Photopigment Bleaching Research Articles

Measuring Rod and Cone Dynamics in Age-Related Maculopathy

A cathode-ray-tube (CRT) monitor-based technique was used to isolate clinically significant components of dark adaptation. The utility of the technique in identifying adaptation abnormalities in eyes with age-related maculopathy (ARM) is described. A CRT dark adaptometer was developed to assess cone and rod recovery after photopigment bleach. The following measures were obtained: cone recovery rate (R(c); in decades per minute) and absolute threshold (Tf(c); log candelas per square meter), rod recovery rate (R(r); decades per minute), and rod-cone transition (rod-cone break [RCB], in minutes). These components were isolated by appropriately selecting stimulus size, stimulus location, pigment bleach, and test duration and by coupling the CRT with judiciously selected neutral-density (ND) filters. The protocol was developed by using 5 young observers and was tested on 27 subjects with ARM in the study eye and 22 age-matched control subjects. The parameters necessary for effective isolation of cone and early phase rod dark adaptation were a 2.6 ND filter (for a standard CRT monitor, 0.08-80 cd . m(-2) luminance output); a 4 degrees foveated, 200-ms, achromatic spot; approximately 30% pigment bleaching; and a 30-minute test duration. These settings returned obvious rod and cone recovery curves in control and ARM eyes that were compatible with conventional test methods and identified 93% of participants with ARM as having delayed dynamics in at least one of the parameters. Cone recovery dynamics were significantly slower in the ARM group when compared with age-matched control subjects (R(c), 0.99 +/- 0.35 vs. 2.63 +/- 0.61 decades . min(-1), P < 0.0001). Three of the 27 eyes with ARM did not achieve RCB during the allowed duration (30 minutes). The remaining eyes with ARM (n = 24) exhibited a significant delay in rod recovery (R(r)(,) ARM, 0.16 +/- 0.03 vs. controls, 0.22 +/- 0.02 decades . min(-1), P < 0.0001) and the average time to RCB (+/-SD) in the ARM group was significantly longer than in the control subjects (19.12 +/- 5.17 minutes vs. 10.40 +/- 2.49 minutes, P < 0.0001). The CRT dark-adaptation technique described in this article is an effective test for identifying abnormalities in cone and rod recovery. Slowed cone and rod recovery and a delayed RCB were evident in the eyes with ARM. The test method is potentially useful for clinical intervention trials in which ARM progression is monitored.

Rodent electroretinography: Methods for extraction and interpretation of rod and cone responses

The flash electroretinogram (ERG) represents a serial ensemble of neural responses that can be used to objectively evaluate retinal function on a layer-by-layer basis. In this review, the seminal concepts of Granit are developed within the modern context to demonstrate how the ERG waveform can be decomposed to isolate the activity of individual neural populations and their circuitry. The contribution of rods and cones to the ERG waveform can be precisely defined with simple methods that yield the veridical cone response, which allows identification of rod-isolated components. This knowledge will afford an enhanced capacity to understand retinal development and ageing as well as to interpret the effects of insult, genetic manipulation and disease processes on photoreceptor and neuron-specific components. This review integrates conclusions drawn from a large body of past work and presents new data that enables the provision of detailed methodology for ERG assessment in rodents. Emphasis is placed on protocols that allow efficient acquisition of useful information for the major ERG components with minimal complexity. In particular, specific guidelines for the isolation of rod and cone contributions from the full-field ERG in rodents are provided. This is complemented with detailed and novel methodology for determining parameters that describe individual neuronal generators of rod and cone responses. The effect of stimulus energy on the kinetics of ERG response recovery and photopigment bleaching and regeneration are also discussed. The guidelines presented here are applicable to a wide range of investigations of retinal disease in rodent models.

