Dark-adapted Eye Research Articles

Characterization of protein phosphatases type 1 and type 2A in Limulus nervous tissue: their light regulation in the lateral eye and evidence of involvement in the photoresponse.

The activities of both protein phosphatases and protein kinases are responsible for the transient changes in the levels of phosphorylation and probably the functions of protein intermediates involved in the biochemical and physiological mechanisms underlying the photoresponse in photoreceptor cells from both vertebrate and invertebrate organisms. Of the known protein serine/threonine phosphatases, various forms of type 1 (PP 1) and type 2A (PP 2A) protein phosphatases are responsible for dephosphorylating many of the known phosphoproteins including those involved in photoreceptor cell function. In this report, we provide biochemical evidence for both PP 1- and PP 2A-like activities in the visual and nonvisual tissue of the horseshoe crab, Limulus polyphemus, that membrane and soluble forms of both enzymes are present, and that the activities of both enzymes are greater in light- than in dark-adapted lateral eyes. These activities were characterized using glycogen phosphorylase a, a substrate for both PP 1 and PP 2A, and various protein phosphatase inhibitors, including okadaic acid. We also report that okadaic acid, at concentrations required to inhibit PP 1, inhibited physiological functions of photoreceptor cells from the ventral eye, causing a delayed reduction of the resting membrane, and slowing and reducing light responses.

Response linearity and kinetics of the cat retina: the bipolar cell component of the dark-adapted electroretinogram.

The electroretinogram (ERG) of the dark-adapted cat eye in response to brief ganzfeld flashes of a wide range of intensities was recorded after intravitreal injection of n-methyl DL aspartate (NMDLA, cumulative intravitreal concentration of 1.3-3.9 mM) to suppress inner-retinal components, and after intravitreal DL or L-2-amino-4-phosphonobutyric acid (DL-APB, 1-3 mM; L-APB, 1.2 mM) and 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX, 40-60 microM), to suppress all post-receptoral neuronal responses. Rod PII, the ERG component arising from rod bipolar cells, was derived by subtracting records obtained after APB and CNQX from post-NMDLA records. When we measured the derived response at fixed times after the stimulus, we found that PII initially increased in proportion to stimulus intensity without any sign of a threshold. The leading edge of PII at early times after the stimulus, when the response was still small, was well described by V(t) = kI(t-td)5 where k is a constant, I is the intensity of the stimulus, and td is a brief delay of about 3 ms. Correspondingly, the time for the response to rise to an arbitrary small criterion voltage Vcrit was adequately fitted by tcrit = td + (Vcrit/kI)1/5. The time course of the leading edge of the PII response can be interpreted to indicate that the mechanism generating PII introduces three stages of temporal integration in addition to the three stages that are provided by the mechanism of the rod photoreceptors. This finding is consistent with the operation within the rod bipolar cell of a G-protein cascade similar to that in the rods.

Effect of 2-amino-4-phosphonobutyrate on ERG OFF-response after glycinergic and GABAergic blockade

Superfusion with 200 μM 2-amino-4-phosphonobutyrate (APB) of dark and chromatically adapted frog eyecups caused marked potentiation of the ERG OFF-response ( d-wave). Blockade of the glycinergic synapses by strychnine did not change this effect at all. Blockade of the GABAergic synapses by picrotoxin slightly diminished the effect of APB in chromatically-adapted eyes with isolated cones' activity, and did not change it in dark-adapted eyes. The results indicate that the action of APB on ERG OFF-response does not depend significantly on GABAergic and glycinergic neurotransmission in frog retina.

