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Two modes of shedding of rod disc membranes were observed by electron microscopy in bullfrog retinas illuminated for various periods from 10 min to 2 hr. One mode is "autonomous shedding" whereby rods shed disc packets directly into the subretinal space. Most of the discarded disc packets are subsequently brought into contact with villous apical processes of pigment epithelial (PE) cells and are ultimately engulfed by these cells. When some of the shed disc membranes remain in the subretinal space, it appears that these remnants may be phagocytized by ameboid phagocytes. The other mode is "cooperative shedding" whereby rods shed disc packets with the participation of pigment epithelial ensheathment. Shedding of a disc packet from a rod tip, and enclosing of the rod tip by a broad, sleeve-like apical process of a PE cell, take place simultaneously. The separated disc packets may be immediately engulfed by the PE cells without risk of failure. Both villous and sleeve-like types of apical processes of PE cells in the bullfrog lack pigment granules, in contrast to the finger-like apical processes that do contain pigment granules. Villous and sleeve-like processes therefore probably belong to the same category as the leaf-like apical processes of PE cells in mammalian retinas.

The morphology of the retinal photoreceptors has been studied in the domestic ferret by electron microscopy. 2 types of photoreceptor are present and are differentiated morphologically into the classical categories of rods and cones. Rods are longer, more slender and more numerous than the cones. The rod outer segment is uniform in diameter along its entire length while the cone outer segment tapers distally. Cone outer segments are short and large apical processes from the pigment epithelium extend some distance to wrap around them. Both rods and cones possess a connecting cilium and a large cross-striated fibril which begins near the ciliary basal body and extends through the outer segment to end near the external limiting membrane. Cone nuclei are more vesicular than rod nuclei. They form a single layer immediately beneath the external limiting membrane while rod nuclei form several layers beneath the cone nuclei. The synaptic region of the cone cells is larger and more elaborate than that of the rods. Synapses in both rods and cones appear to be formed by both superficial and invaginated contacts.

Mg2+-ATP was found to produce a 15 to 30% drop in the turbidity of suspensions of broken retinal rod outer segments from the toad Bufo marinus, prepared by washing or flotation in sucrose. This in vitro process has a half-time of about two minutes and appears to be irreversible. It is not affected by the bleaching of rhodopsin. Direct measurements show that the drop in turbidity is not due to swelling of the disc internal space measured in outer segments recovered by centrifugation. Instead, the total packed volume of the outer segments increases following incubation in Mg2+-ATP. Under the specific conditions of these experiments, the total pellet volume increase was 26 +/- 22% (13 experiments) when corrected for the percent of rhodopsin recovered in the centrifugal pellet. The magnitude of the ATP effect on turbidity suggests that the majority of the discs are involved in some kind of structural change. Vanadium in the +5 oxidation state (vanadate) is an inhibitor of the Mg2+-ATP effect on turbidity at a half-maximal concentration of 0.2 to 0.4 microM, and inhibition is rapidly reversed by norepinephrine, which complexes vanadate. A Mg2+-ATPase activity in extensively washed outer segment membranes, previously shown to be activated as much as twofold following light exposure of the membranes, is not sensitive to vanadate at the concentrations which block the ATP-dependent change of turbidity.

Glycoconjugates, including glycolipids, glycoproteins, and proteoglycans, are present in the plasma membrane of photoreceptor cells and in the interphotoreceptor matrix surrounding photoreceptor cell ellipsoids and outer segments. Although the precise function of these molecules is unknown, they may be important in mediating photoreceptor-pigment epithelial cell interactions, outer segment membrane assembly, and/or disc shedding. Lectins, affinity ligands for defined carbohydrate sequences, have proven particularly useful in studying the glycoconjugate composition of the interphotoreceptor matrix. The peanut lectin selectively binds to domains of the interphotoreceptor matrix surrounding cone ("cone matrix sheaths"), but not rod inner and outer segments. This is evidence for the existence of chemical and structural heterogeneity within the interphotoreceptor matrix. The studies described herein utilized ultrastructural pre-embedding histochemical labeling to assess whether, in addition to the surrounding interphotoreceptor matrix, peanut lectin binding is associated directly with that plasma membrane of cone inner and outer segments. This study confirms that ferritin-conjugated peanut agglutinin binds to cone matrix sheaths, and, in addition, provides ultrastructural evidence for the presence of binding to the plasma membrane surrounding cone inner and outer segments. The data suggest that cone membrane-associated peanut agglutinin-binding molecules may differ from those located within cone matrix sheaths.