Simultaneous measurement of current and calcium in the ultraviolet‐sensitive cones of zebrafish

In rods and visible cone photoreceptors, multiple measurements cannot be made of intracellular Ca2+ concentration from the same cell using fluorescent dyes, because a single exposure of the measuring light bleaches too large a fraction of the rod or cone photopigment. We have therefore identified and characterized UV-sensitive cones of the zebrafish, whose wavelength of maximum sensitivity is at 360 nm which is far enough from the wavelength of our measuring light (514.5 nm) so that it has been possible to make multiple determinations of photocurrent and Ca2+ concentration from the same cells. We show that for a limited number of measurements, for which the bleaching of the cone photopigment is too small to affect flash kinetics, the outer segment Ca2+ concentration closely follows the wave form of the flash response convolved with the dominant time constant for Ca2+ removal by Na+-Ca2+-K+ exchange. For a larger number of measurements, significant acceleration of the response kinetics by pigment bleaching inevitably occurs, but the Ca2+ concentration nevertheless rises and falls in approximate agreement with the flash wave form. During exposure to steady background light, the Ca2+ concentration falls in proportion to the steady-state current for dim backgrounds at all times and for bright backgrounds at steady state. At early times following the onset of bright backgrounds, however, the Ca2+ concentration is markedly higher than expected from the current of the cone. We show this to be the result of light-dependent Ca2+ release by bright background light, which can be abolished by pre-exposure of the cone to the membrane-permeant acetoxymethyl ester of the Ca2+ chelator BAPTA. Our results therefore demonstrate that the cone outer segment Ca2+ concentration is predominantly a function of the rate of influx and efflux of Ca2+ across the plasma membrane, but that a release of Ca2+ in bright light most probably from buffer sites within the cell can transiently elevate the Ca2+ concentration above the level expected from the open probability of the light-dependent channels.

Human cone light adaptation: From behavioral measurements to molecular mechanisms

The ability of the cone visual system to regulate its sensitivity from twilight to bright sunlight is an extraordinary feat of biology. Here, we investigate the changes in visual processing that accompany cone light adaptation over a 5 log10 unit intensity range by combining measures of temporal sensitivity made in one eye with measures of the temporal delay between the two eyes in different states of adaptation. This combination of techniques, which provides more complete information than has been available before, leads to a simple model of steady-state light adaptation. At high light levels, visual sensitivity is maintained mainly by photopigment bleaching. At low-to-moderate light levels, it is maintained by trading unwanted sensitivity for speed and by an additional process that paradoxically increases the overall sensitivity as the light level rises. Each stage of the model can be linked to molecular mechanisms within the photoreceptor: The speeding up can be linked to faster rates of decay of activated molecules; the paradoxical sensitivity increases can be linked to faster rates of molecular resynthesis and to changes in channel sensitivity; and the sensitivity decreases can be linked to bleaching. Together, these mechanisms act to maintain the cone visual system in an optimal operating range and to protect it from overload.

Evaluation of retinal function using the Dynamic Focal Cone ERG

The development of effective means of assessing visual function in retinal disease holds the key to improved understanding of pathogenesis, and better monitoring of treatment outcomes. In diseases such as age-related macular degeneration, in which the primary locus of dysfunction is the outer retina, tests which provide a direct measure of the functional integrity of the photoreceptor/retinal pigment epithelium (RPE) complex are of great importance. Recovery of retinal function following adaptation to a bright light requires the healthy function of photoreceptors, RPE, Bruch's membrane and choroidal circulation, making an assessment of this recovery a potentially useful clinical tool. However, current techniques are either subjective in nature, or are influenced by post-retinal processing of visual information. This report describes a novel technique, the 'Dynamic Focal Cone Electro-retinogram (ERG)', which allows direct, objective assessment of the recovery of macular function following photopigment bleach. A series of 41 Hz ERGs was recorded, and ERG amplitude was plotted as a function of time following cessation of the bleach. Normative data was collected from 10 healthy subjects. For all subjects, there was no measurable ERG immediately after the bleach, but the amplitude had returned to a pre-bleach level within 4 min. The amplitude recovery data were adequately described both by an exponential recovery function and by a model based on a rate-limited recovery process. We conclude that this technique provides a clinically applicable, objective measure of outer retinal recovery.

Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Light adaptation and photopigment bleaching in cone photoreceptors in situ in the retina of the turtle

Light adaptation and photopigment bleaching in cone photoreceptors were studied in the intact, superfused retina of the turtle (Pseudemys scripta elegans). A new method for measuring changes in the photopigment of cones is described. Action spectrum measurements indicate that the signals arise from the red-sensitive cones. Measurements of steady-state bleaching are well described by the monomolecular bleaching equation with a half-bleaching constant of about 5.5 log photons sec-1 microns-2. Quantitative data on light adaptation were obtained by intracellular recording from 15 red-sensitive cones over nearly 8 decades of background illumination obtained from a helium-neon laser (632.8 nm). The steady-state membrane potential, Rs, and the rate of photoisomerization of the photopigment, Pi, rose in parallel with background illumination and then stabilized over the upper 4 decades of illumination. These results are described by the relation Rs = k Pi0.27, and suggest that about 5 x 10(6) photoisomerizations sec-1 lead to the closure of half the cone's light-sensitive channels in the steady state. A full range of decremental and incremental flashes was used to investigate stimulus-response relations. Cones tended to generate responses of approximately constant amplitude to flashes of constant contrast over a substantial range of contrast (< or = 3 x) and background illumination (approximately 3-4 decades). This suggests that a substantial component of contrast constancy in vertebrate vision may originate in cones. Over nearly 7 decades, the small-signal step sensitivity was found to conform closely to Weber's law (sensitivity is inversely proportional to background illumination). Thus, Weber's law extends into the ultra-high-intensity realm, some 3 decades higher than previously known for vertebrate cones. Over the upper 3-4 decades of illumination, Weber's law behavior can be explained by the depletion of photopigment (reduced probability of the photon catch). There remains a substantial low-intensity domain for which light adaptation and Weber's law behavior are presumably mediated by other mechanisms within the cone. These might be the calcium- and/or cGMP-dependent mechanisms recently suggested by others.

Light-activation of Teleost Rod Photoreceptor Elongation

Rod photoreceptors in the retinas of teleost fish undergo changes in cell length in response to changing ambient light intensities. In the dark rods shorten and in the light rods elongate. These movements are mediated by actin-dependent processes which occur in the ellipsoid and myoid of the inner segment. As an approach to examining the underlying intracellular signaling pathways that link light absorption to actin-dependent motility in the inner segment, we have investigated the quantitative aspects of the light stimulus required to activate elongation in isolated rod inner/outer segments (RIS-ROS) of the green sunfish (Lepomis cyanellus).The intensity thresholds and strength-duration characteristics of the light stimulus required to activate teleost rod elongation were found to differ from those reported to activate vertebrate rod membrane hyperpolarization. In response to brief pulses of light, RIS-ROS elongated in a graded manner, both as a function of increasing light pulse intensity and light pulse duration. Half maximal activation of light-induced RIS-ROS elongation was produced by a stimulus of roughly 6 × 1015 photons cm-2, which is calculated to bleach approximately 20% of the photopigment molecules in green sunfish rod outer segments. This degree of photopigment bleach is approximately 6-7 orders of magnitude greater than that required to elicit half maximal changes in membrane potential in other vertebrate rod preparations. Furthermore, the reciprocal relationship between light pulse intensity and duration in eliciting an equal elongation response held for relatively long light pulse durations. There was an approximately equal elongation response to light pulse durations of 15, 120, and 600 sec, that delivered roughly equal quanta. Over these light pulse durations, the temporal integration function is described by a single slope of -0·9 on log-log coordinates of stimulus intensity and duration. The integration time of the rod elongation response lasts for light pulse durations at least as long as 600 sec, which is approximately 3 orders of magnitude longer than that reported for rod membrane hyperpolarization. The high threshold and long integration time of the photoreceptive mechanisms mediating rod elongation indicate that the intracellular signals that regulate motility dramatically diverge from those regulating rod membrane hyperpolarization. A further implication of these results is that vertebrate rod photoreceptors possess different intensity thresholds and temporal processing mechanisms in the light-activation of distinct cell functions such as membrane hyperpolarization and cell elongation.

