47

J. Physiol. (1979), 295, pp. 47-68 With 9 text-ftgvre8 Printed in Great Britain

RECOVERY OF CAT RETINAL GANGLION CELL SENSITIVITY FOLLOWING PIGMENT BLEACHING

BY A. B. BONDS* AND CHRISTINA ENROTH-CUGELL From the Departments of Electrical Engineering and Biological Sciences, and the Biomedical Engineering Center, Northwestern University, Evanston, Illinois 60201, U.S.A.

(Received 21 November 1978) SUMMARY

1. The threshold illuminance for small spot stimulation of on-centre cat retinal ganglion cells was plotted vs. time after exposure to adapting light sufficiently strong to bleach significant amounts of rhodopsin. 2. When the entire receptive field of an X- or Y-type ganglion cell is bleached by at most 40 %, recovery of the cell's rod-system proceeds in two phases: an early relatively fast one during which the response appears transient, and a late, slower one during which responses become more sustained. Log threshold during the later phase is well fit by an exponential in time ('r = 11-5-38 min). 3. After bleaches of 90 % of the underlying pigment, threshold is cone-determined for as long as 40 min. Rod threshold continues to decrease for at least 85 min after the bleach. 4. The rate of recovery is slower after strong than after weak bleaches; 10 and 90 % bleaches yield time constants for the later phase of 11-5 and 38 min, respectively. This contrasts with an approximate time constant of 11 min for rhodopsin regeneration following any bleach. 5. The relationship between the initial elevation of log rod threshold extrapolated from the fitted exponential curves and the initial amount of pigment bleached is monotonic, but nonlinear. 6. After a bleaching exposure, the maintained discharge is initially very regular. The firing rate first rises, then falls to the pre-bleach level, with more extended time courses of change in firing rate after stronger exposures. The discharge rate is restored before threshold has recovered fully. 7. The change in the response vs. log stimulus relationship after bleaching is described as a shift of the curve to the right, paired with a decrease in slope of the linear segment of the curve. INTRODUCTION

The effects upon retinal ganglion cell behaviour of steady backgrounds too weak to cause any significant steady-state bleaching (field-adaptation; Rushton, 1965) have been studied extensively in cat and other species (e.g. Donner, 1959; Cleland & * Present address: School of Optometry, University of California, Berkeley, Calif. 94720, U.S.A.

0022-3751/79/4970-0281 $01.50 © 1979 The Physiological Society

A. B. BONDS AND C. ENROTH-CUGELL Enroth-Cugell, 1968; Barlow & Levick, 1969b; Sakmann & Creutzfeldt, 1969; Enroth-Cugell & Shapley, 1973; Green, Dowling, Siegel & Ripps, 1975). The 'raised threshold and slow recovery that follows exposure to lights that bleach a substantial fraction of the eye's rhodopsin' (bleaching adaptation; Rushton, 1965) can also be observed at the ganglion cell level (e.g. Barlow, Fitzhugh & Kuffler, 1957; Lipetz, 1961; Donner & Reuter, 1965; Green et al. 1975; Rodieck & Rushton, 1976). These changes have not, however, been investigated as thoroughly as those due to field adaptation. Of particular interest is a quantitative comparison between ganglion cell behaviour and pigment kinetics, as this may provide some hints as to the actual mechanisms responsible for changes in sensitivity. So far, any inference in the cat regarding the fraction of rhodopsin bleached and the properties of the ganglion cell have necessarily been based on our knowledge about pigment behaviour in man. However, we now have reliable information about rhodopsin kinetics in the cat (Bonds & MacLeod, 1974) obtained under the same experimental conditions as those used in this laboratory for study of retinal ganglion cells. This paper will deal primarily with the time course of bleaching adaptation and how it is related with the regeneration of rhodopsin; a second paper (Bonds & Enroth-Cugell, 1979) will treat in greater detail spatial aspects of bleaching as manifested at the ganglion cell level. 48

METHODS

Preparation. Eighteen adult cats were used, lightly anaesthetized with I.v. urethane. Doses ranged from 30 to 50 mg/kg. hr (preceded by a 200-300 mg loading dose) over a maximum of 15 h. Most of the detailed results stem from twelve cats. Both the surgical aspects of the preparation and the physiological controls observed during each experiment are detailed elsewhere (Enroth-Cugell & Pinto, 1972). After bleaching good eye stabilization over long periods was essential, and was attained with gallamine 50 mg/kg. hr combined with diallyl-bisnortoxiferine 250 #sg/kg .hr. These drugs were usually supplemented by bilateral sympathectomy and, as a further precaution, most experiments were repeated on cats whose eyes had been glued to fixed rods using Eastman 910 cement (e.g. Enroth-Cugell & Shapley, 1973). Recording and data processing. Each action potential, recorded with a tungsten micro-electrode in the optic tract, triggered a standard 0 5 msec pulse which fed two integrating networks. The first had a 5 see time constant and drove a chart recorder which displayed the 'averaged' mean spike frequency. This record was used to follow the maintained discharge when the animal was in the dark and the average discharge during stimulation. It also provided valuable clues to the stability of the preparation, such as fluctuations in mean firing rate due to residual eye movements or deterioration of the animal's general condition. The second integrating network had a time constant of 10 msec and acted as a frequency-to-voltage converter which fed a digital averaging computer used to produce pulse density tracings (Enroth-Cugell & Robson, 1966). Response magnitude was measured using a digital pulse counter. It accumulated the total number of impulses during the first 200 msec after onset of a 250 msec flash; after extinction of the flash it paused, then subtracted the number of impulses within the 200 msec interval immediately preceding the next flash. The counter thus displayed the number of extra impulses occurring during the first 200 msec of each flash. By averaging over twenty stimulus presentations, a reliable response measure was obtained. In later experiments a PDP 11 computer was used in the same way as the digital counter. For monitoring ganglion cell recovery after a bleach a criterion response between five and ten extra spikes per counting period was chosen. When responses are sustained this criterion level corresponds to a peak magnitude of 25-50 impulses/sec, which falls within the linear response range (Barlow & Levick, 1969a). The stimulus strength required to elicit a small criterion response will (for convenience) be referred to as a threshold stimulus. Sensitivity, as used here, implies the ratio of the magnitude of the criterion response and the strength of the threshold

GANGLION CELL DARK ADAPTATION

49

stimulus. This corresponds not to sensitivity as widely determined by listening to a cell, but to the inverse of Barlow & Levick's (1969a) quantum to spike ratio. Stimulator. Because of the high illuminances required for bleaching, a Maxwellian view stimulator was used. This entails problems when ganglion cell activity is recorded in the optic tract; usually receptive fields more than a few degrees away from the visual axis cannot be stimulated properly without tedious optical alignment and adaptation for use with either eye is cumbersome.