The postnatal development of the area centralis of the kitten retina was followed with the electron microscope. After birth, small but well developed photoreceptor outer segments were observed. Cone pedicles formed triads and bipolar cells formed dyads; so, potentially, cone pathways to the ganglion cell were present at this time. By the start of the second week, rod spherules formed shallow invaginations containing both central and lateral processes and rod bipolar cells formed dyads. Thus, potentially, rod pathways to the ganglion cell were present. By the end of the third week, the area centralis was sufficiently well developed to take advantage of the clearing of the optical media which occurs in postnatal development. Maturity was attained during the fifth week.

The protein(s) that constitute(s) the cGMP-regulated channel in vertebrate photoreceptors has been solubilized from rod outer segment membranes and reincorporated into the membrane of calcium-containing liposomes. The properties of the reconstituted channel protein were determined by studying the cGMP-stimulated efflux of Ca2+ from these liposomes. Among several detergents tested the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) proved to be the most suitable. Solubilization of channel activity was found to be optimal at a detergent concentration of about 18 mM. The presence of Ca2+ ions and phospholipids during solubilization greatly increased the channel stability. The reconstituted channel shared most but not all properties with the channel in situ. It is cooperatively activated by cGMP with an EC50 of 19 microM. The cooperativity as determined from Hill plots was n = 2.7. Unlike the cGMP-sensitive channel in the native membrane of isolated discs and excised patches of plasma membrane it is not blocked by l-cis-diltiazem. Reconstitution of this channel protein(s) may serve as a valuable tool for identifying the polypeptide composition and to study structural and functional aspects of the purified protein(s).

125I-calmodulin gel overlay techniques have been used to identify calmodulin-binding proteins in teleost retina, in a rod fragment preparation which contains rod inner and outer segments (RIS-ROS), and in RIS-ROS cytoskeletons. We have previously shown that teleost rods change length in response to changes in light conditions, that rod movement is mediated by the actin filaments in the rod inner segment, and that both Ca2+ and cAMP appear to be involved in regulating rod movement. We report here the development of a rod fragment preparation (RIS-ROS), which retains the movable part of the rod, for use in biochemical analysis of rod motility. Gel overlay studies indicate that isolated whole retinas have six prominent calmodulin-binding proteins, migrating at 240 K, 190 K, 150 K, 61 K and a doublet at 18/19 K. In contrast, detached RIS-ROS have three different prominent calmodulin-binding proteins, migrating at 330 K, 33 K, and 31 K. RIS-ROS cytoskeletons have been produced by extraction with Triton X-100; they contain both actin filament bundles and microtubules associated with the connecting cilium. RIS-ROS cytoskeletons have 3 prominent calmodulin-binding proteins migrating at 240 and 18/19 K. These proteins produce faint bands in gel overlays of intact RIS-ROS, but prominent bands in overlays of whole retina. The 240 K protein of RIS-ROS cytoskeletons co-migrates with the 240 K calmodulin-binding subunit of rat brain fodrin. We suggest that the rod 240 K calmodulin-binding protein may be a spectrin-like protein which participates in Ca2+- and calmodulin-regulation of rod motility.

Shedding of outer segment discs and their phagocytosis by retinal pigment epithelial cells (RPE) in the guinea pig starts to occur during in utero development. The number of phagosomes in the fetal RPE varies in a cyclical manner somewhat analogous to that observed in mature animals. The present study addresses the question of whether the cycles in fetus and mother are independent or linked. Pregnant guinea pigs were subjected to 10 days of alteration of their lighting schedules beginning on the 46th day of gestation. Dams and fetuses were sacrificed on day 56, and large phagosomes in the RPE of each were counted. A 10-hour phase advance was employed, ie, from 0800 (lights on): 2000 (light off) in controls, to 2200 (on): 1000 (off) in experimental animals. Counts of large phagosomes in the posterior retina were compared in both control and experimental nulliparous, gravid, and fetal animals. A significant burst of shedding occurred within one hour after light onset in the experimental groups comprised of either nulliparous or pregnant animals. The latter also showed a smaller rise in phagosome numbers at the original time of onset of illumination. In contrast, no shift in the time of maximum shedding activity was observed in fetuses in the experimental regime. Their activity was similar to that present in the original 0800:2000 cycle. Whereas maternal RPE shedding cycles can be reentrained in a 10-day period, those in the 46- to 56-day fetal retina are refractory to significant change. Based on this finding, it is likely that the shedding cycles in the mother and fetus at this age are under independent control, and, therefore, that shedding in the fetus is not mediated by maternal borne factors.