Light and dark adaptation of visually perceived eye level controlled by visual pitch

The pitch of a visual field systematically influences the elevation at which a monocularly viewing subject sets a target so as to appear at visually perceived eye level (VPEL). The deviation of the setting from true eye level average approximately 0.6 times the angle of pitch while viewing a fully illuminated complexly structured visual field and is only slightly less with one or two pitched-from-vertical lines in a dark field (Matin & Li, 1994a). The deviation of VPEL from baseline following 20 min of dark adaptation reaches its full value less than 1 min after the onset of illumination of the pitched visual field and decays exponentially in darkness following 5 min of exposure to visual pitch, either 30 degrees topbackward or 20 degrees topforward. The magnitude of the VPEL deviation measured with the dark-adapted right eye following left-eye exposure to pitch was 85% of the deviation that followed pitch exposure of the right eye itself. Time constants for VPEL decay to the dark baseline were the same for same-eye and cross-adaptation conditions and averaged about 4 min. The time constants for decay during dark adaptation were somewhat smaller, and the change during dark adaptation extended over a 16% smaller range following the viewing of the dim two-line pitched-from-vertical stimulus than following the viewing of the complex field. The temporal course of light and dark adaptation of VPEL is virtually identical to the course of light and dark adaptation of the scotopic luminance threshold following exposure to the same luminance. We suggest that, following rod stimulation along particular retinal orientations by portions of the pitched visual field, the storage of the adaptation process resides in the retinogeniculate system and is manifested in the focal system as a change in luminance threshold and in the ambient system as a change in VPEL. The linear model previously developed to account for VPEL, which was based on the interaction of influences from the pitched visual field and extraretinal influences from the body-referenced mechanism, was employed to incorporate the effects of adaptation. Connections between VPEL adaptation and other cases of perceptual adaptation of visual direction are described.

Investigations of pigment granule transport systems in Gonodactylus oerstedii (Crustacea: Hoplocarida: Stomatopoda)

Pigment granules within retinular cells of the stomatopod crustacean, Gonodactylus oerstedii, undergo rapid, radial migrations in response to changes in ambient lighting. Light stimulates centripetal migration of pigment granules towards the microvillar rhabdomeres where they absorb and scatter incoming light, analogous in function to the closure of a pupil. In the dark, pigment granules disperse centrifugally away from rhabdoms, thereby opening the pupil. Two populations of microtubules in retinular cells of G. oerstedii are appropriately oriented for participation in pigment granule migration. We investigated the possibility that microtubules are involved in pigment granule migration by subjecting animals to low temperature (which can depolymerize some microtubules) and determining the effects of low temperature on pigment granule position and microtubule density within retinular cells. When temperature was decreased, pigment granules in previously dark-adapted eyes aggregated around rhabdoms, in the light-adapted configuration. Lowering the temperature also decreased the density of palisade microtubules, which extend longitudinally in retinular cells along the subrhabdomeric palisade vacuole. These changes reversed when temperature increased. We present a model for pigment granule migration based on the idea that the position of pigment granules in retinular cells is dependent upon the presence of intact palisade microtubules.

Visual pigment, dark adaptation and rhodopsin renewal in the eye of Pontoporeia affinis (Crustacea, Amphipoda)

The benthic amphipod Pontoporeia affinis lives in the Baltic sea and in northern European lakes in an environment where very little light is available for vision. The eyes, consisting of 40–50 ommatidia, are correspondingly modified. Microspectrophotometric recordings on isolated eyes show the presence of at least two kinds of screening pigments in the ommatidia with maxima at 540–580 nm and 460–500 nm. Difference spectra obtained from the rhabdoms after exposure to red and blue light, respectively, give evidence of a single rhodopsin with its maximum at 548 nm and a 500-nm metarhodopsin. In ERG recordings sensitivity in the dark-adapted state, after saturating exposures to blue and to red light, stabilizes at levels determined by the rhodopsin concentration. No change is observed during 10–14 h after the beginning of dark adaptation. However, using animals pre-exposed with a strong red light and then kept in darkness, it is found that after a delay of 20–40 h sensitivity of the dark-adapted eye begins to increase and finally, after 5–6 days reaches a level corresponding to 100% rhodopsin. Thus, a slow renewal of rhodopsin appears to occur in darkness, where a photoisomerization of metarhodopsin is excluded.

The vision of the snail: the spectral sensitivity of the dark-adapted eye.