--s

M2

Fig. 1. Schematic of the Maxwellian view stimulator. The light source is indicated by the hexagon. S, electromagnetic shutters; F1..., neutral density wedges; M1.. image masks in planes conjugate with the cat's retina. Arrows are to indicate that the mirror is pivoted on orthogonal axes.

The instrument used was designed and built (in collaboration with Dr Peter Lennie) to alleviate these problems and is described in detail in Enroth-Cugell, Hertz & Lennie (1977). Its Maxwellian lens was of unusually wide aperture and provided a total usable field of 45°. Furthermore, the image of the source filament was focused at the mirror M (Fig. 1), at the pivot axis, which prevented motion of the filament image formed in the cat's pupil as the position of the retinal image of stimuli was changed within the 45 0 field by rotating the mirror. The unattenuated illuminance in each channel was measured using Westheimer's (1966) method, which does not take into account whether a human or a cat will be behind the viewing lens. The troland for the cat will thus represent a slightly higher retinal illuminance than for the human due to the shorter posterior nodal distance and more transparent media of the cat's eye. The first channel delivered 6-6 log scotopic td which corresponds to 2-0 x 1012 equivalent quanta (500 nm)/deg2 sec, or 3-2 x 1012 equivalent quanta (560 iim)/deg2 at the cornea (obtained as described in Enroth-Cugell et al. 1977). The second channel delivered 6-1 and the background channel 6-4 log scotopic td. To make comparison with literature on dark-adaptation, densitometry and ganglion cells easier, bleaching illuminations will be given in trolands while test stimuli, which except where stated were always blue-green, will be expressed in equivalent quanta at the cornea. Neutral wedge densities and the photopic and scotopic equivalent densities of the blue-green (Ilford 623 or 603, dominant wave-length of each 490 nm) and red (Ilford 608, transmits above 620 nm) filters were calibrated with a Gamma 2020 photometer. The values for the colour filters agreed well with those determined with the same stimulator in experiments on retinal

50

A. B. BONDS AND C. ENROTH-CUGELL

ganglion cells driven by rods or cones respectively (see Enroth-Cugell et al. 1977). The photopic equivalent neutral density of the 608 and 603 filters were 2-0, of the 623 filter 1P5. The scotopic equivalent neutral density of the 608, 603 and 623 filters were 3-2, 1-3 and 0 7, respectively. White light only was used for bleaching. All results are from on-centre cells, which after strong bleaches were quite active. Off-centre cells remained silent for periods of up to half an hour after bleaching which frustrated continuity of data collection. This paper is based on data from forty-five cells; detailed results were obtained from eighteen of these. Nine were classified as Y-cells (probably brisk transient), five as X-cells (probably brisk sustained) (Enroth-Cugell & Robson, 1966; Cleland & Levick, 1974); four cells were not classified. Some cells were not subject to the null test (Enroth-Cugell & Robson, 1966) but rather to a combination of tests such as: (1) windmill test (Cleland, Dubin & Levick, 1971), (2) time course of responses to square wave modulated small stimulus spots (Jakiela, Enroth-Cugell & Shapley, 1976), and (3) relation between the shape of the centre's sensitivity profile and centre diameter as obtained from area-sensitivity curves (Enroth-Cugell, 1977). Except where noted, responses originated primarily or entirely within the centre. The magnitude of such responses depends upon the effective flux of the stimulus, i.e. upon the product of stimulus illuminance and area weighted by local sensitivity. This concept can be simplified by assuming the receptive field centre to consist of a radially symmetric region of uniform sensitivity equal to the measured maximal local sensitivity. The area (At) or diameter (DJ) of this equivalent centre may be estimated from area-threshold curves by noting the diameter at the intersection of the sloping part of the curve and a horizontal line drawn through the minimum illuminance required for a criterion response (e.g. Kirby & Enroth-Cugell, 1976, p. 468). Effective flux is then the actual flux if the stimulus is contained entirely within the equivalent centre, or the stimulus illuminance times the equivalent centre if the stimulus extends beyond it. Centre size will be given in terms of Dt, and stimulus strength in effective flux calculated using the conventions described above. RESULTS

This paper is concerned with bleaching adaptation as observed at the ganglion cell level and with the cell's recovery while the retina remained in the dark (darkadaptation). Three findings will be emphasized. (1) When the entire receptive field of an X- or Y-type ganglion cell is bleached to at most 40 %, recovery proceeds in two phases: an early, relatively fast one in which the response appears transient, and a late, slower one in which the response becomes more sustained. (2) The relationship between the elevation of log threshold and the amount of pigment bleached is not simple or easily understood. (3) After bleaching, the straight line segment of the response vs. log stimulus curve both shifted to the right and decreased in slope (see Fig. 9). Bleaching the entire receptive field In these experiments the entire receptive field was bleached with a 150 diameter uniformly illuminated field. Ideally the amount bleached should be determined in each ganglion cell experiment. Since, however, it is not practical to combine densitometry with lengthy single-unit recording, Bonds & MacLeod's (1974) data were used to determine amounts bleached. No bleach exceeded 60 sec. Time course at recovery of rod-threshold after 10-90 % bleaches. For each cell, the centre size (Dt) and the centre's sensitivity profile were first determined after full dark-adaptation. A 0.20 blue-green flashing spot was placed at 0.50 intervals along one vertical and one horizontal diameter through the middle of the receptive field and threshold at each locus was assessed by listening. Repeating this after recovery from a bleach provided a reliable check whether the eye had moved. Next, the

GANGLION CELL DARK ADAPTATION

51

stimulus, which in these experiments was either 0-2 or 1.00 in diameter, was directed onto the middle of the receptive field and flashed on for 250 msec every 800 msec. Using either the spike-counter or the computer (as described in Methods) the strength was adjusted to elicit a criterion response. With the stimulus still flashing, the bleaching light was turned on for a period appropriate to bleach some desired fraction 100

100 E min

15min

C.,

DON

ON E

E 100

B

1

0()

D Dark adapted

1 1 min

0 5 sec

Fig. 2. Peristimulus time histograms obtained at times as indicated in Figure after extinction of a light which bleached 40 % of the pigment. The vertical axis gives the cell's firing rate in impulses sec-'. Bin width, 5 msec, 40 sweeps. The response at 3 min has a transient component which fades with time (see this page) On-centre Y-cell, 32/8.

