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Four monoclonal antibodies obtained from the fusion of mouse myeloma cells with lymphocytes of mice immunized with bovine rod outer segment disc membranes were shown to bind to the surface of sealed discs. Radioimmune labeling of rod outer segment membrane proteins separated by sodium dodecyl sulfate gel electrophoresis indicated that two monoclonal antibodies (3D6 and 4B4) were against rhodopsin. Limited proteolysis of rod outer segment membranes with trypsin and Streptomyces griseus protease indicated that the 3D6 antibody bound to the trypsin-sensitive region close to the carboxyl-terminal end of rhodopsin. The 4B4 antibody bound at a trypsin insensitive, but S. griseus protease-sensitive internal region of rhodopsin accessible on the cytoplasmic surface of discs. Two other monoclonal antibodies (3D12 and 4B2) were found to bind to different regions of the Mr = 220,000 concanavalin A binding glycoprotein of rod outer segment disc membranes. Proteolysis studies indicated that these antibodies also bound to a Mr = 140,000 fragment which does not contain the concanavalin A binding site. Immunoferritin-labeling studies for transmission electron microscopy confirm the location of the 3D6 and 4B2 antigens on the cytoplasmic or interdisc surface of disc membranes.

We have studied sterol distribution in the retinal pigment epithelial (RPE) microvillous and outer segment disc membranes of rats with inherited retinal degeneration (RCS; RCS-p/+) and of normal genetic controls (RCS-rdy+, RCS-rdy+-p/+) by using the polyene antibiotic filipin, which binds specifically to 3-B-hydroxy-sterols, and freeze-fracture techniques. Retinas were perfusion-fixed, incubated with filipin in the same fixative, and prepared routinely for freeze-fracture electron microscopy. In the normal retina, the distribution of filipin binding sites on both RPE microvillous and outer segment disc membranes changes during development. Prior to outer segment elongation and the onset of phagocytosis (10 days postnatal), filipin sterol complexes are homogeneously distributed in both microvillous and outer segment membranes. With the onset of phagocytosis (2 weeks postnatal and later) filipin binding in both tissues forms a proximal-to-distal gradient, and binding sites decrease as distance from the cell body increases. In the normal RPE microvillous membranes, binding sites are numerous proximally and sparse on the distal tips. In the normal outer segment disc membranes, binding sites are often present on the basal discs, but are sparse on the intact apical discs prior to shedding. As the discs are cast off and engulfed by the RPE, however, filipin binding increases on both disc and phagosome membranes. In the dystrophic retina, the distribution of filipin binding sites differs from the normal. First, in the microvillous membranes, the proximal-to-distal gradient in filipin binding is rarely present at 2 weeks postnatal and becomes prominent only after the buildup of membranous debris has begun (3-5 weeks postnatal). Second, as the photoreceptors degenerate and the membrane debris disappears (4 months postnatal), filipin binding on the microvillous membranes becomes relatively sparse and homogeneous. Third, filipin binding on the intact disc membranes does not change with outer segment elongation, and numerous filipin binding sites are present on both apical and basal outer segment disc membranes. Fourth, large aggregates of filipin binding sites occupy the vast expanses of particle-free areas of debris membranes which accumulate between the photoreceptors and the RPE. These changes in the amount and distribution of filipin binding sites in the dystrophic retina add to the evidence that the disease process involves outer segment as well as RPE membranes and suggest that alterations in cholesterol distribution could contribute to the phagocytic defect.

Isolated Xenopus laevis retinas were incubated with 3H-labeled mannose or leucine in the presence or absence of tunicamycin (TM), a selective inhibitor of dolichyl phosphate-dependent protein glycosylation. At a TM concentration of 20 micrograms/ml, the incorporation of [3H]mannose and [3H]leucine into retinal macromolecules was inhibited by approximately 66 and 12-16%, respectively, relative to controls. Cellular uptake of the radiolabeled substrates was not inhibited at this TM concentration. Polyacrylamide gel electrophoresis revealed that TM had little effect on the incorporation of [3H]leucine into the proteins of whole retinas and that labeling of proteins (especially opsin) in isolated rod outer segment (ROS) membranes was negligible. The incorporation of [3H]mannose into proteins of whole retinas and ROS membranes was nearly abolished in the presence of TM. Autoradiograms of control retinas incubated with either [3H]mannose or [3H]leucine exhibited a discrete concentration of silver grains over ROS basal disc membranes. In TM-treated retinas, the extracellular space between rod inner and outer segments was dilated and filled with numerous heterogeneously size vesicles, which were labeled with [3H]leucine but not with [3H]mannose. ROS disc membranes per se were not labeled in the TM-treated retinas. Quantitative light microscopic autoradiography of retinas pulse-labeled with [3H]leucine showed no differences in labeling of rod cellular compartments in the presence or absence of TM as a function of increasing chase time. These results demonstrate that TM can block retinal protein glycosylation and normal disc membrane assembly under conditions where synthesis and intracellular transport of rod cell proteins (e.g., opsin) are not inhibited.

