Mary E. Rayborn

The emergence, localization, and maturation of neurotransmitter systems during development of the retina in Xenopus laevis. III. Dopamine

The uptake, synthesis, and release of dopamine was studied in retinas of Xenopus laevi. In the tadpole and adult retina, 3H‐dopamine is accumulated by cells located in the inner nuclear layer. Retinas preloaded with 3H‐dopamine release this compound in response to high K+ concentrations in the medium. This release is probably Ca++‐dependent as it is inhibited by Co++ in the medium. Adult retinas are also capable of synthesizing 3H‐dopamine from 3H‐tyrosine. The appearance and maturation of these dopaminergic properties were followed during retinal development. Our data indicate that synthesis of dopamine can first be detected as early as stage 35/36 whereas uptake of dopamine first occurs at stage 43. K+‐stimulated release of preloaded 3H‐dopamine from putative dopaminergic neurons is, however, not evident until stage 46. These results show that similar to the development of GABA‐ergic and glycinergic properties, the uptake, synthesis, and release mechanisms for dopamine emerge at different stages during retinal differentiation in Xenopus Laevis.

Retinal attachment to the pigment epithelium. Linkage through an extracellular sheath surrounding cone photoreceptors.

The interphotoreceptor matrix, which occupies the so-called subretinal space in normal human eyes, was examined with electron microscopy after treatment with Cuprolinic Blue, a dye that selectively stains sulfated polyanions. A cylindrically shaped, extracellular ensheathing domain, composed of Cuprolinic Blue-positive particles, was observed surrounding each cone photoreceptor examined. Equivalent structures were not present around rods. Surrounding peripheral cones, the unit particles which comprised the sheath, were punctate in shape whereas in foveal and foveolar cones the particles were more elongate. A matrix domain completely enveloped each cone photoreceptor, beginning at the outer limiting membrane, extending beyond the tip of the cone outer segment, and ending at the apical surface of the pigment epithelium. Proximal to the pigment epithelium, specialized extensions of apical microvilli were present in the lumen of the matrix sheath and shorter microvilli appeared to be embedded in the sheath wall. When retinas were experimentally detached from the pigment epithelium and washed extensively before fixation and staining, the cone matrix domains remained associated with the cones, and broken fragments of pigment epithelial cell processes were present embedded in the distal tip of the cone matrix sheath wall. These observations indicate that the cone matrix sheath is insoluble and is closely associated with both the cone photoreceptor and the apical surface of the pigment epithelium. The implication of these findings is that the cone matrix sheath is a bridge through which the retina and pigment epithelium are physically attached.

Rod outer segments elongate in constant light: Darkness is required for normal shedding

Abstract Shedding of large rod outer segment fragments is greatly reduced when Rana pipiens are maintained under moderate levels of continuous light. This inhibition of shedding results in an increase in outer segment length, since disc addition continues even as shedding stops. Some shedding will occur when constant-light frogs are subjected to a brief period of darkness, suggesting that darkness alone may stimulate shedding. Massive shedding, resulting in a reduction to normal outer segment length, occurs when constant-light animals are maintained for as little as 30 min in darkness and are then returned to constant light. This shedding response will occur even after both optic nerves are severed, and after pinealectomy or hypophysectomy.

The effect of light on the quantity of phagosomes in the pigment epithelium

Abstract The effect of light on the number of phagosomes within the pigment epithelium was studied using Rana pipiens tadpoles dark-adapted for various intervals before light stimulation. Two populations of phagosomes were observed. One consists of large packets of rod outer segment discs 4–10 μm long and 5 μm wide. Light stimulation following various periods of darkness results in a steady increase in the density of these large phagosomes reaching a peak 2–3·5 times above control levels after 2 hr of light. The maximum number of large phagosomes was obtained in animals dark adapted for 3 days before light stimulation. The second population of phagosomes consists of small packets of outer segment discs 1–2 μm in diameter. The number of these phagosomes does not change with different conditions of illumination. Electron microscopic analysis indicates that the small phagosomes are lost from the rod outer segment tips as small whorls of 5–30 discs.

Localization of proteoglycan within the extracellular matrix sheath of cone photoreceptors

The interphotoreceptor matrix of the human retina was examined histochemically by staining with a cationic copper phthalocyanin dye, Cuprolinic Blue, in a critical electrolyte concentration method which allowed staining of sulfated polyanions. In the presence of Cuprolinic Blue, a dense punctate matrix component was visualized covering the cone from the outer limiting membrane of the retina to the tip of the cone outer segment and extending through the matrix to the apical surface of the pigment epithelium. The size and appearance of the stained material are consistent with its being proteoglycan and its distribution is consistent with its being a component of the cone extracellular matrix sheath. A fine reticular pattern was observed in the presence and absence of Cuprolinic Blue.

Phosphoinositide metabolism in the retina: Localization to horizontal cells and regulation by light and divalent cations

Abstract: Isolated retinas from Xenopus laevis incorporated greater amounts of [3H]inositol and 32Pi into phosphoinositides when incubated in light than did control retinas incubated in the dark. Inositol was primarily incorporated into phosphatidylinositol (83–86%), while phosphate labeled the polyphosphoinositides (72–79%). The incorporation of radioactive glycerol, serine, choline, or ethanolarnine into retinal lipids was unaffected by light. Following incubation with [3H]inositol, the cell type involved in the light response was identified by light and electron microscope autoradiography to be the horizontal cell. These results are consistent with a classic phosphatidylinositol effect in the retina. An interesting feature of this response is that the stimulus (light) is received in the photoreceptor cell and the effect is manifest in the horizontal cell.