of the underlying pigment. Following the extinction of the light, the cell remained quite unresponsive to test flashes for 1-4 min (although there was always a maintained discharge) depending upon bleaching duration. In order to insure that the cell's threshold was determined by its rod-system, the blue-green stimulus was limited to a maximum of 3-8 x 107 equivalent quanta (560 nm)/sec (see p. 56). Once recovery had proceeded to the point that this stimulus generated a criterion response the experimenter continuously adjusted the neutral density to maintain the response just at this level. The log of the stimulus strength was plotted versus time after extinction of the bleaching light. During recovery from a bleaching exposure, response wave forms of both X- and Y-cells changed in a consistent pattern. Shortly after extinction of the bleaching light, responses were clearly transient with an initial peak which decayed rapidly to a level only slightly above the pre-stimulus discharge (Fig. 2A). At stimulus off-set there was only a rather brief dip in the discharge frequency which thus quickly returned to the pre-stimulus level. This 'nose' and short off-dip (or occasionally small off-overshoot) is usually characteristic of ganglion cell responses to which both the centre and the surround contribute. As dark-adaptation proceeded, responses became distinctly more sustained from 5-10 min after bleaching, this process taking longer after stronger exposures. When the retina became fully dark-adapted, the

52 A. B. BONDS AND C. ENROTH-CUGELL response appeared very nearly square (Fig. 2B, C and D). A decreasing transience at stimulus onset, but without the quick return to the pre-stimulus firing rate or overshoot at offset, can also be seen in central responses as the level of lightadaptation due to steady backgrounds decreases (Yoon, 1972; Jakiela et al. 1976). 8

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Fig. 3. Rod-threshold recovery for an on centre X-cell. A 150 diameter white bleaching spot was centred on the receptive field 3 times, to bleach 10, 20 and 30 % of the underlying rhodopsin. After each exposure, the course of dark-adaptation was followed by tracking (as described in the text, p. 48) the stimulus strength required for the cell, driven by its centre to produce a criterion response. A. 1 diameter blue-green 250 msec flash stimulated the receptive field middle. When the cell was fully dark-adapted, threshold for this stimulus was 3-15 log quanta (500 nm)/sec (represented by the horizontal axis). The continuous curves are exponentials obtained as described in the text (below), with time constants of (from left to right) 11-5, 13-7 and 17-2 min. The stimulus flux on the ordinate was calculated from illuminance in scotopic trolands by using the conversion given on p. 49 in the text. Cell 37/3; Dt 0.73°.

Fig. 3. shows the recovery of one X-cell whose receptive field was bleached 3 times, removing about 10, 20 and 30 % of the underlying pigment. The data in all three cases appear to describe a simple exponential decay, but the process is more strictly bimodal. Application of an iterative FORTRAN computer routine (STEPIT; Chandler, 1965) designed to minimise the squared difference between the data and the function A + B (exp (- t/T)) yielded much better fits for this and all other cells if based only on the data points from the later recovery phase, during which the response was nearly all sustained. The early points then fell well above the computed fits. This behaviour suggests recovery in two stages, and probably reflects the need for stronger stimulation to compensate for the transience of the response during the early phase. Another general rule emerged from the experiments of this type. In Fig. 3 the exponentials fit to the later part of the recovery curves had time constants which

53 GANGLION CELL DARK ADAPTATION increased with the fraction of pigment initially bleached. The 10, 20 and 30 % bleaches were best fit by time constants of 11-5, 13-7 and 17-2 min respectively. To explore this further, eleven additional cells (four X-cells, seven Y-cells) were subjected to full-field bleaching from 10 to 90 % (90 % requiring 6-6 log td instead of the usual 5-4 log td). Because of the time required for complete dark-adaptation, it was unusual to be able to complete more than two runs on a given unit; many receptive 40 30~~~~~~~

30~~~~~ ~~ 20 *-20

.001 ~~~ 0

10~~

20

40

60

80

100

Bleach (%)

Fig. 4. Dependence of time constant of ganglion cell recovery upon fraction of rhodopsin initially bleached. Results are from a total of eleven cells (four X-type, seven Y-type). In all cases the bleaching field was 150 in diameter. The test spot was 0-2 or 10 and located in the receptive field middle. Experimental procedures were otherwise as in Fig. 3, including curve fitting to obtain the time constants. Filled circles represent individual runs on four different cells. Open circles are all from the same cell as in Fig. 3. The vertical bar indicates the standard deviation for all runs (7 cells) 'where the initial bleach was 40 %.

fields were exposed to one bleach only, of which 40 % (5.4 log td, 25 sec) was the most common. We always found that, as described above, (1) recovery of rod log threshold after moderate bleaches (up to approximately 40 %) proceeded in two phases (although after stronger bleaches, the early rod phase was masked by the cone system) and (2) in all cases the time constant for the later phase increased as fractions of bleached pigment increased. In Fig. 4 our results on the relation between the time constant of the late recovery phase and initial fraction bleached are summarized. One cell (not included in Fig. 4) recovered significantly more rapidly than the others; however, we have reason to believe this was due to optical misalignment of the bleaching beam. Of the remaining population, the smallest value of T was 11 5 min for a 10% bleach (5.4 log td, 5 see), the largest 38 min for a 90 % bleach (6.6 log td, 60 see) and the mean from seven cells for a 40 % bleach (5.4 log td, 25 see) was 22 min. The pattern of longer r with increased bleaching is best illustrated by results from a single cell which was bleached and allowed to recover 5 times (open circles, Fig. 4). In this case, the strongest bleach was delivered first to guard against a spurious result should recovery be retarded by repeated bleaches. When the same bleach (40 %)