The spectral sensitivity of the dark-adapted eye of the snail Helix lucorum L. was investigated in semi-intact "optic cupoptic nerve-cerebral ganglion" preparations. The data of the recording of the electroretinogram in response to monochromatic radiation of varied intensities were used to plot the spectral sensitivity functions. The averaged spectral sensitivity function is well described by a Dartnall nomogram for the photopigment rhodopsin with a sensitivity peak at 496 nm.

Screening-Pigment Migration in the Octopus Retina Includes Control by Dopaminergic Efferents

ABSTRACT The extent of screening-pigment (SP) migration in the intact octopus retina and the amplitude of the early receptor potential (ERP) correspond with the degree of adaptation to light or darkness. The light-adapted retina has SP granules concentrated in an apical layer, at the tips of the photoreceptor rhabdoms and supporting cells, and the ERP is barely detectable. In the fully dark-adapted retina, the SP granules are mostly at the base of the rhabdoms, and the ERP is at its maximum. Retinae at intermediate stages, between the fully dark-and light-adapted states, show corresponding intermediate stages of SP migration and ERP amplitude. A series of experiments demonstrates the effects on SP migration of the efferent nerves, which form a subset of fibres in the optic nerves. When the optic nerves to one half of the retina have been severed, there is a dramatic difference in the distribution of SP in areas of the retina (of the dark-adapted eye) connected with severed or intact nerves: apical versus basal, respectively. On incubation of a light-adapted retina with 5 μmol l−1 dopamine, but not with other catecholamines or other putative neurotransmitter substances, SP migrates basally and the ERP is significantly larger than for controls. In octopuses treated with reserpine, SP stays in an apical location and the ERP remains very small, regardless of the state of adaptation and of whether the optic nerves are intact. It is concluded that dopaminergic efferents from the optic lobes effect dark-adaptational SP migration in the cephalopod retina. The arrival in the retina of efferent signals that effect adaptational changes through the mediation of dopamine is a remarkable analogue of the vertebrate system.

Extracellular dopamine concentration in the retina of the clawed frog, Xenopus laevis.

Dopamine reaches targets in the outer retina of the clawed frog (Xenopus laevis) by diffusion from a network of dopaminergic cells and processes located predominantly at the junction of inner nuclear and inner plexiform layers. We obtained values for the steady-state release, uptake, and extracellular concentration of dopamine in the retina by a combination of HPLC (with electrochemical detection), scintillation spectroscopy, and fast-scan cyclic voltammetry. Vitreal concentrations of dopamine varied from 564 +/- 109 nM in light-adapted eyes near the time of subjective dawn to 156 +/- 12 nM in dark-adapted eyes. The data are consistent with a simple model for steady-state dopamine diffusion from an appropriately sited thin-sheet source. This model was used to generate a profile of extracellular dopamine concentration as a function of retinal depth. The model predicted an increase in the dopamine concentration from the vitreous to the layer of dopaminergic cells, remaining constant from that layer to the distal tips of the photoreceptors. This prediction was borne out by comparing fast-scan voltammetric measures of dopamine at the distal tips of the receptors with the vitreal concentrations determined by HPLC using electrochemical detection.

Neuromodulation of pigment movement in the RPE of normal and 6-OHDA-lesioned goldfish retinas.

The role of dopamine as the endogenous signal-initiating light-dependent changes in the distribution of pigment granules in goldfish retinal pigment epithelium was investigated. In normal retinas, light adaptation resulted in the dispersion of pigment granules. This effect of light was mimicked by the intraocular injection of dopamine or serotonin, which is thought to increase endogenous dopamine release, into dark-adapted eyes. The effect of light, dopamine, or serotonin on dark-adapted retinas was blocked by the dopamine receptor antagonists haloperidol and sulpiride. However, lesioning the endogenous source of retinal dopamine, by prior intraocular injection of 6-hydroxydopamine (6-OHDA), did not block the dispersion of pigment granules in light-adapted retinas. No significant differences in pigment dispersion were noted between unlesioned and lesioned light- or dark-adapted retinas. However, the effect of light on pigment dispersion was no longer blocked by haloperidol or sulpiride in 6-OHDA lesioned animals. Dopamine and serotonin mimicked the effect of light when injected into lesioned dark-adapted eyes, but their effects were also not blocked by haloperidol or sulpiride. These results suggest that dopamine, acting on D2 receptors, is sufficient to induce pigment migration in unlesioned animals. In 6-OHDA-lesioned animals, however, pigment migration is mediated by a receptor mechanism other than D2.