The temporal sequence of ultrastructural changes induced in the rat rod photoreceptor by 80 lux light-stress has been studied. The changes seen were compared with those produced by a much dimmer (3 lux) illumination. Some of the early signs of abnormality were (1) degradation of some disk membranes at the tips of outer segments, (2) disaggregation and detachment of ribosomes, (3) lighter matrices in swollen mitochondria, (4) disappearance of the Golgi apparatus, (5) proliferation of autophagic bodies in the inner segments, and (6) appearance of perimitochondrial membrane whorls in the synaptic terminals. No single change could be identified that would inexorably lead to cell death. The overall picture, however, suggested that an inability of the cell to maintain its anabolic balance is responsible for the pyknosis that occurs when the 80 lux exposure exceeds 12-15 h. All changes were reversible when exposure duration did not exceed 12 h, the normal length of the light cycle for these rats.

In rod outer segments (ROSs) of the Congo eel salamander (Amphiuma), we have observed a system of cytoplasmic filaments that interconnect the lamellar domains of adjacent disks. In longitudinal sections, these filaments occur in pairs, spaced 18-20 nm apart. Each filament is 9-11 nm in length, and perpendicular to the disk membrane. Such filament pairs display extended axial alignment through the lamellar domains of successive disks, indicating the involvement of trans-membranous elements. In cross-sectional views, these filaments appear as a row of paired, punctate densities with a periodic spacing of 14-16 nm. Such views indicate that these filaments are organized on a two-dimensional crystalline lattice, oriented perpendicular to the disk lamellae. The linear interdisk densities of the terminal loop complex appear to be identical to these cytoplasmic filaments: both are equal in length, and both are organized as two-dimensional assemblies with equivalent orientations and lattice parameters. These shared features suggest a common subset of molecular components and lattice determinants. Where the paired filament assemblies intersect the disks, one observes a characteristic dilatation of the intradisk space. In these regions, the disk is 3-6 nm wider than adjacent lamellar regions, and tends to approximate the width of the terminal loop expansion along the disk perimeter. Within such dilatations, we have not been able to demonstrate crescentic densities corresponding to those found within the terminal loop region. Paired filament arrays are not randomly located within the lamellar domains, but lie parallel to the local disk perimeter. Both single and multiple arrays are commonly observed. Single arrays are recessed about 80 nm from the disk edge. When more than one array is present, the additional arrays lie parallel to the first and are spaced at intervals of about 70 nm. Thus, in three dimensions, the multiple arrays appear as regularly spaced sheets of filaments, parallel to the local disk perimeter and perpendicular to the lamellar domains. The characteristic interval between such filament arrays suggests the presence of 'spacer' molecules that organize the two-dimensional filament lattices in the third dimension. Such a spacing mechanism may also account for the minimum width observed for disk lobule segments defined by extended parallel incisures. The potential role of paired filament arrays in the axial propagation of incisures is discussed.

Adult rhesus and pigtail monkeys were exposed to a uniform field of light from daylight fluorescent lamps to determine the initial site and characteristics of structural retinal damage induced by continuous exposure and the threshold intensity required to produce the effects. Electron microscopic examination revealed that the initial site of damage is the photoreceptor outer segments. Damage remained restricted to the outer segments at intensities as high as 24,700 lux and with 12 hr exposure periods repeated for up to 4 days. Rods were swollen at the distal tip and showed disc membrane separation. Come outer segments were affected at the proximal end with vesiculation and membrane rearrangement. The threshold intensity for morphological changes to comes in the macula for a single 12 hr exposure was between 195 and 361 mu W/cm2 at the retina (400 to 700 mm; uncorrected for ocular transmittance), whereas the threshold for changes in rods was higher, at between 361 and 615 mu W/cm2. These levels correspond to between 5900 and 10,800 lux (550 to 1000 ft-cd) for comes and 10,800 to 19,400 lux (1000 to 1800 ft-cd) for rods of monkeys with fully dilated pupils. The paramacular areas of the retina were less sensitive to damage than macular areas of the same animal. No paramacular changes were observed in animals exposed to 10,800 lux or less. The patched (control) eyes of each monkey remained structurally normal in both the macula and paramacula at all exposure levels. These results demonstrate the sensitivity of the adult primate retina to damage by relatively moderate levels of light.