54

A. B. BONDS AND C. ENROTH-CUGELL was delivered to seven cells (three X, four Y) we found no correlation between T and cell type or centre size. Because the response wave form tended to become less transient even during the later recovery stages a 200 msec counting period might have affected the finding that stronger bleaches lead to longer time constants (see, e.g. legend 6 of Barlow & Levick, 1969a). On the cell shown in Fig. 3 we also used a 40 msec counting period when analysing the results for the 20 and 40 % bleaches and the difference was only about 0X2 log units over all of the later recovery period. This change was in the direction of increasing fitted time constants, with the stronger bleach still yielding a longer time constant. Extrinsic factors may also have contributed to a retardation of the recovery curves. van Norren & Padmos (1977) have shown that cone dark-adaptation in monkey is retarded by urethane anaesthesia (1 g/kg over approximately 3 h). It seems most unlikely that it would be an important cause of the pattern seen in our Fig. 4. First, the broadest range of time constants (14.9-29-4 min) for a particular fraction bleached (40 %) came from two cells in the same cat and the shorter time constant was observed later in the experiment, after the cat had been given additional urethane. Secondly, Barlow et al. (1957), recording from cats which were decerebrated in ether narcosis more than 3 hr earlier, found dark-adaptation curves with slower time courses than any of ours. It is also unlikely that the stimulus itself bleached enough to perturb the darkadaptation curves. The maximum stimulus used in this first series of moderate bleaches was 2-5 log scotopic td, sustained for only a few minutes (see uppermost points in Fig. 3). This illumination, according to Bonds & MacLeod (1974), causes an equilibrium bleach of only 2 %. If bleaching by the stimulus would have been important, early recovery would have been slower and not faster than late recovery. It is interesting to note that, at least in these experiments, the time constants for late dark-adaptation never went below about 10 min, which is to a first approximation the rate at which pigment regenerates under similar conditions (Bonds & MacLeod, 1974). This may imply that the regeneration of pigment is a necessary (but not sufficient) condition for recovery of the sensitivity of centre-driven retinal ganglion cells. Recovery of threshold when the ganglion cell was driven by its cone-system. We claim that in the experiments so far, sensitivity was tested under such conditions that responses were primarily due to the rod-system. Had initial thresholds been due to the cone-system, they would lie below those predicted by extrapolation back from later levels assuredly determined by rods. In Fig. 3, however, thresholds lie conspicuously above such backward extrapolations. Moreover, in those experiments the equivalent photopic flux of the blue-green stimuli never exceeded 3-8 x 107 quanta (560 nm)/sec, and remained at this level only briefly during the beginning of the early recovery phase. Such a stimulus strength is near the middle of the distribution of minimum thresholds for central responses of cone-driven ganglion cells using Stiles' two-colour technique (Enroth-Cugell et al. 1977), so it is unlikely that these stimuli ever generated significant cone inputs. Consequently, in the experiments described so far, no cone-plateau with subsequent rod-cone break was observed. In the cat, even at locations of minimum

GANGLION CELL DARK ADAPTATION 55 rod:cone ratio, rods outnumber cones ten to one (Steinberg, Reid & Lacy, 1973). This may be why, when after as much as 40 % of the rhodopsin within a ganglion cell's receptive field was bleached, its rod-system remained more sensitive (to bluegreen light) than its cone-system, at least during the interval when threshold measurement was practical (see p. 51). In the experiments to be described now the maximum 10

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Fig. 5. Visualization of the cone plateau. The receptive field of this X-cell was bleached twice to 90 % using a 150 diameter field and recovery was followed using a 1° centred red test (open symbols) after the first, then a 10 blue-green test (filled symbols) after the second bleach. In each case the criterion responses were produced by the centre mechanism. Cell 33/1; Dt = 0.90.

bleaching capacity of the Maxwellian stimulator was used (1) to visualize the coneplateau as a check of previous estimates of the stimulus flux needed to drive ganglion cells through their maximally sensitive cone-system; (2) to obtain additional data for describing the relationship between the amount of pigment bleached and loss of sensitivity; and (3) to generate dark-adaptation curves suitable for comparison with those of Barlow et al. (1957) who clearly documented the cone contribution to the cells' response during recovery. The general experimental procedure was the same as before except that the bleaching illuminance was higher (6.6 log scotopic td) and that recovery was tracked first with a blue-green, then after a second bleach with a red stimulus. Fig.-5 shows results from a Y-cell which had twice suffered a 90 % full-field bleach. Consider first the recovery curve tracked with the red test (open symbols). The fall of log threshold is initially exponential, best fit with a time constant of 3-5 min. This asymptotes to the long cone plateau, which lasts about half an hour. It ends with the rod-cone break at about 45 min, followed by the rod portion ofthe curve; recovery

A. B. BONDS AND C. ENROTH-CUGELL 56 is completed after about 85 min. By tracking recovery with the blue-green test spot after the second bleaching exposure (filled symbols) the behaviour of the rod system is shown more fully. Cone recovery is exposed more briefly than with a red test flash, and has about the same time constant as for the red. Both recovery curves were plotted in scotopic units; as a result the two rod branches coincide. The separation between the two cone plateaus is of the order of 2-0 log units, because the red (Ilford 608) test is 1.2 log units more effective for cones than rods and the blue-green (Ilford 603) about 0-8 log units less effective for cones than rods (see Methods). From about 25 to 50 min. rod log threshold drops at a fixed velocity of about 0 09 log u./min. Past 50 min the rate decreases exponentially, altogether being compatible with a first-order process with rate-limited kinetics. Complete recovery of rod sensitivity again takes about 85 mi. The weakest stimulus which produced a criterion response during the coneplateau was 3*8 x 107 (7.58 log) equivalent quanta (560)/sec, as indicated by the right-hand ordinate which is valid for the red recovery curve only (open circles). This is the upper limit of the strongest test stimulus used in the previous series of experiments, thus supporting our claim that these involved only scotopic thresholds. A second Y-cell receptive field was exposed to a 90 % full-field bleach and the same experiment as in Fig. 5 repeated. This time, stimulus flux at threshold during the cone plateau was 6-2 x 107 (7.79 log) quanta (560)/sec and again, the recovery period required for total dark-adaptation was in excess of 80 min.

Relation between ganglion cell threshold and amount of rhodopsin in the bleached state. So far ganglion cell thresholds have been described, without any attempt to relate the time course of recovery with that of rhodopsin regeneration. To do so would be difficult because of the complexity of the pattern of regeneration in the cat. Here we present three qualitative observations and one attempt to produce a more quantitative correlation. (1) Briefly summarized, Bonds & MacLeod's (1974) measurements of regeneration in the cat after a 1 min bleach show the following: for exposures bleaching 40 % or above, immediately after extinction of the bleaching light there is almost no regeneration of pigment during the first 8-9 min, although for the strongest bleaches, this plateau is preceded by a brief transient increase in pigment corresponding to about 150% of the total. Following the plateau, a second phase commences during which pigment regeneration seems to be rate-limited (Michaelis-Menten kinetics) and regeneration is virtually completed after 35 min. There are thus two phases of regeneration, just as there are two phases in the recovery of the ganglion cell's rod log threshold in the experiments described above. But in those experiments the general finding was that in all cases, when up to about 40 % of the pigment within a cell's receptive field was bleached, recovery of rod threshold proceeded at maximum rate just during the time when no rhodopsin is regenerated, i.e. during approximately the first 10 min. This was probably also true when more (than 40 %) pigment was bleached, although the cone-system then masked recovery of the rod-system (see Fig. 5). (2) The time constant of the late exponential phase of ganglion cell recovery always increased as the amount of bleached pigment increased. The complexity of the

CANGLION CELL DARK ADAPTATION 57 rhodopsin regeneration pattern in the cat after a 1 min bleach prevents fitting an exponential with a single time constant to the experimental points. However, as they increased bleaching energy, A. B. Bonds & D. I. A. MacLeod (unpublished observation) noticed no systematic retardation of rhodopsin regeneration similar to the increase in time constant for recovery shown in Fig. 4. 9 60

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Fig. 6. Comparison of time course of ganglion cell threshold recovery and rhodopsin regeneration after identical bleaching exposures (60 %; 5-4 log td for 60 see). The left vertical axis and filled circles show densitometry data from Bonds' & MacLeod's work (for wave-length 546 nm; this particular experiment previously unpublished). The right vertical axis indicates the flux required for a 1° diameter centred blue-green flash to elicit a threshold response (filled triangles). The large open triangle shows the time after extinction of the bleaching light at which the cell reached its pre-bleach darkadapted threshold, about 70 min. Cell 38/4, Y-type; Dt = 2.250.