Background and bleaching adaptation in luminosity type horizontal cells in the isolated turtle retina.

1. The effects of background illumination and bleached photopigment on luminosity type horizontal cells were studied in the isolated turtle retina. 2. Background illumination, which produced less than 60% bleaching, hyperpolarized and desensitized the horizontal cells to a degree which depended upon the background intensity. The desensitization of horizontal cells by these backgrounds is described by a Weber-Fechner type relationship. This desensitization primarily reflects the activation of a 'gain reduction' mechanism and cannot be accounted for by 'response compression'. 3. Following the termination of these backgrounds, horizontal cell sensitivity partially recovered but did not return to the pre-background, dark-adapted level. This desensitization was attributed to the presence of bleached photoproducts which were produced by the background exposure. 4. Application of very bright backgrounds caused the horizontal cells to initially hyperpolarize, and then to gradually depolarize towards the dark-adapted level along an exponential time course which appeared to reflect the decreased quantal catching associated with very high levels of photopigment bleaching. 5. From the time constant of the exponential decay of horizontal cell potential during the bright background illumination, the photosensitivity to bleaching of the cone photopigment was determined to be 4.5 x 10(7) effective quanta (633 nm) microns-2. 6. After termination of bright backgrounds which bleached more than 99% of the cone photopigment, the horizontal cell sensitivity increased linearly with time and after 25 min reached a level which was about 15% of the pre-background sensitivity. 7. Bleached photopigment reduces light sensitivity via at least two different mechanisms. For moderate degrees of bleaching (less than 95%), the presence of bleached photoproducts plays the major role in sensitivity control, producing a desensitization which is logarithmically related to the fraction of bleached pigment. During extensive bleaching (greater than 99%), the contribution of reduced quantal catching to sensitivity control becomes apparent and produces an additional loss in sensitivity which is linearly related to the fraction of unbleached pigment present.

Direct psychophysical estimates of the cone-pigment absorption spectra

The absorption spectra of the long- and medium-wavelength-sensitive cone photopigments were derived by determining the spectra that best accounted for either the individual differences in the Stiles-Burch 10 degrees color matches [Opt. Acta 6, 1 (1959)] or the changes in color matches at high light levels due to photopigment bleaching [Vision Res. 20, 23 (1980)]. The estimates were made by finding the best-fitting coefficients for an 11th-order polynomial function of wavelength, with no requirement that the resulting sensitivities be consistent with the color-matching functions. The estimates are independent of the scaling effects of any inert screening filters and therefore directly reflect the photopigment sensitivities. The spectra implied by the differences in the matches are similar to the absorption spectra of Smith et al. [Vision Res. 16, 1087 (1976)], which were used as initial estimates. However, the peak sensitivity of the required long-wavelength-sensitive pigment is shifted toward slightly longer wavelengths.

Foveal cone photopigment bleaching in central serous retinopathy

Color-matching techniques were used to follow the course of central serous retinopathy in thirteen patients. During periods of neurosensory detachment?, cone optical densities were low. In some patients optical densities remained low following clinical resolution of the disease. In addition, abnormalities of bleaching were observed for one patient in one eye that appeared clinically unaffected during a period of detachment of the fellow eye. Analysis of the data indicates that for longitudinal measurements of optical density, standard Nagel anomaloscope matches can provide an accurate estimate of changes in foveal optical density, although they cannot provide a measure of the absolute optical density.

Color-prediction discrepancies and differential chromaticity thresholds with photopigment bleaching.

The differential chromaticity thresholds of three color stimuli have been determined from metameric matches carried out during the cone-plateau period after photopigment bleaching. The results are compared with some recently published ones [E. Hita, L. Jiménez del Barco, and J. Romero, J. Opt. Soc. Am. A 3, 1203 (1986)] obtained under similar experimental conditions but without prior photopigment bleaching. The possible effects of rod intrusion on color-prediction discrepancies are discussed in the light of both sets of results. Under our experimental conditions, with foveal fields of 3 deg, luminance within the low photopic level, and sets of primary colors produced by filters of differing spectral bandwidth, the rod intrusion effect can be ruled out as a possible cause of color-prediction discrepancies, although it does appear to produce variations in the sizes of the discrimination ellipses and in their orientation.