Chloroquine retinopathy was produced experimentally in the eye of the albino corydoras (one of the tropical fish) by daily administration of chloroquine (0.1 mg per os). The enucleated eyes were examined from the 14th day to 3 months after the beginning of drug administration under light and electron microscopy. The first change of retina was the appearance of membraneous cytoplasmic body (MCB) in the cytoplasm of ganglion, amacrine, bipolar and horizontal cells. MCB might be degenerated lysosome. They showed lamellar figures or crystalline lattice-like structures. Secondarily, these MCB appeared in the inner segments of photoreceptor cells. The outer segments of rod cells disappeared, and then those of cone cells. Although photoreceptor cells were diminished in number in advanced degeneration, the cells of inner nuclear layer and ganglion cells were maintained in number. The presence of MCB dose not mean death of cells. The retinal pigment epithelial cells contained MCB in its cytoplasm only in severe degenerative cases, and did not show other remarkable changes. MCB also appeared in the cytoplasm of pericytes of retinal vessels. Chloroquine is considered to damage directly photoreceptor cells most severely.

We compared the toxicity of the aminoglycoside antibiotics (tobramycin, amikacin, netilmicin, and kanamycin) by ophthalmoscopy, light and electron microscopy, and electro-retinography after intravitreal injection in rabbits in doses ranging from 100 to 3,000 micrograms. The earliest manifestations of toxicity were confined to the outer retina with each drug, with lamellar lysosomal inclusions in the retinal pigment epithelium as the earliest finding. However, the aminoglycosides displayed marked differences in the threshold dose required to produce toxic reactions, permitting the following ordering of toxicity: (most toxic) gentamicin greater than netilmicin = tobramycin greater than amikacin = kanamycin (least toxic).

A recessively inherited retinopathy in collies aged 8 to 189 days was studied by light and electron microscopy. The disease is produced when the outer segments of rods and cones fail to develop normally. Retinal pigment epithelial changes found in several litters appeared to form a separate disease entity. We compared the collie retinopathy with other canine models and the collie ectasia syndrome.

Sequences of X-ray diffraction patterns were obtained from dehydrating, artificially oriented multilayers of isolated, bovine rod outer segment disks. A direct-phase analysis was applied to highly hydrated specimens to determine sequences of low resolution (approx. 30 A) electron density profiles of the disks as dehydration proceeded. The profiles were found to evolve smoothly as the multilayer lattice simultaneously shrank and became increasingly ordered. The bilayer profiles were largely invariant under dehydration and the evolution of the diffraction consistent with simple decreases in fluid spacings. The specimens were observed to phase separate into characteristic primary and a secondary lattices when the multi-layer became too dehydrated. The small unit cell size of the secondary lattice was suggestive of a lipid phase. Large changes in the diffraction patterns from phase separated specimens were observed upon bleaching of the specimen. The changes were consistent with a reversible disordering of the primary lattice.

The external surface of rod outer segments contains receptors for the Jack Bean lectin, concanavalin A. Using isolated intact bovine rod outer segments, we have modified the density of the outer segment plasma membranes by means of polystyrene beads carrying covalently linked concanavalin A. After hypotonic lysis the bulk of the disk membranes can be removed and a plasma membrane fraction is isolated. The plasma membrane preparation contains 1.5% of total outer segment rhodopsin and 2.7% of total outer segment protein. it shows very little contamination with inner segment plasma membrane. Contamination with disk membranes appears to be low as well. Fatty acid analysis reveals that the plasma membranes are more saturated than the highly unsaturated disk membranes. Gel electrophoresis shows the presence of at least six additional polypeptides besides besides rhodopsin.