The retinal pigment epithelium (RPE) interacts with the photoreceptors, which it faces across the subretinal space. In these interactions the RPE acts as three types of cell - epithelium, macrophage, and glia. This review briefly describes selected interactions between the RPE and photoreceptors in ion and water transport, Vitamin A transport, phagocytosis of shed portions of outer segments, ensheathment of photoreceptors outer segments, and electrical responses. The electrical interactions can be recorded at the cornea in the c-wave, fast oscillation, and light peak of the DC electroretinogram (DC-ERG) and electrooculogram (EOG). Each response reflects photoreceptor-RPE interactions in a distinct way. The three responses taken together provide perhaps the best opportunity to learn how pathophysiological conditions alter the interactions between the RPE and photoreceptors.

Microtitration plates were used to culture simultaneously multiple, small (6 mm diameter) explants of bovine retinal pigment epithelium (RPE). Evaluation of tissue by light microscopy and by scanning and transmission electron microscopy after various incubation periods up to 6 h showed that RPE maintained in this system retains near normal morphology. Initially, the explanted RPE lacks apical microvilli, but during the first 2-3 h in culture recovery of apical microvilli occurs. The results suggest that the system is suitable for short-term maintenance of RPE for experimental purposes. Moreover, the ability to culture up to 16 explants from one bovine eye aids statistical evaluation of RPE behaviour under varying experimental conditions.

In addition to rhodopsin, the disk membranes of rod outer segments (ROS) contain a large integral membrane protein (mol. wt 290,000). This protein was previously localized by immunocytochemistry to the margins and incisures of disks in frog red ROS by specific antibody applied to thin sections of bovine serum albumin embedded retinas (Papermaster et al., 1978b, J. Cell Biol. 78, 415-425). Upon further study of the reactions of this antibody with outer segments of other photoreceptor classes in frog retina, labeling of the short incisures and margins of green ROS and margins of cone outer segment lamellae is also observed. Thus the large protein participates in the structure of the edges of disks and lamellae of all photoreceptors in the frog. In addition, labeling of the inter-incisure surface of all photoreceptor classes was observed at high antibody concentration. In order to interpret this labeling, the effect of dilution on labeling density was determined and double reciprocal plots (Markham and Benton, 1931, J. Am. Chem. Soc. 53, 497) were employed to evaluate the relative affinity and heterogeneity. There was considerable deviation from linearity in the plots of labeling disk interiors compared to the relatively linear plots of disk incisure labeling which suggests that the interior sites contain a weakly cross-reacting antigen or that the serum contains a lower concentration of antibody weakly reactive with another antigen.

The ultrastructure of single and twin cone photoreceptors in the retina of the walleye was analyzed by scanning and transmission electronmicroscopy. The outer segment disks resemble those of other vertebrates. An accessory outer segment arises from the inner segment, makes frequent contacts with the outer segment proper, and may thus provide a bridge for signal transmission from outer to inner segment. A palisade of some 30 calycal processes surrounds and may provide structural support for the outer segment. The region of apposition between the inner segments of twin cones consists of a space of some 28 nm with no indication of gap junctions. The proximal quarter of the inner segment displays a profusion of some 50 lateral fins which increase the inner segment surface by 3-4 X and do not contact fins of neighboring cones. The fins surround a profusion of Müller cell microvilli and probably promote metabolic exchange between cones and Müller cells. Apart from differences in size and the presence of apposed inner segments, twin and single cones appear to be morphologically similar.

Advanced thioridazine retinopathy was observed clinically and histopathologically in a 61-year-old man with progressive, severe loss of vision who had received thioridazine in high dosages 18 years previously. The ocular fundi showed multiple confluent, nummular areas of hypopigmentation and choroidal atrophy posterior to the equator. Atrophy and disorganization of the photoreceptor outer segments appeared to be the initial degenerative event, followed by loss of the retinal pigment epithelium and choriocapillaris.