(3) A third general observation on threshold recovery and pigment regeneration is exemplified in Fig. 6. where a cell's recovery (filled triangles) after a 60 % bleach is shown together with a curve for rhodopsin regeneration (filled circles) after an identical exposure (from A. B. Bonds & D. I. A. MacLeod's work, unpublished). Although the rhodopsin was completely regenerated at 30 min the ganglion cell still had 1-9 log units to go before its pre-bleach dark-adapted threshold was reached (shown as open triangle), some 70 min after extinction of the bleaching light. Whenever the initial fraction of rhodopsin bleached exceeded 35-40 %, ganglion cell threshold was still above its dark-adapted value by 0-5-2-3 log units at 35 min, while full rhodopsin regeneration never exceeded 35 min in Bonds & MacLeod's densitometry experiments. Since the proportionality between ganglion cell log threshold and fraction of pigment still in the bleached state thus clearly changes during dark-adaptation, a unique quantitative relationship between the two appears impossible. Other electrophysiological results (e.g. Dowling, 1960, 1963) and the psychophysical work of Rushton (e.g. 1961 a) and Rushton & Powell (1972a), on the other hand, support a relationship wherein the log threshold throughout dark-adaptation remains proportional to the fraction of unregenerated rhodopsin i.e.

log (01A)

=

aB,

(1)

A. B. BONDS AND C. ENROTH-CUGELL where 0 represents the threshold during dark-adaptation, A the threshold in complete dark-adaptation, B is the fraction of bleached rhodopsin and a is a constant of proportionality. There are, however, conditions when this overall linear relationship (often referred to as the Dowling-Rushton relationship, e.g. Pugh, 1975b) does not 58

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20

40 100 60 80 Bleach (%) Fig. 7. Correlation between projected threshold at the moment of extinction of the bleaching light and the fraction of rhodopsin bleached at that moment. The threshold was estimated by extrapolating backwards the exponential curve fitted to the threshold recovery data (see p. 52), and the fraction bleached was estimated from Fig. 2 of Bonds & MacLeod (1974). These results stem from sixteen dark-adaptation curves obtained from three X- and five Y-type ganglion cells. Bars denote the standard deviation of threshold elevation when multiple dark-adaptation curves were traced for more than one cell after the same bleaching exposure, and adjacent numbers indicate the sample size. In the inset, open circles show the same type of data as the main Figure, but are from a single cat ganglion cell. The filled circles are from psychophysical experiments in man and have been derived from the work of Pugh (1975) as described in the text (p. 59).

hold. For example, the log of the psychophysical threshold following bleaches of less than 10% lies substantially above the expected level just after extinction of the bleaching light (Rushton & Powell, 1972a). Moreover, some of Pugh's data (1975a, b) on threshold elevation in man violates the Dowling-Rushton relationship. We have tried to show some consistent quantitative relationship between ganglion cell log threshold and fraction bleached. Since this proportion is continually changing in time, we chose to examine the relationship at termination of the bleach (time zero). Ideally, one should measure threshold at just that moment; however, the cell's responsiveness was then often so low that a threshold stimulus would have caused additional bleaching. The log of threshold elevation at time zero was instead estimated by extrapolating backwards the exponential fitted to the later phase of rod-recovery

GANGLION CELL DARK ADAPTATION

59

(p. 52). Beside the obvious advantage that it provided more points upon which to base a fit, it was also not complicated by the receptive field re-organization which seemed to take place during the early phase, as evidenced by a changeover from somewhat transient to sustained responses. A summary of results from sixteen dark-adaptation runs from eight cells (three X- and five Y-cells) is presented in Fig. 7. If these data, averaged over an ensemble of cells, were in fact obeying eqn. (1) it should be possible to fit them with a straight line through the origin. But a leastsquares straight line yielded a Y-intercept indicating a threshold elevation of 2'8 log units for no bleach whatsoever. A straight line was also drawn through the points obtained from five runs on one individual cell (open circles in inset) and in that case, too, the line did not pass through the origin but indicated a 2-4 log unit increase above dark-adapted threshold for zero bleach. The use of a shorter (40 msec) analysis period would reduce this figure, but by 0 3 log units at the most. If the extrapolations were based on exponentials fit either to the early phase alone or to the entire recovery curve (without distinction of phases) the effect would be to elevate the Y-intercept still more. We have no experimental points for bleaches below 10 %. Since it is reasonable to believe that no pigment bleached corresponds to no threshold elevation, one must assume that for bleaches below 10 % the slope of the curve changes rapidly becoming very steep for the smallest bleaches. A corresponding plot (filled circles in inset) was constructed from Pugh's psychophysical data on man (1975a, b). The fraction bleached was taken from Table 1 of his first paper and the threshold elevation at time zero was derived from the data in Table 1 of his second paper. The behaviour assumed for the cat curve for bleaches below 10 % is indeed exhibited by these human data. The slope of the curve is quite steep for the weakest bleaches, and decreases continuously for stronger exposures. Thus both our ganglion cell data and Pugh's human experiments support Rushton & Powell's (1972b) finding that weak bleaches have a proportionally more profound threshold elevating effect than stronger ones.) Pugh's data also show, as in cat, that a straight-line fit is acceptable for points above 10% bleach, but again such a line would not pass through the origin, demonstrating an over-all non-linear relationship.

