Real-time hexose monophosphate shunt activity in light- and dark-adapted rabbit retinas

Correlation proton nuclear magnetic resonance spectroscopy was performed on light- and dark-adapted rabbit retinas to elucidate real-time hexose monophosphate shunt (HMPS) activity. Light significantly stimulated retinal HMPS initially, which consumed 17% more glucose than that in dark-adapted retinas. The glucose consumption eventually declined to 16% of total, the baseline level, after 60 min of light exposure. In contrast, dark-adapted retinas showed an initial HMPS activity of 29% total glucose consumption, which declined to the basal level after 40 min. Lactate production appeared stable in both sets of retinas. Tert-butyl hydroperoxide (1 mM) also stimulated the shunt; however, a combination of light-adaptation and tert-butyl hydroperoxide did not stimulate the shunt additively. These data indicate that the retina has limited HMPS capacity.     

Cyclic nucleotide levels in light- and dark-adapted ground squirrel whole eyes

Concentrations of cyclic AMP and cyclic GMP were measured in whole eyes from ground squirrels. Light- and dark-adaptation had no apparent effect on the level of either cyclic nucleotide. However, ischemia markedly elevates the level of cyclic AMP, but not cyclic GMP. These data suggest that regulation of cyclic nucleotides in cone-dominant retinas differs from that in rod-dominant retinas.     

S-crystallin and arginine kinase bind F-actin in light- and dark-adapted octopus retinas

PURPOSE: Rhabdomere microvilli dramatically reorganize in conditions of light and dark. This reorganization involves remodeling of the microvillus actin cytoskeleton. We are using the rhabdomeric retina of Octopus bimaculoides to identify actin-binding proteins that may be involved in this remodeling. METHODS: Octopus were light-/dark-adapted, retinas separated into dorsal and ventral halves, and homogenized. Actin-binding proteins were recognized using F-actin overlay blot assays and selected proteins from the overlays were identified using N-terminal sequencing methods or mass spectroscopy. Protein concentrations were quantified and compared by statistical analysis. RESULTS: Total protein gels of light-/dark-adapted, ventral/dorsal halves were almost identical except for a protein band at 26 kD. The relative amount of this protein in the dark was almost double that found in the light. The levels of other proteins did not vary significantly between the light and dark. F-actin overlays also showed matching patterns of actin-binding proteins except for the 26 kD protein. Although the 26 kD protein from light-adapted retinas transferred to the blotting membranes, it did not bind F-actin while the 26 kD protein on overlays from dark-adapted retinas always demonstrated F-actin binding. Besides the 26 kD protein, other proteins at 200 kD, 80 kD, 40 kD appeared on the overlays. These proteins and the 26 kD protein were sequenced and identified as hemocynanin, transitional ER ATPase, arginine kinase and S-crystallin, respectively. CONCLUSIONS: The amount of S-crystallin present in the octopus retina is significantly greater in dark-adapted retinas and it binds to F-actin. In the light, the level of S-crystallin is greatly reduced and there is no apparent F-actin binding. No other studies, to our knowledge, show that S-crystallin binds to the actin cytoskeleton or that its expression is regulated by light. Arginine kinase may provide energy for cytoskeletal remodeling as it may in other neural tissues.     

Localization of (3H2)vitamin A in mouse retina.

Following intraperitoneal injection of [³H₂]vitamin A acetate, the accumulation of labeled material in mouse retinas was studied by autoradiographic techniques. Significant accumulations of label were observed in the pigment epithelium and photoreceptor cells. Isotope continued to accumulate for 2 days, then achieved a steady state which was maintained through 9 days. The major localization within the photoreceptor cells was in the outer segments; inner segments and nuclei showed lesser amounts of label but were significantly above background level. Although the concentration of label varied markedly from one region of the photoreceptor cells to another, the distribution within each region was homogeneous. Photoreceptor outer segments gave no evidence of localized banding of labeled material. A metabolic pathway for vitamin A within mouse photoreceptor cells involving inner segments and nuclei is suggested.     

Perceptive field size in fovea and periphery of the light- and dark-adapted retina

The sizes of perceptive fields between 0 and 70° eccentricity were measured in the light- and dark-adapted human retina using the Westheimer paradigm. The results indicate that at photopic background luminances, centre size increases from a foveal diameter of less than 9′ to 1° at 10° eccentricity, then more slowly to 1.75° at 70° eccentricity. Total perceptive field size, on the other hand, increases more rapidly from 1° in the fovea to about 6.5° at 70° eccentricity. With dark-adaptation, the perceptive field surround becomes almost ineffective and centre size expands at all eccentricities by about a quarter of its photopic size. These results are compared to previous estimates of perceptive field sizes obtained with other methods and their relationship to the retino-cortical mignification factor are discussed.     