Color matching at high illuminances: the color-match-area effect and photopigment bleaching.

We evaluated whether a self-screening hypothesis can account for changes in red-green color matches with changes in retinal illuminance and changes in the size of the matching field. The dependence of the color match on field size measured at moderate illuminances was not present at high illuminances. For color matches made with normal pupil entry, there was no need to postulate any factors other than self-screening to account for the changes with either illuminance or field size. The self-screening model allowed us to estimate the optical density of the foveal cones and the retinal illuminance that caused half of the photopigment to be bleached at equilibrium. These estimates were in quantitative agreement with previous estimates made using other techniques. We also found that the change in a color match with increasing illuminance was inconsistent with first-order kinetics.

Cone response latency and log sensitivity: Proportional changes with light adaptation

Field adaptation causes proportional changes in cone response latency and log sensitivity for (1)psychophysical reaction times to threshold stimuli, (2)intracellular recordings of turtle cone responses, (3)local electroretinogram recordings of monkey cone responses and (4)theoretical impulse response functions derived from psychophysical flicker thresholds. To a good approximation, this relationship holds for all levels of cone adaptation except those that cause significant photopigment bleaching. For L and M cones, the proportionality constant between latency and log sensitivity appears to be cone-specific and largely independent of spatiotemporal stimulus parameters. For S cones, the proportionality constant depends to some extent on influences from other cone types.

Primate cone sensitivity to flicker during light and dark adaptation as indicated by the foveal local electroretinogram

During light and dark adaptation the primate foveal local electroretinogram (LERG) was monitored in response to yellow sinusoidally flickering stimuli. Since the foveal LERG is dominated by the late receptor potential (LRP) under these stimulus conditions, we were most likely monitoring cone photoreceptor activity. (1) Relative threshold vs intensity ( tvi) functions were obtained for stimuli flickering at 40, 20 and 5 Hz. Each of these functions achieves a unit slope at moderate to high intensities. Changes in phase relations between the stimulus and response occur only at those adaptation levels where photopigment bleaching is relatively ineffective as an adaptational mechanism. (2) The time course of dark adaptation was monitored for stimuli flickering at various frequencies between 5 and 50 Hz. At frequencies between 5 and 20 Hz, the time course of recovery of log relative sensitivity follows a nonexponential course; a break at 1.5 min sometimes occurs in the 5 Hz function. At higher frequencies the functions are exponential with time constants smaller than found for photopigment regeneration. These results are inconsistent with a simple linear relation relating log sensitivity and photopigment regeneration. (3) A Crawford type of transform indicates a non-equivalence between the effects of prior bleaching and steady-state light adaptation. (4) Absolute amplitude sensitivity functions obtained at various times after a bleach have low and high frequency branches. After compensating for the reduction in photon catch due to photopigment depletion the high frequency sensitivity changes little during dark adaptation, but the low frequency sensitivity changes markedly.1

The effects of background illumination on the photoresponses of red and green cones.