Bleaching with fields smaller than the receptive field centre Most of the experiments in which the bleached area was smaller than the receptive field centre will be described in a second paper (A. B. Bonds & C. Enroth-Cugell, in preparation) which is devoted specifically to spatial aspects of the effect of bleaching. Here we are concerned only with the more general properties of the ganglion cell's maintained discharge and its stimulus-response curve after a bleach. Maintained discharge following a bleach. Healthy on-centre ganglion cells have a steady maintained discharge even when dark-adapted. In this study mean maintained frequency (in the dark) varied between about 5 and 40 impulses/sec from cell to cell. When pigment within the receptive field was removed by bleaching, this discharge changed substantially. While the general pattern of change over time was reasonably consistent, the actual timing of the individual increases and decreases depended upon the strength of the bleach. Consider a cell in which 12 % of the pigment within a 0.20 diameter area in the middle of its receptive field was bleached (Fig. 8 A, B). An initial large increase caused

A. B. BONDS AND C. ENROTH-CUGELL

60

B

A

80

.)_

0) C,

Cell 15/2

20

30 45 15 Seconds after bleach

a

2

4 6 8 1 Minutes after bleach

C

Cell 12/2

a.CA

20

25

Minutes after bleach

D

1isec

1 .1- LI LL-I -IIl

I1

011 01

O

-I.1 1tLLtlL 1 I-

Fig. 8. Maintained firing in the dark following a bleach. In each case, the bleaching period is denoted by a heavy bar on the abscissa; impulse frequency is 'averaged' by a smoothing network having a 5-sec time constant. A and B, a bleaching field 0.20 in diameter was centred on the receptive field sufficiently long to bleach 12 % of the underlying rhodopsin. The data in A and B are the same, but plotted on different time scales. The frequency rise after extinction of the adapting light in A was more abrupt than shownl here, as smoothed by the 5-sec time constant. The temporary drop in the maintained frequency at about 6 min was seen in six cells, and when such a dip occurred at all it did so after every bleach (in this case, after twelve bleaches). No change was observed in the cell's response properties during the dip. Cell 15/2, Y-type; Dt = 6°. C, maintained discharge after exposure to a bleaching light of similar geometry, but kept on long enough to bleach 60 of the rhodopsin. The major differences in the postbleach behaviour is that all events are slower (same time scale as in B). Cell 12/2, Y-type; Dt = 1-60. D, retouched spike train obtained two minutes after a 40 % bleach to show regularity of discharge. Cell 38/3, Y-type: D, = 2.24°.

GANGLION CELL DARK ADAPTATION 61 by the onset of the bleaching light decayed almost immediately to just below the pre-bleach firing level (when the bleaching light remained on for more than a few seconds, the dip sometimes approached zero impulses/sec). Extinction ofthe adapting light was marked by a second, much smaller increase in firing (barely noticeable in Fig. 8A) immediately followed by a more protracted rise in frequency. This climb was nearly linear (see legend, Fig. 8) with a duration of 20-30 sec. Then followed a more gradual decay to the dark-adapted firing level. This can be observed in Fig. 8B where the time scale is in minutes instead of seconds. If, by increasing bleaching illuminance, area or exposure time, a larger fraction of the pigment within a receptive field was bleached, the temporal pattern of the changes was extended. An example of this (from another cell) is shown in Fig. 8C. The diameter of the bleaching spot was again 0.20 but this time 60 % of the pigment lying beneath it was bleached. The burst at extinction (marked by arrow) of the adapting light was larger, and the rise in firing rate which followed was retarded to a slow exponential function with a time constant of about 3 min and peaking at 6 min. Hughes & Maffei (1965) using diffuse bleaching lights (and diffuse flashes), noted similar fluctuations in firing rate of cat on-centre ganglion cells after extinction. We also found, as did Hughes & Maffei, that there seemed to be little correlation between the cell's threshold and the maintained discharge; the discharge level was usually restored long before threshold had returned to its dark-adapted value. After the transients due to extinction of the adapting light, in all cases the maintained discharge displayed remarkable constancy of the inter-spike intervals, as exemplified in Fig. 8D. Except for the very slow modulation of spike interval, as indicated by the changes in mean firing rate, this regularity was sustained until somewhat after the point in time where the mean rate started its descent towards the firing level in the dark. Such regularity is not typical of cat retinal ganglion cells under most conditions of adaptation, although there is a tendency for the variance of the discharge to be reduced with increased field-adaptation (Barlow & Levick, 1969b), and Enroth-Cugell & Pinto (1972) noticed that placing a steady, small bleaching spot in the receptive field middle rendered the cell's discharge remarkably regular. Similar firing regularity has also been noted in retinal ganglion cells of the skate. Again, however, this occurred while the adapting light was still on (Dowling & Ripps, 1970). Stimulus-response relationships. Sakmann & Creutzfeldt (1969) measured incremental stimulus-response curves for cat retinal ganglion cells at different levels of field-adaptation, and showed that increasing steady backgrounds caused the curves (response vs. log stimulus) to shift to the right. We performed the same type of measurements at different times following a bleach to establish what effect bleaching has on the cell's stimulus-response relationship and how it changes during dark-adaptation. For reliability, a stimulus-response curve should be based on at least four points. In these experiments, however, the averaging of responses to produce a single point took a minimum of 40 sec. Since the sensitivity of a ganglion cell's centre changes quite rapidly during the first few minutes following the removal of the bleaching light, it is impractical to construct an entire family of curves during a single period of dark-adaptation. The curves were therefore synthesized in the following manner.

A. B. BONDS AND C. ENROTH-CUGELL With the retina well dark-adapted, an initial averaged response to one stimulus strength was obtained. Then, as this stimulus cycled continuously, a small bleaching spot was applied to the receptive field middle. Following its extinction, averaged responses to the fixed stimulus were obtained continually at 40-sec intervals until the response had stabilized (see below). The stimulus strength was then incremented 62

A

quanta (500)/sec Cl213Log Cl2135-4 100

120 100 - 80 .60

B ---

52-2 5*0 ~5 ' 0~~~~~~~~-

2 4 Minutes after bleach C

140 120

E

Cell 21/3

,100

-

~80

-

~60

-

6

140 9

120

d

~100

D

Cell 24/6

e *d

80-

.60 E -40-

-

a

40

a

20 -20 0

I

I

I

I0

4-8 5 0 5-2 5-4 4-8 5-0 5-2 5-4 5-6 Log effective flux (quanta (500)/sec)

Fig. 9. Stimulus-response curves at different times during dark-adaptation. A. moderate bleach (35-40 %; 5-4 log td, 20 see) was applied 4 times with a 0.20 diameter spot to the middle of the receptive field of two different cells. After each bleach, peak response magnitude to a stimulus of fixed illuminance was measured every 40 sec. as indicated in B, and plotted against time in the dark to yield curves as in A for both cells. In each case, the stimulus was a 250 msec flash centred on the receptive field; the diameters were 1.10 for cell 21/3 and 0.20 for cell 24/6, and the different test illuminances were adjusted so that each cell was exposed to the same four (effective) stimulus fluxes. Stimulus-response curves for (a) 20 see, (b) 60 sec, (c) 100 sec, (d) 140 sec, (e) 180 sec, (g) 260 sec and (h) 340 sec (constructed by taking vertical 'slices' through the response V8. time curves as in A are shown in C and D (filled symbols). The open symbols represent stimulus-response curves measured in the dark-adapted conditioning. Note that compared with the dark-adapted curves those measured after bleaching are both shifted to the right and have a lesser slope. Cell 21-3, X-type; Dt = approx. 0.90. Cell 26/6, Y-type; Dt = 2-8.