Proceedings: Autoradiographic and radiobiochemical studies on the incorporation of (6-3H)glucosamine into frog rhodopsin

Our previous work on rhodopsin biosynthesis has dealt with the incorporation of amino acids into newly synthesized rhodopsin. The present work adds to this story by dealing with the intracellular site of incorporation of [³H]glucosamine into the polypeptide and the subsequent distribution of the glycoprotein within the cell.Freshly dissected retinas from dark-adapted frogs were incubated in the dark in oxygenated Ringer's bicarbonate medium containing d-[6-³H]glucosamine. At selected intervals from 5 min to 6 hr, retinas were retrieved from the incubation medium and killed by aldehyde fixation. Following this, light and electron microscope autoradiograms of the tissue were prepared. In additional experiments, retinas were incubated for varying periods in a similar medium and rhodopsin was extracted, purified and analyzed for glucosamine incorporation.Glucosamine is first bound within the rod photoreceptor in the ribosome-rich myoid region, where the first glycosylation presumably occurs. By 20 min, the labeled product appears in the Golgi apparatus, where additional glycosylation presumably occurs. Finally, by 2 hr, the fully glycosylated product reaches the base of the outer segment, where it is inserted into the growing disc membrane. Biochemical analysis shows that the purified rhodopsin is heavily labeled by this time, and that the labeled precursor is incorporated chemically unchanged.The data imply that rhodopsin is sequentially glycosylated first on the ribosomes and then in the Golgi apparatus, and that the glycoprotein is complete in terms of its sugar moiety prior to its insertion into the growing disc membrane.     

A new template for rhodopsin (vitamin A1 based) visual pigments.

A new template curve for rhodopsin visual pigments is presented. The template was constructed from absorbance spectra of purified bovine rhodopsin with an abscissa transform of lambda max/lambda. Absorbance curves generated from the template are compared with absorbance spectra of visual pigments measured in situ in single cells by microspectrophotometry (msp). The template was found to agree well with msp data which included visual pigments with lambda max values ranging from 450 to 570 nm. A cubic polynomial is given by which the new template may be fitted to the long wavelength limb of visual pigment data. A Chebyshev polynomial is also given which models the new template with a high degree of accuracy. This polynomial provides a convenient method for the computer generation of visual pigment absorbance curves.     

Identification and immunolocalization of actin cytoskeletal components in light- and dark-adapted octopus retinas.

Photoreceptors in the octopus retina are of the rhabdomeric type, with rhabdomeres arising from the plasma membrane on opposite sides of the cylindrical outer segment. Each rhabdomere microvillus has an actin filament core, but other actin-binding proteins have not been identified. We used immunoblotting techniques to identify actin-binding proteins in octopus retinal extracts and immunofluorescence microscopy to localize the same proteins in fixed tissue. Antibodies directed against alpha-actinin and vinculin recognized single protein bands on immunoblots of octopus retinal extract with molecular weights comparable to the same proteins in other tissues. Anti-filamin identified two closely spaced bands similar in molecular weight to filamin in other species. Antibodies to the larger of the Drosophila ninaC gene products, p174, identified two bands lower in molecular weight than p174. Anti-villin localized a band that was significantly less in molecular weight than villin found in other cells. Epifluorescence and confocal microscopy were used to map the location of the same actin-binding proteins in dark- and light-adapted octopus photoreceptors and other retinal cells. Antibodies to most of the actin-binding proteins showed heavy staining of the photoreceptor proximal/supportive cell region accompanied by rhabdom membrane and rhabdom tip staining, although subtle differences were detected with individual antibodies. In dark-adapted retinas anti-alpha-actinin stained the photoreceptor proximal/supportive cell region where an extensive junctional complex joins these two cell types, but in the light, immunoreactivity extended above the junctional complex into the rhabdom bases. Most antibodies densely stained the rhabdom tips but anti-villin exhibited a striated pattern of localization at the tips. We believe that the actin-binding proteins identified in the octopus retina may play a significant role in the formation of new rhabdomere microvilli in the dark. We speculate that these proteins and actin remain associated with an avillar membrane that connects opposing sets of rhabdomeres in light-adapted retinas. Association of these cytoskeletal proteins with the avillar membrane would constitute a pool of proteins that could be recruited for rapid microvillus formation from the previously avillar region.     