Actin has been localized in Rana pipiens retinas that were fixed and embedded in aldehyde cross-linked BSA. Thin sections were reacted sequentially with (a) affinity-purified antiactin antibodies induced in rabbits; (b) biotinyl-sheep anti-rabbit antibodies; and (c) avidin-ferritin conjugates. As expected, antiactin labeling density was high in the apical pigment epithelial cell processes and in the calycal processes of photoreceptors. Actin was also localized in a new site. The connecting cilium that joins the inner and outer segments of both rods and cones was heavily labeled by antiactin at its outer segment (OS), or distal, end. In this region of the cilium, the plasma membrane evaginates to form new OS disks and these basal disks were labeled in some instances. Below the new disks in rods, the cytoplasm of liplike expansions of the distal cilium was also heavily labeled. The plasma membrane and interior of the connecting cilium and the remainder of the OS were unlabeled. These findings suggest that actin may participate in the vectorial transport of opsin and other intrinsic membrane proteins that are incorporated into newly forming OS disks. The results also implicate actin in the membrane expansion involved with OS disk formation.

Biochemical and immunochemical studies indicate that rod outer segments (ROS) of bovine photoreceptor cells contain a Mr 240,000 polypeptide related to the alpha-subunit of red blood cell (RBC) spectrin. With the use of sodium dodecyl sulfate gel electrophoresis in conjunction with the immunoblotting technique, monoclonal antibody 4B2 was found to bind to a Mr 240,000 polypeptide in ROS that is distinct from the prominent Mr 220,000 concanavalin A binding glycoprotein. The Mr 240,000 polypeptide is highly susceptible to degradation by endogenous proteases. It does not appear to be an integral membrane protein but is tightly membrane associated since it can be partially extracted from ROS membranes with urea in the absence of detergent. The 4B2 antibody cross-reacted with RBC ghosts and bovine brain microsomal membranes. Radioimmune assays and immunoblotting analysis of purified bovine RBC spectrin further revealed that the 4B2 antibody predominantly labeled the alpha-chain of RBC spectrin having an apparent molecular weight of 240,000. Polyclonal anti-spectrin antibody that bound to both the alpha- and beta-chain of RBC spectrin predominantly labeled a Mr 240,000 polypeptide of ROS membranes. Two faintly labeled bands in the molecular weight range of 210,000-220,000 were also observed. These components may represent variants of the beta-chain of spectrin that are weakly cross-reacting or present in smaller quantities than the alpha-chain.(ABSTRACT TRUNCATED AT 250 WORDS)

In recent theories of visual transduction, an active form of rhodopsin plays a decisive role. If metarhodopsin II is considered to be a likely candidate for this role, its properties and reactions are of interest. A scheme describes how the various metarhodopsins react with each other. In the electrophysiological experiments, the dark currents and the photocurrents generated by the plasma membrane of the outer segments are analyzed. While the dark currents are computed from voltage gradients and the interstitial resistance in the receptor layer, the photocurrents are directly from single cells under voltage-clamp conditions.

The plasma membrane and disk membranes of bovine retinal rod outer segments (ROS) have been purified by a novel density-gradient perturbation method for analysis of their protein compositions. Purified ROS were treated with neuraminidase to expose galactose residues on plasma membrane-specific glycoproteins and labeled with ricin-gold-dextran particles. After the ROS were lysed in hypotonic buffer, the plasma membrane was dissociated from the disks by either mild trypsin digestion or prolonged exposure to low ionic strength buffer. The dense ricin-gold-dextran-labeled plasma membrane was separated from disks by sucrose gradient centrifugation. Electron microscopy was used to follow this fractionation procedure. The dense red pellet primarily consisted of inverted plasma membrane vesicles containing gold particles; the membrane fraction of density 1.13 g/cc consisted of unlabeled intact disks and vesicles. Ricin-binding studies indicated that the plasma membrane from trypsin-treated ROS was purified between 10-15-fold. The protein composition of plasma membranes and disks was significantly different as analyzed by SDS gels and Western blots labeled with lectins and monoclonal antibodies. ROS plasma membrane exhibited three major proteins of 36 (rhodopsin), 38, and 52 kD, three ricin-binding glycoproteins of 230, 160, and 110 kD, and numerous minor proteins in the range of 14-270 kD. In disk membranes rhodopsin appeared as the only major protein. A 220-kD concanavalin A-binding glycoprotein and peripherin, a rim-specific protein, were also present along with minor proteins of 43 and 57-63 kD. Radioimmune assays indicated that the ROS plasma membrane contained about half as much rhodopsin as disk membranes.

