precisely like a suitable background. Thus in some way the free opsin must signal to the 'adaptation pool' and change its organization more or less as a luminous background does. What is this 'free opsin' signal and by what path does it reach the 'pool'?

SUJMARY 1. Threshold measurements were made by a rod monochromat (C. B. B.) in various states of adaptation using various criteria for threshold. 2. The criteria used were either the detection of 1 sec flashes subtending an angle that ranged between 60 and 5', or the resolution of gratings of various pitches. 3. The conditions of adaptation were either dark adaptation following exposure to a bright light that bleached about 50 % of the rhodopsin, or increment threshold where the test flash fell upon a background of variable luminance. 4. For any particular test flash used, it was possible to find the background that raised the threshold to the same value that it had at any given moment of dark adaptation. In this way a dark adaptation curve could be plotted not as log threshold against time but as log equivalent background against time. DARK ADAPTATION IN A ROD MONOCHROMAT 627 5. The dark adaptation curve plotted as log threshold against time has a shape that depends greatly upon the kind of test flash used, and therefore cannot represent directly the regeneration of rhodopsin. When plotted as log equivalent background against time the shape is the same no matter what kind of test is used. This, then, is the excitability measurement that related directly to the amount of rhodopsin bleached. 6. This confirms the conclusions of Crawford (1947), and extends them as follows. (a) We bleached 50 % ofthe rhodopsin; Crawford only bleached 0-2 %. (b) Our subject (C. B. B.) had no cones and the rod threshold was studied over a range of 6 log units. (c) We used as threshold criteria, in addition to the detection of lights, the resolution of gratings. 7. Despite this increase in the range ofinvestigation Crawford's principle of 'equivalent backgrounds' remained valid. Our thanks are due to Mr Clive Hood, who built the equipment and assisted in the experiments. W.A. H.R. acknowledges with thanks a grant from the U.S. National Institute of Neurological Diseases and Blindness (NB 03014-04).

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