Distribution of tubulin, kinesin, and dynein in light- and dark-adapted octopus retinas

Cephalopod retinas exhibit several responses to light and dark adaptation, including rhabdom size changes, photopigment movements, and pigment granule migration. Light- and dark-directed rearrangements of microfilament and microtubule cytoskeletal transport pathways could drive these changes. Recently, we localized actin-binding proteins in light-/dark-adapted octopus rhabdoms and suggested that actin cytoskeletal rearrangements bring about the formation and degradation of rhabdomere microvilli subsets. To determine if the microtubule cytoskeleton and associated motor proteins control the other light/dark changes, we used immunoblotting and immunocytochemical procedures to map the distribution of tubulin, kinesin, and dynein in dorsal and ventral halves of light- and dark-adapted octopus retinas. Immunoblots detected alpha- and beta-tubulin, dynein intermediate chain, and kinesin heavy chain in extracts of whole retinas. Epifluorescence and confocal microscopy showed that the tubulin proteins were distributed throughout the retina with more immunoreactivity in retinas exposed to light. Kinesin localization was heavy in the pigment layer of light- and dark-adapted ventral retinas but was less prominent in the dorsal region. Dynein distribution also varied in dorsal and ventral retinas with more immunoreactivity in light- and dark-adapted ventral retinas and confocal microscopy emphasized the granular nature of this labeling. We suggest that light may regulate the distribution of microtubule cytoskeletal proteins in the octopus retina and that position, dorsal versus ventral, also influences the distribution of motor proteins. The microtubule cytoskeleton is most likely involved in pigment granule migration in the light and dark and with the movement of transport vesicles from the photoreceptor inner segments to the rhabdoms.     

Phagocytosis of light- and dark-adapted rod outer segments by cultured RPE cells: a reassessment

Rod outer segments (ROS) isolated from adult rat retinas are phagocytized by cultured rat retinal pigment epithelial (RPE) cells. Using a double immunofluorescent labeling procedure, we have compared the binding and ingestion of ROS isolated at different times of the day. After 2 hr of incubation, approximately 98% of the ROS are ingested, while 2% are still attached to the RPE cell surface, irrespective of the time of day or lighting conditions under which the ROS are isolated. These findings differ from those reported earlier, using a radioactive method for quantitating ROS phagocytosis (Hall, 1978).     

Visual function and rhodopsin levels in humans with vitamin A deficiency

Details of rod and cone dysfunction in vitamin A deficiency have been studied in two subjects with primary biliary cirrhosis and one with Crohn's disease, all of whom presented with symptoms of night blindness. Visual function in the mid-peripheral retina was monitored with two-color adaptometry and rhodopsin levels were measured by fundus reflectometry. Initially all three subjects had no measurable rod function and delayed cone adaptation. In one case the dark-adapted cone threshold was also elevated. Oral supplementation with vitamin A restored visual function to normal within 8 days in all subjects. During supplementation, cone function was restored more rapidly than that of rods, though the pattern of recovery was similar for each receptor type. Final thresholds improved first, though the rates at which they were reached were abnormally slow. As recovery continued, adaptation kinetics returned to normal. When rod adaptation was delayed, the regeneration of rhodopsin was also abnormally slow. When rod final threshold was 2 log units higher than normal, rhodopsin regeneration was incomplete, reaching about 70% of the normal level. The initial stages of visual dysfunction during onset of vitamin A deficiency were studied in one subject, and were found to mirror the pattern seen during recovery: rod adaptation was initially slower than normal, but reached completion. Cone adaptation remained normal until rod function was almost absent.     

应用~3H－TdR掺入法标记活体角膜内皮细胞的初步报告

用液氮冷冻兔和猴角膜，造成角膜内皮创伤。在ＤＮＡ合成期前房注入氚标记的胸腺嘧啶核苷（３Ｈ—ＴｄＲ），使之掺入角膜内皮细胞核内。这种标有３Ｈ—ＴｄＲ的内皮细胞将在体内长期存活。再用放射自显影法显示出标记细胞。用此法可获得带有稳定标记的角膜内皮细胞的动物模型，用来观察穿透性角膜移植术后角膜内皮的动态变化。     

Evaluation of light- and dark-adapted ERGs using a mydriasis-free,portable system: clinical classifications and normative data

Purpose

The full-field electroretinogram (ff-ERG) is a widely used clinical tool to evaluate generalized retinal function by recording electrical potentials generated by the cells in the retina in response to flash stimuli and requires mydriasis. The purpose of this study was to determine the intra-visit reliability and diagnostic capability of a handheld, mydriasis-free ERG, RETeval (LKC Technologies, Gaithersburg, MD, USA), in comparison with the standard clinical ff-ERG by measuring responses recommended by the International Society for Clinical Electrophysiology of Vision (ISCEV).