A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.

We used specific antibodies in immunoblot studies of membrane fractions derived from bovine retina, and in immunohistochemical studies of sections of rat retina to determine the distribution of two guanine nucleotide binding proteins Go (a G-protein of unknown function discovered in the brain) and transducin, in retina. Both Go and transducin were readily detected in membranes derived from whole retina, and in crude rod outer segment membranes. Purification of rod outer segment membranes by sucrose density gradient centrifugation resulted in enrichment in transducin and depletion of Go immunoreactivity. Transducin-alpha immunoreactivity was localized to photoreceptor inner and outer segments and the outer nuclear layer. In contrast, Go-alpha immunoreactivity was localized in the inner and outer plexiform layers and ganglion cell layer. The results indicate that Go unlike transducin, is not associated with rod outer segment membranes and is therefore unlikely to function in phototransduction. Go is, however, relatively abundant in neural layers of retina where it may be involved in signal transduction.

Rod photoreceptors show a discrete band of labeled molecules at the outer segment base shortly after the administration of radioactive protein precursors (Young, 1967). The band signifies the insertion of radiolabeled protein, primarily opsin, into rod disc membrane (Basinger, Bok and Hall, 1976). In contrast, the autoradiographic labeling pattern in cones is characteristically diffuse (Young, 1971), although cones as well as rods are now thought to replace their disc membranes continually (Anderson, Fisher, and Steinberg, 1978). Using grain count analysis in conjunction with electron microscope autoradiography, we demonstrate for the first time that the initial cone outer segment (COS) labeling pattern in the retinas of cone-dominant rodents is not random or diffuse after intraocular injection of [3H]-fucose--a specific glycoprotein precursor. Thirty minutes after [3H]-fucose injection, the Golgi complex is heavily labeled in both rods and cones. At 1.5 hr, labeling first appears in the periciliary region and in the basal portion of the COS. We detected a basal concentration of fucosylated molecules up to 12 hr after injection; thereafter, we found no differential distribution of labeled product molecules in COS. In rods, we observed no band of labeled protein at any of the time points sampled. The low level of labeling in rods is uniformly distributed between the basal and apical portion of the outer segments. Although the identity of the fucosylated material remains unknown, the early labeling of the Golgi complex--followed by labeling over the periciliary region and the basal portion of the outer segment--suggests that newly synthesized membrane protein is transported from sites of synthesis in the inner segment and inserted into disc membrane at the COS base. However, in contrast to rods, the protein is apparently capable of diffusing longitudinally throughout the interconnected membrane network.

After experimental retinal detachment in the cat, a number of morphological changes take place in retinal and RPE cells. Following reattachment, the ultrastructural relationship between the photoreceptors and the RPE is re-established, but it does not return to the predetachment state even after short detachment episodes coupled with prolonged recovery periods. All of the reattached retinae show some degree of abnormality, ranging from subtle changes in photoreceptor ultrastructure to dramatic degenerative effects in the outer retina. Abrupt transitions in morphology from one reattached area to an adjacent area are not unusual. Photoreceptor recovery varies widely between animals, and between adjacent regions within the same retina. Ensheathment of outer segments by RPE apical processes is abnormal. In some reattached areas rod outer segment dimensions and disc structure are near normal as is the displacement rate of rod outer segment discs. In others, especially in areas of RPE or Müller cell proliferation and hypertrophy, the outer segments are shortened or absent completely, and there is a reduction of cell bodies in the outer nuclear layer. In some retinae, recovery in cones is inferior to that in rods. At short detachment durations (less than 1 wk) morphological recovery in the reattached retina is optimal while at long intervals (greater than 1 month) recovery is poor. The changes at the photoreceptor-RPE interface identified in the reattached cat retina probably have adverse effects on visual recovery when they occur within the human macula.