(or decremented) by 0*2 log units, a new initial response was recorded, the same bleach was applied and the measuring process repeated, ultimately producing the family of curves shown in Fig. 9A. The stimulus-response curves for different times following the bleach were then constructed by taking vertical 'slices' through these curves at a given time and replotting the response versus the logarithm of the stimulus as shown in Fig. 9C and D. In each case, the bleaching spot was 0.20 diameter, had an illuminance of 5*4 log td and was centered on the receptive field for 20 sec. Since in the different experiments

GANGLION CELL DARK ADAPTATION 63 stimulus diameter varied from 0-2 to 1.10, stimulus strength is presented as the logarithm of the effective flux (see p. 50). The use of a small (0.20) bleaching spot in these experiments accelerated considerably the recovery in the dark compared with that seen after large area bleaches of the same energy (cf. Fig. 3). Even so, following the first bleach, in some experiments the response magnitude had not re-attained exactly its dark-adapted level, although it had very nearly stabilized after half an hour. In order to record dark-adaptation of the same cell several times, the experiments were performed allowing a minimum of 5 min between the stabilization of the response and the next bleach. With this procedure, the response did recover its prebleach (albeit somewhat less than dark-adapted) amplitude between all subsequent runs. When plotted on semilogarithmic co-ordinates, the stimulus-response relationship of retinal ganglion cells resembles a sigmoid (cf. Sakmann & Creutzfeldt, 1969). Although we could not collect enough points to describe the curves completely, those points we did collect seemed to fall on the straight portion of the sigmoid and were fitted by straight lines using the method of least-squares. This experiment was completed successfully on one X-cell and four Y-cells. Representative results are shown in Fig. 9C and D from which it is clear that a decrease in slope was accompanied by a small shift to the right, as evidenced by the point at which the fitted lines intersect the abscissa. In the rightward shift, the behaviour resembles the results of Sakmann & Creutzfeldt (1969) for field adaptation. This shift in combination with the slope change (in some instances greater than a factor of two) is in agreement with what has been seen in single receptors (Gekko) following a substantial bleach (Kleinschmidt & Dowling, 1975). Moreover, Dodt & Echte (1961) found that in the rat, the slope of the curve plotting e.r.g.-amplitude vs. log-stimulus strength is less over the first 30 min of dark-adaptation than after long periods in the dark and in light-adaptation. One equation which yields straight lines on semilogarithmic co-ordinates (as seen in Fig. 9C and D) is R = A log (BS), where S represents the stimulus, R the response, A a constant which controls the slope of the line, and B another constant which determines the intercept. If one models the control of ganglion cell sensitivity as a concatenation of serial processes, B corresponds to a linear attenuation before a logarithmic transformation and A a linear attenuation after such a transformation. The parallel shift of the stimulusresponse relationship in the presence of a steady background described by Sakmann & Creutzfeldt is therefore consistent with only a change in gain prior to the stage of transformation, while the results seen here after bleaching implicate a combination of this process with an additional stage of attenuation of a different nature. DISCUSSION

Early dark adaptation. It is evident from the pattern of change of the response wave form together with the bimodal progression of recovery (Figs. 2 and 3), that there are two stages (at least) in the recovery of ganglion cell rod threshold after moderate bleaches. During the first, the response is somewhat transient and the

A. B. BONDS AND C. ENROTH-CUGELL threshold elevation is 'excessive' (i.e. the experimental points fall above the curve, Fig. 3). This fades within 5-10 min after bleaching and during the second stage the response is more sustained, and recovery follows a simple exponential in time. Divisions of rod recovery after bleaching into a rapid early phase and a slower later phase have been reported for humans (Hecht, Haig & Chase, 1937; Rushton & Powell, 1972a, b; Pugh, 1975b), rats (Dodt & Echte, 1961; Dowling, 1963) and frogs (Donner & Reuter, 1965, 1968). Such a dichotomy has also been noted in receptors (Kleinschmidt & Dowling, 1975; Green et al. 1976). The initial rapid descent of rod threshold has been called neural adaptation (e.g. Dowling, 1963), receptor adaptation (Grabowski & Pak, 1975) and network adaptation (Green et al. 1976), but to avoid causative implications we will call it early dark-adaptation (Baker, Doran & Miller, 1959). The common denominator in all cases is the extreme but rapidly diminishing elevation of rod threshold after bleaching. It is possible that the transience seen in the ganglion cell response during the early phase is due to the adaptation of the receptors in the centre to such a great degree that signals from the normally weaker surround become temporarily significant and, through antagonism, erode what sustained component that might arise from the centre. An observation bearing on early dark-adaptation in the cat was made by Steinberg while working with ganglion cell discharges (Steinberg, 1969a) and S-potentials (Steinberg, 1969b, c). Very bright test flashes caused the return of either the ganglion cell discharge or the S-potential to its resting level to slow; Steinberg called this the 'rod after-effect'. For bleaching exposures, the 50 % return time of the potential was as long as 95 see (a decay constant of 2-3 min), which is consonant with the times seen here for early dark-adaptation. The 'rod after-effect' has subsequently been noted in the rods themselves, of Necturus (Normann & Werblin, 1974), axolotl (Grabowski & Pak, 1975) and gekko (Kleinschmidt & Dowling, 1975). Each shows hyperpolarization after strong illumination, with the rate of recovery extending to minutes. In the cases where pigment regenerated (Steinberg, 1969b; Kleinschmidt & Dowling, 1975) the change in membrane potential reflected the events of the early recovery phase, but did not parallel the overall return of log threshold, as the membrane potential had recovered long before dark-adapted threshold was reestablished. Early dark-adaptation thus apparently involves some process which delays the restoration of the rod membrane potential after adaptation by a strong 64

light. The decline of the surround contribution to the ganglion cell response during early dark-adaptation necessarily indicates a spatial re-organization of the receptive field. This may help explain the different time courses of dark-adaptation seen when using stimuli of different sizes in both psychophysical (e.g. Crawford, 1947; Arden & Weale, 1954; Blakemore & Rushton, 1965) and physiological tests (Barlow et al. 1957; Donner & Reuter, 1965). The question of differential adaptation of centre and surround as well as spatial consequences of bleaching adaptation will be treated in a further paper (Bonds & Enroth-Cugell, 1979). Late dark-adaptation. The time course of late rod dark-adaptation in the cat has been studied often, but comparison of results is complicated by inconsistencies in the definition of the bleaching exposures. Barlow et al. (1957) describe recovery of cat ganglion cell thresholds requiring almost 200 min after an exposure to '44 ft-c (on