1. The photoresponses of light- and dark-adapted red and green cone photoreceptors were recorded intracellularly in the retina of the turtle, Pseduemys scripta elegans. Background illumination produced similar effects on both types of cones. 2. In response to the onset of a prolonged, steady background illumination the cone initially hyperpolarized to a peak which then sagged back to a steady-state polarization that was typically about one half the initial peak amplitude. This sag was observed for all backgrounds studied (dim as well as bright). 3. A resensitization was observed concomitantly with this sag; both the maximum increment and decrement responses grew in amplitude as light-adaptation proceeded. After about 2--3 min of background illumination, the amplitudes of these responses stabilized. 4. The dark-adapted cone produced graded responses to test pulses over a range of intensities spanning about 3.5 log units. The amplitudes of these responses were well fit by the relationship V = I.Vm/(I + sigma). 5. After 2--3 min of background illumination, 500 msec test pulses either brighter or dimmer than the background intensity were substituted for the background. The light-adapted intensity-response curves constructed from this data were similar to the dark-adapted curve but were shifted horizontally and slightly vertically, so that they still spanned about 3.5 log units of intensity. Thus, in the light-adapted cone, graded responses were elicited by a range of bright test pulses which would have produced saturated responses when delivered to the dark-adapted cone. 6. The 'off response' observed at the offset of the background became faster as the background intensity was increased. It also became faster with time following the onset of any particular background intensity. 7. It was concluded that cone sensitivity during any state of light-adaptation is determined by two mechanisms; response compression resulting from the instantaneous non-linearity between 'internal transmitter' concentration and membrane potential and a more active 'cellular adaptation' mechanism which is manifest as a shift in the intensity-response curve. In the steady-state condition of light-adaptation, most of the sensitivity changes are a result of the cellular adaptation mechanism. 8. Photopigment bleaching caused by the backgrounds, negative feed-back from horizontal cells and voltage dependent mechanisms in the cones could not account for this cellular adaptation. These effects of background illumination were interpreted in terms of the 'internal transmitter' hypothesis of phototransduction.

Effects of chromatic adaptation on color discrimination and color appearance

Color discrimination and color appearance were examined at a number of chromaticities along an equal luminance line running from red to green as a function of adaptation to several lights along the same line. Like K. J. W. Craik's results for brightness discrimination [ J. Physiol. 92, 406–421]. color discrimination proved best for chromaticities nearest the adapting light with a worsening of discrimination (relative to neutral adaptation) for more distant chromaticities. This result is explicable in terms of a sliding of the “working range” of red-green opponent cells along an axis of chromaticity. Consistent with this interpretation are the systematic changes in color appearance brought about by preexposure to chromatic light. An ancillary part of the investigation revealed that a flickering adapting light produces lesser adaptive shifts than steady light of the same time average luminance. This latter result rules out the possibility that photopigment bleaching, under these conditions, plays more than a minimal role in controlling the sensitivity of the cones or more proximal neurons.

The photopigment bleaching hypothesis of complementary after-images: A psychophysical test

Normal observers color-adapted simultaneously to two spatially distinct sequences of light stimulation, equivalent in their bleaching effects but complementary to one another in hue. Following the adapting period, observers reported on the color appearance of the two adjacent after-images seen against a neutral background. The nonequivalence of the two after-images argues against the explanation of complementary after-images based on photopigment bleaching; in addition, the fact that the two after-images are mutually complementary further restricts the class of (neural) adaptation hypotheses.

Net Electric Charge on Photopigment Molecules and Frog Retinal Receptor Disk Membrane Structure

The photopigment molecules in frog retinal receptor disk membranes protude some 50-65% of their molecular diameter ( approximately 42 A) into the aqueous surface layer of the disk membrane, depending on whether the photopigment is bleached, while the remainder is embedded in the lipid core of the membrane. In order to determine whether the presumably polar groups covering this surface protruding into the aqueous phase possessed net electric charge, we collected X-ray diffraction data from the photopigment molecules in wet pellets of oriented disk membranes as a function of the pH and ionic strength of the sedimentation medium. The Fourier analysis applied to this data provided average nearest neighbor separations for the photopigment molecules for their planar arrangement in the disk membranes. Changes in the average separation of photopigment molecule nearest neighbors as a function of pH, ionic strength, and photopigment bleaching indicated that photopigment molecules possess negative net electric charge, that this net electric charge occurs in the aqueous surface layer of the disk membrane, and that this net charge is reduced on photopigment bleaching. This polar portion of the photopigment molecule may thereby determine the location of the photopigment molecules relative to the lipid core and other photopigment molecules in the disk membrane. In addition, the orientation (dichroism) of the photopigment relative to an axis normal to the plane of the disk membrane and the bleaching-dependent "sinking" of the photopigment molecule into the lipid core of the disk membrane may be accounted for.