Mannose receptor-mediated phagocytosis in RPE cells and macrophages

A number of significant age-related changes were found to occur in the retinal pigment epithelium (RPE) of normal pigmented ACI rats. Age-related morphological changes in the RPE included: changes in RPE cell height, an increase in lipofuscin content, alterations in the apical microvilli and basal infoldings, and an apparent thickening of the RPE basal lamina. Unusual banded structures were often seen within the basal infoldings of the RPE in senescent but not in young adult rats. Lipid droplets were relatively common in the RPE of senescent rats, but were quite rare in 4- and 11-month-old animals. In addition, contact between the RPE apical microvilli and the photoreceptor outer segments appeared to become less intimate with age.Phagocytosis of rod outer segment discs appeared to be reduced between 4 and 32 months of age. This reduction was greater than could be accounted for solely by a concomitant age-related decrease in the number of photoreceptors per unit retinal length. RPE-choroid acid phosphatase activity (a lysosomal marker), on the other hand, did not appear to change with age.The molar ratio of vitamin A-palmitate to vitamin A-stearate in the RPE-choroid rose significantly with age, from about 1·85 at both 4 and 11 months, to over 2·5 at 28 months. At each age, the palmitate- to stearate-ester ratio was the same in the dark-adapted and light-adapted eyes.

Effects of light and darkness on pH outside rod photoreceptors in the cat retina

We recorded pH in the extracellular space surrounding rod photoreceptors in the dark-adapted eye of the cat and during illumination with double-barreled H +-selective microelectrodes. A pH of 7·17 was recorded in the vitreous at the retinal surface of the dark-adapted eye and this became more alkaline during light adaptation. In dark adaptation, a pH close to 7·00 was recorded in a region of maximal acidity in the extracellular space surrounding rods in the outer nuclear layer (ONL), pH steeply alkalinized as the microelectrode was moved more distally towards the retinal pigment epithelium (RPE), and almost reached the pH of the arterial blood at the apical surface of the RPE. Illumination produced an intraretinal alkalinization that was largest (up to 0·2 pH units) in the ONL, maximal in amplitude at rod-saturating intensities, and that was sustained during steady background illumination. The light-evoked alkalinization was relatively slow in onset, having a time constant ( 1 e ) of 64 sec, and took 8–12·5 min to return to the dark-adapted level after the offset of maintained illumination. These results show that acid production by cat rods is highest in the dark, reflecting a high rate of energy metabolism, and suggest that glycolysis is required to support the dark current. Illumination, by suppressing both glycolysis and respiration, alkalinizes the extracellular space surrounding rods. The substantial change in pH outside rods from dark to light could alter pH dependent properties of the interphotoreceptor matrix.

Changes in sensitivity of the dark-adapted eye during concurrent light adaptation of the other eye.

Thresholds for detection of light by a dark-adapted test eye were measured while the other, non-test eye was either similarly dark adapted or while it was exposed to an intense red adapting field. An interocular effect that depends on the retinal location of the test was found: compared to the threshold during binocular dark adaptation, sensitivity decreased during contralateral light adaptation when the test was presented to the foveola and up to 4 deg above it; but sensitivity increased when the test was between 7 and 12 deg, showing a reversal at 5 deg.

Influence of rod adaptation upon cone responses to light offset in humans: I. Results in normal observers.