The membrane surfaces within the rod outer segment of the toad, Bufo marinus, were exposed by rapid-freezing followed by freeze-fracture and deep-etching. Platinum-carbon replicas of disk membranes prepared in this way demonstrate a distinct sidedness. The membrane surface that faces the lumen of the disk shows a fine granularity; particles of approximately 6 nm are packed at a density of approximately 30,000/micron 2. These dimensions suggest that the particles represent protrusions of the integral membrane protein, rhodopsin, into the intradisk space. In addition, when rhodopsin packing is intentionally perturbed by exhaustive digestion with phospholipase C, a concomitant change is observed in the appearance of the luminal surface granularity. The cytoplasmic surface of the disk rarely displays this rough texture; instead it exhibits a collection of much larger particles (8-12 nm) present at approximately 10% of the concentration of rhodopsin. This is about the size and concentration expected for certain light-regulated enzymes, cGMP phosphodiesterase and GTP-binding protein, which are currently thought to localize on or near the cytoplasmic surface of the disk. The molecular identity of the 8-12-nm particles will be identified in the following companion paper. A further differentiation of the cytoplasmic surface can be seen around the very edge, or rim, of each disk. This rim has relatively few 8-12-nm particles and instead displays short filamentlike structures connecting it to other membranes. These filaments extend between adjacent disks, across disk incisures, and from disk rims to the nearby plasma membrane.

Monoclonal anti-tubulin antibodies were used to label microtubules in the connecting cilia and outer segments of retinal photoreceptors isolated from Rana pipiens. In paraformaldehyde-fixed rods from frogs maintained on diurnal light cycles, the anti-tubulin labeling of ciliary microtubules (mean length = 27 micron) typically extends to slightly over half the length of the outer segments (mean length = 46 micron). Rod outer segments from frogs kept in constant darkness for 3-4 weeks are longer (mean length = 53 micron) than rod outer segments from frogs maintained in cyclic lighting. However, the distribution of fractional lengths of anti-tubulin labeling of ciliary microtubules is the same for both lighting regimens. Incubating retinas in 1.0 mM colchicine prior to outer-segment fixation has no effect on the length of immunolabeling of ciliary microtubules, suggesting that post-mortem elongation artifacts are not significant. Incubating retinas in 10 microM taxol prior to fixation significantly increases the length of stained ciliary microtubules, suggesting that taxol either promotes post-mortem assembly of microtubules, or that taxol reduces post-mortem disassembly. The mean position of the end of anti-tubulin-labeled ciliary microtubules does not correspond to the position of disk shedding for any of the experimental conditions employed.

A mouse monoclonal antibody was produced against salamander retinal membranes. It binds to the retina as well as to a wide variety of other salamander tissues and is called Sal-1. Because retinal neurons dissociated from the mature salamander retina adhere poorly to standard substrates, cells were plated onto coverslips previously treated with Sal-1. This previous treatment resulted in a dramatic increase in cell-substrate adhesion. At low concentrations, the antibody had no detectable effect on the light response and fine structure of freshly dissociated rod cells. After several weeks in culture, retinal neurons continued to be attached to Sal-1-treated coverslips and appeared healthy. Furthermore, many cells had extended elaborate cell processes and achieved morphologies characteristic of mature neurons. These results show that antibodies can be used as substrates for the culture of mature neurons. This technique may also prove useful for in vitro studies of a variety of cell systems.

A systematic electron microscopic study was made of the structure of foveal cones of Macaca spp. Transverse sections of inner (IS) and outer segments (OS) were made in sequence, from the pigment epithelial zone (PEZ) to the outer limiting membrane (OLM). The smallest diameters of hundreds of cone sections were measured from electron micrographs with a Zeiss particle-size analyzer, and analyzed statistically. Some details are also included about Cebus photoreceptors. It is claimed in the literature that foveal cones are rod-like (cylindrical) and untapered. Our study shows the foveolar cone to be a tapered structure. There has been some confusion between the foveola, which is rod-free, and the fovea, which has a high concentration of cones, but is not rod-free. Within the fovea, as the ratio of cones to rods falls from infinity to 1, with distance from the central bouquet of cones, the cone center-to-center distances increase, the inner segment diameters increase, and the number of cones/sq mm decreases. The tapered calycal processes are more massive in M. irus than M. mulatta, and the lateral fins are better developed. Lateral fins are not present in the foveola. The cones are arranged in straight lines.