GANGLION CELL DARK ADAPTATION

65

the retina) for 30 minutes'. The rod-cone break is seen at 60 and 100 min for blue and red test spots, respectively. Dodt & Elenius (1960) studied rod dark-adaptation in cats using the B-wave of the e.r.g. Their bleaching exposure (unspecified) was delivered via Maxwellian view. Using a blue test, the rod-cone break was found at about 12 min and the entire recovery lasted 70 min, suggestive of about a 60 % bleach as seen here (e.g. Fig. 6). Daw & Pearlman (1969) describe a cone plateau lasting 30 min (ganglion cell threshold) using a white test after a 5 min exposure to about 5x6 log td. Rodieck & Rushton (1976) found that after an 85 % bleach, rod log (ganglion cell) threshold fell linearly in time (cf. our Fig. 6) at a rate of about 1 log unit/tO min. This falls between 1 log unit/13-5 min (about 90 % bleach) and 1 log unit/9 min (60 % bleach) seen here. Thus, while figures do not compare exactly, there is reasonable agreement between these studies with respect to the rate of cat late rod dark-adaptation. A behavioural assessment of dark-adaptation in the cat (La Motte & Brown, 1970) shows the time required for recovery to be shorter than expected from the physiological results. Pre-adaptation was to 750 ft.-Lb. (dilated natural pupil; about 5-5 log td) for 5 min. Using a blue test, the rod cone break appeared after about 10 min, and full recovery was reported at about 35 min. Cat dark-adaptation may well be slower in general when measured under the conditions of physiological experiments; alternatively, the bleaching exposure in the behavioural study may not have been as effective as was expected. Relationship of pigment regeneration and dark-adaptation. Ever since it became possible to measure the time course of dark-adaptation and rhodopsin regeneration in man in the same experiment (Rushton, 1961 a), a good deal of evidence supportive of eqn. (1) (p. 57) has accumulated. In normal subjects the time domains for accurate densitometry and threshold measurements overlap minimally but even when extensive overlap is achieved by using as a subject a rod-monochromat, a linear relationship between log threshold and fraction of bleached pigment is described (Rushton, 1961b; Blakemore & Rushton, 1965; Alpern, 1971a, b). Electrophysiological evidence for this relationship seems strong as well. Dowling (1963) reports linearity between log threshold and pigment in rats over about a 4 log unit range. Dowling & Ripps (1970) have also tracked dark-adaptation in the skate eyecup after a flashed bleach and find both e.r.g. B-wave and ganglion cell log thresholds to be linear with pigment regeneration after the first 20 min. In isolated retina preparations (e.g. Weinstein, Hobson & Dowling, 1967; Baumann & Scheibner, 1968), where pigment does not regenerate, it is seen that the log of the difference between the final threshold reached and the original dark-adapted threshold is related linearly with the pigment bleached. The situation is complicated by the fact that in even the supportive cases mentioned above, the relationship is by no means general. For example, changing the size of the test flash used to determine a psychophysical dark-adaptation curve does not simply shift the curve vertically; it changes the shape of the curve as well (Arden & Weale, 1954; Rushton, 1961 a; Blakemore & Rushton, 1965). Test stimulus geometry most certainly has negligible effect on pigment regeneration. The period necessary for ganglion cell dark-adaptation in cat (80 min at least for strong bleaches in the present study), compared with the rather shorter interval 3

PHY 295

A. B. BONDS AND C. ENROTH-CUGELL required for pigment regeneration (30-35 min, Weale, 1953; Bonds & MacLeod, 1974) is incompatible with the linearity between log visual threshold and unregenerated pigment expressed in eqn. (1). While the evidence cited above in support of eqn. (1) is abundant, a lack of correspondence between threshold recovery and pigment regeneration during dark-adaptation in man has also been found. Pugh reports (1975b) that although the time constant for pigment regeneration in man remains fixed at about 6-7 min, recovery of log threshold after a strong bleach can have a time constant as high as 12 min and he has even observed a figure of 25 min when a very small test spot was used (personal communication). Similar departures in frog have been noted in physiological experiments. Although the proportionality holds when using the e.r.g. (Baumann & Scheibner, 1968), it fails for ganglion cell thresholds (Donner & Reuter, 1965). In axolotl isolated retina, rod receptors show proportionality of final log threshold with fractions bleached only between 10 and 5000 (Grabowski & Pak, 1975). Pigment regeneration in the rat requires 5-6 h (Tansley, 1931; Lewis, 1957) at which time, after a strong bleach, rat e.r.g. threshold is still elevated 2 log units (Dodt & Echte, 1961). In 1939, Granit, Munsterhjelm & Zewi described an experiment in which rhodopsin regeneration in the cat was measured via pigment extractions, together with determinations of e.r.g. response during dark-adaptation. Since they measured response amplitude rather than sensitivity, their findings cannot be compared directly with those discussed here. They found, however, that regeneration of rhodopsin took only about 30 min, while the electrical response after the same bleaching exposure continued to rise for at least an hour. Their primary conclusion was that 'the rise in sensitivity, as measured electrically during dark-adaptation, is not a simple function of the curve depicting visual purple regeneration... (but must be) due to some other secondary or intermediate process'. We are in full agreement with their statement. While eqn. (1) does appear to describe elevation of threshold after bleaching in some situations, it is not universally applicable. The exceptions to its validity are sufficiently numerous that at least a secondary mechanism must be invoked for a complete explanation of elevation of threshold following bleaching.

66

We want to thank many colleagues for helping us with this manuscript, in particular Ed Pugh, Donald MacLeod and Larry Pinto. John Trimble gave invaluable help with additional analyses of data for the revised manuscript. Toxiferine was kindly supplied by Hoffman-LaRoche of Nutley, N.J. This work was supported mainly by N.E.I. grant ROI EY00206, but also by 5K 03 TO-GM 00874 and the Rowland Foundation. Part of the work was done while A. B. Bonds held an N.I.H. post-doctoral fellowship (EY00775).

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