The disc incisures in the bullfrog retinal rod were examined by electron microscopy. The number of disc incisures is proportional to the cross-sectional perimeter of the outer segment. While the diameter of the red rod outer segments varies with their location along the vertical meridian of the retina, the incisure number also changes similarly. It is estimated by regression analysis that a single disc lobule, bordered by two adjacent incisures, has on the average about 1 micron of arc-like rim edge. When red and green rod discs are compared at their middle outer segments in the ventro-central retina, the disc incisures are 1.8 times more numerous and 1.5 times deeper in the red rod (mean incisure number, 27; mean length, 2.7 microns) than in the green rod (15; 1.8 microns). These differences result in the total disc edge of the red rod (mean, 178 microns) being two times longer than that of the green rod (85 microns). The disc incisures emerge at the basal end of the outer segment and on the average become linearly longer toward the middle outer segment. During displacement of discs over the distance of 2.5 microns, which covers about 90 discs, the incisures reach more than 90% of their maximum length of 2.7 micron in the middle outer segment. In addition, the process of incisure elongation has a kind of fluctuation in that the incisure frequently shows a sharp decrease and increase in length accompanied by a change in course in the disc surface.

The ability of cholesterol to move between bovine rod outer segment disk membranes and phospholipid membranes was examined. Disk membranes were incubated with small unilamellar phospholipid vesicles containing varying amounts of cholesterol. Aliquots were removed at specific times, and then the disks and the vesicles were separated by centrifugation and assayed for phospholipid and cholesterol content. When incubated with vesicles containing no cholesterol, the cholesterol to phospholipid ratio in the disk membrane was reduced due to migration of cholesterol from the disks into the vesicles. The cholesterol content of these cholesterol depleted disks could be readily returned to the normal disk cholesterol content by incubation of the cholesterol-depleted disks with small unilamellar vesicles containing high cholesterol. An apparent partition coefficient K was calculated as the quotient of the cholesterol/phospholipid mole ratio in the donor membranes and the cholesterol/phospholipid mole ratio in the acceptor membranes. The value of K was approximately 1 at cholesterol levels below normal disk cholesterol content, for disk membranes and phosphatidylcholine small unilamellar vesicles. Inclusion of phosphatidylethanolamine in the small unilamellar vesicle acceptor raised K, indicating that phosphatidylethanolamine creates an unfavourable environment for cholesterol. The cholesterol to phospholipid ratio of native disks could be increased by incubation with phosphatidylcholine small unilamellar vesicles (donor) which contained higher amounts of cholesterol than the disk membrane acceptor. In these experiments the distribution of cholesterol between disks and small unilamellar vesicles always favored the vesicles. The apparent partition coefficient was 1.7 at several cholesterol levels above the native disk cholesterol content. Liposomes made from lipid extracted from the disk membrane behaved in the same manner as intact disks with respect to cholesterol distribution at equilibrium. The phospholipid content of the disk membrane may be an important factor in determining the cholesterol content of the disk membrane.

The effect of light deprivation on the retinal degeneration induced by taurine deficiency in cats was examined in this study. After 25 weeks of taurine-free diet, taurine levels in plasma, retina, heart, cerebellar and cerebral cortex decrease to 16-25% of normal. The ERG a-wave and b-amplitudes also decreased as a consequence of the taurine-free diet. After 15 weeks of taurine-free diet the b-wave amplitude decreased to 36-41% of normal, and after 25 weeks both the a-wave and the b-wave were undetectable. The structure of the outer segments of photoreceptors, examined by electron microscopy appeared disturbed in the taurine-deficient cats, showing disorientation of the disk membranes, vesiculation and swelling. The time course of taurine deficiency and the retinal degeneration pattern were observed in all cats fed the taurine-free diet, irrespective of the conditions of lighting. These results indicate that the morphological and functional disturbances induced by taurine deficiency in cats are independent of the physiological stimulation of the retina by light.

Complementary replicas of samples prepared for electron microscopy by the freeze-fracture/etch technique are extremely valuable in the interpretation of the exposed surfaces, the nature and location of the membrane fracture plane, and as an aid in the recognition of the potential artefacts of this technique. This paper describes a procedure for the preparation of complementary replicas of thin samples sandwiched between copper foil strips and frozen ultra-rapidly in the absence of chemical pretreatments. In this procedure, the copper foil support bearing the replica is floated on the surface of a chromic acid solution, resulting in the controlled dissolution of the copper metal. The replica which remains at the surface of the chromic acid is then stabilized against fragmentation during subsequent cleaning and rinsing steps by placing a 50 mesh gold grid on top of the replica. To minimize agitation of the replica/grid, all cleaning steps are performed in a single depression plate well. The clean replica/grid is picked up from below on a thin Formvar film, dried, and then separated from the extra film. Careful placement of the gold grid on the replicas and low magnification electron micrograph montages of the complementary grids facilitate the location of complementary regions and simplify examination of complementary specimen areas at higher magnification.