Polyacrylamide gel and immunoelectrophoresis were used to measure the specific activity of nascent inner segment opsin following injections of labeled amino acids into frogs at different times during the diurnal cycle. Animals injected just after light onset showed the greatest incorporation of label into opsin, while animals injected during the night period had the lowest levels of isotope incorporation. As determined by rocket immunoelectrophoresis, there was a 13-fold decline in specific activity in animals injected during the late night hours in comparison with animals injected just after light onset. In contrast, rod outer segment rhodopsin-specific activity did not vary as greatly. Animals subjected to a 12-hour light-12-hour dark diurnal cycle incorporated only twice the radiolabeled amino acids into their rhodopsin compared with animals injected and maintained in constant dark for 24 hours. Taken together, these experiments suggest that adult Rana pipiens accumulate opsin within their inner segments during the dark phase of the diurnal cycle and that alterations of this pool effect changes in inner segment opsin specific activity. Differences in outer segment rhodopsin specific activity are probably due to averaged metabolic differences between constant dark and light-dark animals.

Male Sprague-Dawley weanling rats maintained on a controlled dietary zinc intake had an accumulation of osmiophilic inclusion bodies in the retinal pigment epithelium (RPE) of the zinc-deficient group. At 7 weeks of zinc deficiency there were marked ultrastructural alterations of the RPE. In some instances a deepening of the basal infoldings of the cells of the RPE were observed. There was also vesiculation and degeneration of the photoreceptor outer segments. No changes were observed in the pair-fed, weight-restricted, and ad libitum-fed controls. The possible functions of zinc in the retina are discussed.

A new procedure for assaying the phagocytosis of rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Using an ROS antiserum and a double immunofluorescent labeling procedure, ROS attached to the external surfaces of these cells can be distinguished from those that have already been ingested. We have used this procedure to study the phagocytosis of ROS by PE cells isolated from normal rats and rats with inherited retinal dystrophy (RCS rats). With this approach we have been able to show that the attachment of ROS to the external surfaces of dystrophic PE cells does take place to a normal extent. However, only a small number of these bound ROS are subsequently ingested, demonstrating that the ingestion phase of phagocytosis is defective. After a 4-hr incubation during which ROS are continuously present, normal rat PE cells ingest about 80% of the ROS that have bound to the cell surfaces. In contrast, after this time period, less than 20% of the ROS bound to the dystrophic PE cells have been ingested. These results, as well as the results of pulse-chase experiments in which ROS are rinsed away after two hours and the incubation continued without further addition of ROS, have demonstrated that normal PE cells rapidly ingest most of the bound ROS, whereas the dystrophic PE cells show no such rapid ingestion. Both cell types, however, are able to slowly ingest additional bound ROS with time.

It is shown that electron-phase-contrast images of unstained, melamine-embedded membranes, muscles and collagen fibers favorably compare with images recorded from corresponding preparations which have in addition been stained with heavy metals, with regard to both contrast and resolution of structural detail.

Birefringence variations, seen as regularly spaced altering light and dark rings (bands), have been observed along the length of unfixed, freshly isolated rod outer segments (ROS) of both Rana pipiens and Xenopus laevis. In our hands, the spatial frequency of the banding pattern is from 1.3-1.6u/band in Rana pipiens and 1.8-2.2u/band in Xenopus laevis ROS, both corresponding closely to determinations we made in the same animals of the quantity of new ROS disks added each day. To further probe this correlation, Xenopus laevis were maintained at 16 degrees C to lower the disk renewal rate. A similar correspondence was found, with the banding pattern and renewal rate both at 0.8-1.Ou/day. Further experiments involving Xenopus laevis placed on altered lighting cycles have suggested the existence of two normally superimposed periodicities. The more intense component is driven by the environmental light cycle, and thus may be regarded as diurnal. The less intense component is seen infrequently, suggesting lability, and apparently follows a 24-hour period in both constant light and darkness.

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and 31P nuclear magnetic resonance (NMR) techniques. 31P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25 degrees C and 45 degrees C. The 31P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca2+ and in the presence of 10 mM and 50 mM Ca2+. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca2+ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca2+, however, there was a change in morphology to non-lamellar structures. Removal of the Ca2+ caused the system to reassume the lamellar form.