Methods

This prospective, cross-sectional study included 35 patients recruited at the Hospital for Sick Children (median age?=?17, range 11 months–69 years) who had undergone a clinical ff-ERG according to ISCEV standards. For RETeval (n?=?35), pupils were undilated in most (n?=?29) and sensor strip electrodes were placed under the inferior orbital rim. Stimulus settings on RETeval were equivalent to those used in the clinical ERG. Fifty-seven control participants (median age?=?22, range 8–65 years) underwent undilated RETeval ERG to establish standard values for comparison. Patient waveform components with amplitudes?<?5th percentile, or implicit times?>?95th percentile of normal relative to control data were classified as abnormal for the RETeval system.

Results

The RETeval system demonstrated a high degree of within-visit reliability for amplitudes (ICC?=?0.82) and moderate reliability for implicit times (ICC?=?0.53). Cohen’s Kappa analysis revealed a substantial level of agreement between the diagnostic capability of RETeval in comparison with clinical ff-ERG (k?=?0.82), with a sensitivity and specificity of 1.00 and 0.82, respectively. Pearson’s correlations for clinical ERG versus RETeval demonstrated a positive correlation for amplitudes across the rod (r?=?0.65) and cone (r?=?0.74) ERG waveforms. Bland–Altman plots showed no bias between the mean differences across all amplitude and implicit time parameters of the two systems.

Conclusions

The present study demonstrated that RETeval is a reliable tool with reasonable accuracy in comparison with the clinical ERG. The portable nature of RETeval system enables its incorporation at resource-limited centers where the ff-ERG is not readily available. The avoidance of sedation and pupillary dilation are added advantages of RETeval ERG.

     

3-Dehydroretinal (vitamin A2 aldehyde) in crayfish eye

11-Cis-3-dehydroretinal was found in the eye of crayfish, Procambarus clarkii. The 11-cis-3-dehydroretinal was isomerized to all-trans isomer by light-illumination, as was also 11-cis-retinal. Irradiation with deep-red light (lambda greater than 680 nm) selectively isomerized the 11-cis-3-dehydroretinal. The 3-dehydroretinal was not extracted with petroleum ether from the tissue after freeze-drying. These facts suggest that the 11-cis-3-dehydroretinal is the chromophore of crayfish visual pigment. The 3-dehydroretinal content varied with season, high level in winter and low in summer. The crayfish may have a vitamin A1-A2 visual pigment system similar to those of freshwater fishes and amphibians.     

The relationship between ambient lighting conditions, absolute dark-adapted thresholds, and rhodopsin in black and hypopigmented mice

Significant variation in absolute dark-adapted thresholds is observed both within and between strains of mice with differing ocular pigmentation levels. Differences in threshold within a single strain are related to the Williams' photostasis effect, that is, photoreceptor rhodopsin levels are dependent upon ambient lighting conditions. To examine threshold differences among strains, we equalized rhodopsin levels by maintaining albino mice (c2J/c2J) at 2 x 10-4 cd/m2 (dim light) and black mice at 2 x 102 cd/m2 (bright light). This resulted in ocular rhodopsin levels for albino mice (albino--dim) of 494 +/- 11 pmoles/eye and rhodopsin levels for black mice (black--bright) of 506 +/- 25 pmoles/eye. For comparison, rhodopsin levels in black mice maintained in dim light are 586 +/- 46 pmoles/eye and 217 +/- 46 pmoles/eye in albino mice maintained in bright light. We found similar dark-adapted thresholds (6.38 log cd/m2 vs. 6.47 log cd/m2)) in albino and black mice with equivalent rhodopsin determined with a water maze test. This suggests that dark-adapted thresholds are directly related to rhodopsin levels regardless of the level of ocular melanin. The number of photoreceptors, photoreceptor layer thickness, and outer segment length did not differ significantly between albino (dark) and black mice (bright). These results demonstrate that the visual sensitivity defect found in hypopigmented animals is secondary to abnormal rhodopsin regulation and that hypopigmented animals have either an improper input to the photostasis mechanism or that the photostasis mechanism is defective.