Dark-adapted rods exert a tonic suppressive influence upon cone-mediated sensitivity to rapid flicker, a phenomenon called suppressive rod-cone interaction (SRCI). However, rod dark adaptation has negligible influence upon cone-mediated thresholds measured with more usual psychophysical procedures. The present study separately examined the influences of rod light and dark adaptation upon cone-mediated sensitivity to transient increases or decreases in illumination using sawtooth flicker with rapid-on (ramp-off) or rapid-off (ramp-on) waveforms. In the parafoveal retina, cones alone were stimulated with flicker by spatially superimposing long- and short-wavelength stimuli presented in counterphase and matched in scotopic illuminance. Several different adaptation procedures were used. For higher (greater than 4 Hz) frequencies, sensitivity of cones to both waveforms is nearly identical under any condition of adaptation; sensitivity decreases as rods progressively dark adapt. A considerably different situation exists for slower frequencies (1-4 Hz). Sensitivity of cones to rapid-off flicker is appreciably greater under light-adapted conditions confirming recent observations by Bowen et al. (1989). But as rods progressively dark adapt, sensitivity of cones to rapid-off waveforms decreases considerably while sensitivity to rapid-on waveforms is much less affected; in the totally dark-adapted eye, sensitivity to both waveforms is identical. These results confirm and extend recent physiological observations in amphibian retina (Frumkes & Wu, 1990) suggesting that SRCI specifically involves responses to transient decreases in illumination.

Funduscopic morphology of the light- and dark-adapted eye of the toadfish (Opsanus tau) in vivo

Intraocular funduscopy and photography permitted serial examination and documentation of time-related changes in the living toadfish (Opsanus tau) eye as it went from the dark-adapted to the light-adapted state. In the present study, the fully dark-adapted eye showed virtually no choroidal, pigment epithelial, or retinal vascular detail. The optic nerve was chalky white with indistinct borders. Grayish brown to chalky white retinal streaks radiated from the optic nerve into the periphery. As light adaptation began, the retinal streaks faded, with the retinal vasculature and the underlying choroidal and pigment epithelial detail becoming more discernible. Once the eye became fully light adapted, the typical geographic orange and black mottled appearance of the underlying pigment epithelium and choroid was visible, and the optic nerve head developed a pink hue with a distinct border. Even in the fully light-adapted eye, a vague radiation of lines could be seen extending from the optic nerve. This was superimposed on a cross-hatched mosaic of black dots seen throughout the fundus, not visible in the fully dark-adapted eye. Histological examination of the retina indicated forward migration of pigment in the pigment epithelium in the light-adapted state.

Effect of light on endogenous ligands carried by interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP) is a vitamin A carrier present only in the extracellular material lying between the neural retina and the retinal pigment epithelium of vertebrate eyes. The amount of retinol bound endogenously by IRBP in this interphotoreceptor space is known to increase upon illumination. This finding led to the hypothesis that IRBP may act as a shuttle for vitamin A during the visual cycle that regenerates rhodopsin. In the present work, we separated IRBP from other retinoid-binding proteins in bovine interphotoreceptor matrix preparations by means of size-exclusion chromatography. IRBP's endogenous ligands were retained during this procedure and were then extracted into hexane and analysed by normal-phase HPCL. We found that IRBP carries, in a light-dependent manner, all the retinoid isomers involved in the visual cycle. For dark-adapted eyes the amounts of bound ligands are (in nmol per eye) 0·09 all- trans retinol, 0·11 11- cis retinol, 0·04 all- trans retinal, 0·16 11- cis retinal, and 0·07 retinyl esters. For light-adapted eyes the amount of all- trans retinol was found to increase by a factor of five, and that of 11- cis retinal to decrease by a factor of four. (These eyes contain 3·1 nmol of IRBP, which does not change in amount with lighting conditions.) Thus, the major endogenousligand of IRBP is 11- cis retinaldehyde in the dark and all- trans retinol in the light. The data are consistent with a role for IRBP as a non-selective scavenger and stabilizer of retinoids released from photoreceptors and pigment epithelial cells. However, it cannot be concluded from these data that IRBP is involved in directed transport of retinoids across the interphotoreceptor matrix, since there is no evidence for the appropriate spatial gradients in the ligands bound to the protein.

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH-luc fusion to be observed by a dark-adapted eye and photographed.

Tonic interocular suppression and binocular summation in human vision.