Our experience in the growth of two-dimensional crystals of different proteins is presented. Polyethylene glycol was used to produce two-dimensional arrays of haemocyanin from O. vulgaris and of cholera toxin. The arrays showed a hexagonal close-packed structure of only randomly oriented molecules. The increase in protein concentration probably occurred too quickly to allow complete crystallization. Different two-dimensional arrays of hexameric haemocyanin molecules (from P. interruptus) were obtained by microdialysis through the specimen supporting film. A comparison was made with X-ray data. Two-dimensional tetrameric arrays of molecules, possibly rhodopsin, were seen in samples of bovine retinal rod outer segments in the presence of ammonium sulphate. Two-dimensional crystals of complex I (from bovine mitochondria) were prepared by dialysis in the presence of ammonium sulphate. A three-dimensional reconstruction was made from two tilt-series by computer filtration using the direct SIRT procedure. Finally, the possibility of computer crystallization using correlation techniques in combination with correspondence analysis is discussed.

Normal adult pigmented rats, born and raised in cyclic light for 25- to 27 days and then placed in darkness for up to 24 hr, showed an inverse relation between electroretinogram (ERG) sensitivity and phagosome frequency in the pigment epithelium over the course of a day. Linear regression revealed that a two-fold increase in the frequency of large phagosomes was associated with approximately a one-third decrease in ERG sensitivity. The observed 37-40% decline in ERG sensitivity 1.5 hr after expected light onset was significantly greater than what would be expected from the measured 11% shortening of rod outer segments at that time of day. Possible explanations for this disparity are considered.

Earlier studies have shown that visual function in skate is subserved solely by the rod mechanism and that the retina of this elasmobranch contains only rod photoreceptors. Nevertheless, the skate retina is capable of responding to levels of illumination that extend well into the photopic range, and we have detected in histological sections (usually from younger animals) small, proximally displaced, conelike photoreceptors which possibly represent another class of visual cell. However, ultrastructural and histochemical studies showed that the membranous discs of the outer segments of these cells were isolated from the plasma membrane, and that their synaptic terminals appeared immature and unlike those usually associated with cone receptors. In addition, the pattern of incorporation of 3H-fucose, as revealed by radioautography, was similar for both the rods and the smaller visual cells; i.e., the label was concentrated along the basal discs of the outer segment. When we examined the disc-shedding behavior of the visual cells in skates entrained for 2 weeks or longer to a 12-hour light:12-hour dark cycle, enhanced phagocytic activity was seen only following light onset; there was no significant increase following light offset. On the available evidence, it seems reasonable to conclude that the small visual cells are rods that have recently differentiated, and are growing and being incorporated into the photoreceptor layer of the retina.

The photoreceptor outer segments and retinal pigment epithelium of vitamin E deficient rats were examined by electron microscope using electron histochemical techniques. In rats fed a vitamin E deficient diet for more than 6 months starting 20 days after birth, the photoreceptor outer segment showed vesiculation of the discs near the apical portion of the retinal pigment epithelium. In the retinal pigment epithelium, secondary lysosomes were increased in number. Reaction products of acid phosphatase activity were located in secondary lysosomes in the retinal pigment epithelium and on the disc membranes of the photoreceptor outer segment. These alterations became more prominent with the duration of vitamin E deficiency. It was assumed that the disc degeneration of the photoreceptor outer segment might be due to enhanced activity of lysosomal enzymes in the retinal pigment epithelium and the effect of released lysomal enzymes on the photoreceptor outer segment.

At the Lawrence Berkeley Laboratory Bevalac Facility, iron nuclei were accelerated to an energy of 600 MeV/amu. The beam of iron thus obtained was used to irradiate living biological specimens in order to study possible microscopic tissue damage with the aid of SEM. The experiments involved total head irradiation of live rats which were subsequently returned to their cages to remain for 1 day and 30 days before further examination. After the 1 day and 30 day waits, both eyes were enucleated and placed in chemical fixative followed by ethanol dehydration and critical point drying. Retinas were carefully removed from the eye cups and loaded separately on aluminum stubs which were sputter coated. SEM of the 1 day and 30 day retinas revealed lesions which were not found at all in control retinas. The 1 day and 30 day retinas manifest regions where outer rod segments were missing or rearranged. A single energetic iron nucleus may be capable of generating a retinal lesion which becomes enlarged as biological processes intervene during the 1 day and 30 day waits. Being composed of highly specialized nerve cells, retinas cannot regenerate following irradiation which severely damages the rod cells. Thus one would expect the observed radiation induced retinal lesions to correspond to permanent tissue damage and possible loss of visual acuity in the intact animal.