Characterization of the interphotoreceptor matrix surrounding rod photoreceptors in the human retina

Previous studies have documented the presence of specific lectin-binding domains in the insoluble interphotoreceptor matrix (IPM) isolated from human retina. Peanut agglutinin (PNA) selectively binds to cone matrix domains whereas wheat germ agglutinin (WGA) binds to matrix domains surrounding rods. In the present study, the rod-associated WGA-binding domains are further characterized using lectin-based cytochemistry and polyacrylamide gel electrophoresis in combination with neuraminidase digestion. The lectin-binding patterns of non-neuraminidase-treated samples are similar to those described in previous reports. After neuraminidase treatment, both rod and cone matrix domains demonstrate PNA binding while the binding of WGA to rod matrix domains is reduced. However, the binding of WGA to photoreceptor outer segments is not affected by neuraminidase. Blots of IPM proteins probed with lectins indicate that the WGA-binding macromolecules are represented as a group of high molecular weight glycoproteins, whereas the PNA-binding components are represented as a group of lower molecular weight glycoproteins. The major WGA-binding glycoprotein (147 kDa) shows reduced binding affinity to WGA and increased binding affinity to PNA following neuraminidase treatment. Further, this 147-kDa glycoprotein, although similar in molecular weight to IRBP (interphotoreceptor retinol-binding protein) (141 kDa), is not recognized by the lectin, concanavalin A (Con A), or by an anti-IRBP antibody. Our data suggest that: (1) the major component of the WGA-binding domain demonstrated by polyacrylamide gel analysis is a glycoprotein with a molecular weight of 147 kDa containing galactose residues that are masked by terminal sialic acid residues; and (2) the binding of WGA to photoreceptor outer segments is resistant to neuraminidase, consistent with the earlier reports that WGA-binding domains of photoreceptor outer segments may not be sialyl-containing glycoconjugates.

Some aspects of corneal and scleral differentiation in the primate

Abstract Electron microscopic observations were made of corneal and scleral structure in a series of staged primate fetuses from the time the earliest aggregated collagen fibrils appeared. Average diameters of these fibrils range from 31·2–33·3 nm in the cornea and from 26·0–26·6 nm in the sclera, at 43 days of gestation. Though scleral collagen fibrils lag in the initial phases of aggregation, at 54 days of development their average diameter surpasses that of the corneal collagen diameter by about 50%. The scleral collagen fibrils grow and differentiate rapidly, attaining an average thickness of approximately 100 nm with repeating periods of about 80 nm at 76 days of gestation. The diameter of corneal collagen fibrils remain essentially stable during fetal life; their macroperiod, in contrast to that of the scleral fibrils, is poorly defined till late in gestation. Within the fibrocyte the appearance of the organelles during fibrogenesis, the various forms of their contents, and their locations in relation to the cell membrane are noted. The cisternae of the rough endoplasmic reticulum of all ages contain the flocculent or granular material indicative of protein syntheses. Cisternae have sites of ribosomal detachment, and either closely approach the plasma membrane, or they bleb and vesiculate. During the second trimester of gestation some Golgi vacuoles enclose granular or homogenous substances, others long, parallel filaments, often assembled in bundles. Membrane-enclosed, aggregated fibrils, resembling the extracellular collagen fibrils, occur frequently. Similarities between chick and primate collagen-producing cells are pointed out. Within the scleral fibrocytes, near the sites where the first presumed elastic microfibrils appear, are associated glycogen clumps, polysomes and small granules. Close to the outer side of the plasma membrane individual collagen fibrils and the fine filaments of the extracellular space may participate in the formation of pre-elastic foci.

A Dominantly Inherited Chorioretinal Degeneration Resembling Sectored Retinitis Pigmentosa

A light and electron microscopic study of an eye from a 79-year-old woman diagnosed as having sector retinitis pigmentosa is presented. Prominent bone spicule pigmentation was present bilaterally in the nasal quadrant. Retinal structure in the central fundus around the fovea and extending 2-3 mm peripherally was near normal with all photoreceptors and other retinal neurons present. Extensive degeneration of the retina occurred as one proceeded toward the peripheral regions from 3-5 mm from the fovea in all quadrants. Changes were evident within the entire choroid and were most severe in regions where retinal degeneration was most pronounced. It is likely that the extensive degenerative changes present in the retina were secondary effects that follow alterations in the choroidal blood supply in individuals affected with this disorder.

Membrane assembly in photoreceptor outer segments: Progressive increase in ‘open’ basal discs with increased temperature☆

Abstract Rod outer segment renewal rates of 0·8, 2·1 and 2·5 μm/day were calculated from 3 H-band displacement in autoradiographs following 3 H-leucine injection into Xenopus laevis tadpoles maintained in cyclic lighting (12 hr light: 12 hr darkness) at 16°C, 21°C and 26°C, respectively. Based on disc packing density in rod outer segments, these displacement rates reflect the addition of 29, 76 and 91 discs/day at these temperatures. The accumulation of newly forming ‘open’ membrane discs at the outer segment base was also established for animals maintained at each of the above temperatures under the same lighting schedule. Open discs, though absent at the time of light onset, increase in number following light stimulation to reach a maximum after 6 hr light exposure. The numbers of open discs present at this time were 3·3 ± 0·2; 6·2 ± 0·1; and 9·8 ± 0·7 per rod (mean ± s.d. ) at 16°C, 21°C and 26°C, respectively. Open discs decrease in number thereafter and are rarely observed 10 hr into the light cycle or during the dark phase of the diurnal cycle. Although the maximum number of open discs accumulating at 6 hr after light exposure at each temperature represents only 10% of the total number of new discs added each day, the similarity in the proportional increase in open discs to the increase in renewal rate over the temperature range utilized indicates that the number of open discs provides a useful morphological index for the relative rates of membrane assembly occurring in rod photoreceptors.