1. Spatial sensitivity of human foveal vision was examined using sinusoidally modulated gratings. Our primary concern was the influence of interocular light adaptation upon monocular visibility. 2. Interocular adapting influences depend upon spatial frequency and adapting luminance. Interocular adaptation has a negligible influence upon the sensitivity to 1 cycle/deg gratings. Any visible interocular adapting field improves the sensitivity to intermediate spatial frequencies (2-5 cycles/deg). 3. Brighter interocular backgrounds (greater than 0.1 cd/m2) improve sensitivity to higher spatial frequencies (10-20 cycles/deg). 4. The interocular adapting influences summarized in (2) and (3) above cannot be duplicated by monocular or binocular adaptation. Similarly, monocular or binocular adaptation have negligible influences upon binocular visibility. 5. The interocular adapting effect summarized in (3) above can be duplicated by pressure blinding the contralateral eye. We conclude that monocular spatial sensitivity is subject to a tonic interocular suppression (TIS) from the dark-adapted eye. 6. The spatial sensitivity resulting from binocular viewing is nearly identical to that observed by combining monocular viewing with interocular light adaptation. We suggest that the improvement in sensitivity resulting from two-eyed viewing may be attributable to the removal of TIS instead of to binocular physiological summation.

Light‐adapting migration of the screening‐pigment in crayfish photoreceptors is a two‐stage movement comprising an all‐or‐nothing initial phase

The light-adapting response of the screening-pigment in crayfish retinal photoreceptors, previously described as a monophasic movement, was found to consist of two stages with different properties: (1) a rapid initial expansion that once started proceeds for at least half of the full distance, and (2) a slower and more variable continuation of the movement. The two components were resolved in isolated eyes stimulated under conditions expected to restrict Na+ influx into the photoreceptors. Only the second stage of the response to light was inhibited when Na+ was substituted with choline, or if the normal saline contained amiloride, a diuretic that hinders Na+ entry across many cell membranes. Amiloride in a medium without Na+ delayed, but did not curb, the first stage, whereas the rest of the movement was markedly restrained. Partial replacement of Na+ with Li+ blocked the second stage without affecting the rapid initial shift triggered by light. Exposure of dark-adapted eyes to high Na+ levels or to ouabain in the presence of Na+ in the dark also elicited a two-staged pigment dispersion to the light-adapted position. Low Na+ concentrations or amiloride affected the latency, but not the rate or extent, of the first stage of migration in ouabain-treated eyes. Consistent though less significant results were obtained with cyanide and the Na+ ionophore monensin. These observations suggest a differential control of pigment position over two defined domains along the photoreceptors, probably to integrate a double mechanism of light-adaptation: an all-or-nothing partial shift of the pigment screen as a safety factor against overexposure, followed by a regulated adjustment according to stimulation intensity.

Spectral sensitivity of the horse fly Tabanus nigrovittatus (Diptera: Tabanidae)

Spectral sensitivity functions were calculated from electroretinograms recorded from dark-adapted compound eyes of male and female horse flies (Tabanus nigrovittatus Macquart). Females had a broad sensitivity in the violet to green area of the spectrum; their spectral sensitivity was fitted by a theoretical mixture containing 20% of 440-nm and 80% 520-nm rhodopsins. Older females (8–18 days) were 93 times more sensitive than 1-day-old females. Males showed a narrower sensitivity function with more blue and less green sensitivity. Older males (8–18 days) were the most blue-sensitive of all groups; their spectral sensitivity was best fitted by a mixture containing 10% 440-nm, 70% 480-nm, and 20% 520-nm rhodopsins. Older males that were light-adapted to red light showed an apparent decline in the contribution of the 520-nm rhodopsin to overall sensitivity, as expected if this pigment is present in a separate system. The sensitivity function of 1-day-old males was best fitted by a mixture of 55% 480-nm and 45% 520-nm rhodopsins. The absolute sensitivity of both groups of males was close to that of the older females. All flies had substantial ultraviolet sensitivity, averaging 67% of the sensitivity at the longer wavelength maximum. The role of the differing sensitivities in males and females, and in young and old females, is discussed in relation to the visual behavior and sexual dimorphism of horse flies.