Membrane turnover in outer segments of Rana pipiens red rods (ROS) was studied in tadpoles maintained under cyclic lighting (12L:12D) at 23 degrees, 28 degrees, and 33 degrees C. Large fragments (greater than 2 microns in diameter or length) were shed from the ROS tips shortly after the onset of light. These were phagocytized by the pigment epithelium (PE) which caused an increase in the number of phagosomes greater than 2 microns in size (large phagosomes). Large phagosomes were present in highest numbers 2-4 h after light exposure and were degraded by 8-12 h. The proportion of ROS that shed each day after the onset of the light cycle increased with increment increases in temperatures (23 degrees C-18%, 28 degrees C-33%, 33 degrees C-42% per day), resulting, in a reduction in the average interval of time between repeated sheddings (23 degrees C-5.6 days, 28 degrees C-3 days, 33 degrees C-2.4 days) though the average numbers of disks shed from ROS at the various temperatures were not significantly different (23 degrees C-139.5 +/- 5.7, 28 degrees C-129.4 +/- 7.6, 33 degrees C-129.9 +/- 4.8 disks/shed packet). Phagosomes in the PE that were less than 2 microns in diameter (small phagosomes) were present in relatively constant numbers throughout the day, and their numbers increased at higher temperatures. The absence of a concomitant increase in small phagosomes as large phagosomes were degraded indicates that large phagosomes were not the major source of small phagosomes. When the PE was isolated to culture in the absence of the retina, these small phagosomes were degraded. The rate of disk addition to the ROS base was determined by autoradiography after [3H]leucine injection. The number of disks added per day increased with elevations of temperature (23 degrees C-32.4; 28 degrees C-55.9; 33 degrees C-65.5). The average number of disks added to the ROS between repeated sheddings (23 degrees C-181.4; 28 degrees C-167.7; 33 degrees C-157.2) was greater than the number of disks shed after light exposure. Inasmuch as the ROS show no net increase in length during the tadpole stages utilized, the remaining disks must be lost at some other time. Electron microscope analysis revealed the presence of small groups of disks in curled configurations at the tips of ROS, suggesting possible stages of detachment.(ABSTRACT TRUNCATED AT 400 WORDS)

The retinal pigment epithelial (RPE) layer of a nocturnally active bird, the nighthawk (Chordeiles minor), has been investigated by electron microscopy. This layer is composed of a single layer of low cuboidal cells joined laterally by tight junctions. Basally (sclerally), the epithelial cells display deep infoldings while apically (vitreally) numerous processes enclose photoreceptor outer segments. Internally, the nucleus is large, vesicular and basally located. Smooth endoplasmic reticulum and mitochondria are abundant. Rough endoplasmic reticulum (RER) is scarce but numerous polysomes are present. Myeloid bodies are widespread and often appear to be continuous with the RER membranes. Phagosomes of outer segment material are present but not numerous in the light-adapted condition. Large spindle-shaped melanosomes as well as numerous rounded tapetal spheres are present in the apical region and apical processes of the epithelial cells. The composition of the reflective tapetal spheres is unknown. Bruch's membrane (complexus basalis) in most locations displays a pentalaminate appearance while in some regions it is trilaminate. The endothelium of the choriocapillaris facing Bruch's membrane is extremely thin but only sparsely fenestrated.

We have used the mouse monoclonal antibody 6-11 B-1, specific for acetylated alpha-tubulin, to determine the distribution of acetylated alpha-tubulin in in vitro-assembled microtubules and retinal tissue. Analysis by immunoblots revealed that microtubules assembled from bovine brain extracts contain both acetylated and nonacetylated alpha-tubulin. Immunofluorescence, using 6-11 B-1 and antitubulin B-5-1-2, a monoclonal antibody specific for alpha-tubulin, demonstrated the colocalization of both alpha-tubulin species in neurons of the retina and that acetylated microtubules are relatively abundant in neurons. However, analysis at higher resolution revealed that rod photoreceptors contain spatially distinct microtubule arrays which differ in content of acetylated alpha-tubulin and differ in stability. Acetylated microtubules which composed those of the rod outer segment and connecting cilium were resistant to depolymerization in nocodazole or colchicine. In contrast, the nonacetylated microtubules which composed those of the rod-inner segment were depolymerized in nocodazole or colchicine. Therefore, these acetylated microtubules are more resistant to depolymerization than non-acetylated microtubules.