When biological materials are infiltrated by a water-soluble melamine resin and hardened, they become as hard as glass. This is a prerequisite for extreme thin-sectioning. In this paper, the structural information from unsupported transparent thin sections of beef liver catalase, calf thymus DNA, horse spleen ferritin, insect muscle and rat microtubules is compared to that of normal thin sections. While ferritin molecules (12 nm diameter), microtubule subunits (8 nm long axis) and catalase crystals (8 nm subunit diameter) appear to become mechanically damaged in a 10 nm section (as measured by resectioning), DNA-molecules (3 nm diameter) are satisfactorily preserved during sectioning. Remarkably, for electron phase contrast imaging of unstained cross-sectioned insect muscle, a minimum section thickness of about 30-40 nm is required.

In the previous article the authors reported that the ingestion phase of phagocytosis is defective in cultured dystrophic rat pigment epithelial (PE) cells. When these cells are challenged with isolated rod outer segments (ROS), attachment of ROS to the PE cell surfaces occurs to a normal extent. However, only a small number of these bound ROS are subsequently ingested. This raised the possibility that the contractile protein actin might not function normally in the dystrophic rat PE cells, since actin is intimately involved in the ingestion mechanism in other phagocytic cells. Utilizing actin antibodies and the technique of indirect immunofluorescence, we have studied the distribution of actin in cultured normal and dystrophic rat PE cells. Results show that the arrangement of actin fibers in the dystrophic cells appears normal both before and during the attachment of ROS to the cell surfaces. With the additional use of an ROS antiserum to label externally bound ROS, it is also possible to show that actin is involved with the ingestion of ROS by both normal and dystrophic PE cells. Thus, it appears that actin can function normally in dystrophic PE cells, but that the ingestion mechanism becomes activated at only a few sites of ROS attachment. The results of a scanning electron microscope study support this conclusion and also show the presence of a saucer-shaped elaboration of the PE cell plasma membrane beneath attached ROS. These may correspond to the actin feltworks seen with immunofluorescence microscopy at sites of ROS attachment.

Cytoplasmic membranes of rod outer segments from frog retina intact rods in retina were stained with fluorescent dye fluoresceinmonomercur acetate. The dye is covalently bound to proteins of cytoplasmic membrane and doesn't penetrate into the cells. Upon isolation of the purified outer segments with the labeled cytoplasmic membranes the cells were disrupted and fractionated in density sucrose gradient. Cytoplasmic membranes possess floating densities different from those of disk membranes and thus providing a mean for separating them from the latter. The main peptides of cytoplasmic membranes are 56, 53, 45, 30 and 28 kDa proteins.

A form of retinal degeneration in the Wistar-Furth strain of rat is described. The changes observed was classified as either mild or severe, depending on the severity of the dystrophy seen by light and electron microscopy. In the mild stage, there was a slight decrease in the number of photoreceptor nuclei in the outer nuclear layer (ONL) and there were pyknotic nuclei in the inner nuclear layer. A few photoreceptor outer and inner segments showed focal swellings. The basal infoldings of the retinal pigment epithelium (RPE) were distributed irregularly, creating spaces that contained collagen-like fibrils. However, the interepithelial tight junctions remained impermeable to intravenously injected peroxidase. In severely dystrophic retinas, there was a further decrease in the width of the ONL, and the photoreceptor outer and inner segments were degenerated. In extreme cases, the photoreceptor cell disappeared completely and the inner retina abutted the RPE. Further changes were also noted in the basal membrane of the RPE; in some regions the membrane was totally flat while in others it formed exaggerated infoldings. In addition, the RPE was frequently vascularized by vessels that originated from the retina. The endothelium of those intra-RPE vessels located near Bruch's membrane was frequently fenestrated. Alterations also occurred in the retinal capillaries; some were degenerated, while others showed increased tortuosity or focal thickenings of the basal lamina. In addition, there were focal increases in the amount of stroma between Bruch's membrane and the choriocapillaris.

Electron density profiles of disk membranes isolated from bovine retinal rod outer segments have been determined to 12 A resolution by analysis of the X-ray diffraction from oriented multilayers, in the absence of lipid phase separation. Data were collected on both film and a two-dimensional TV-detector; both detectors yielded identical patterns consisting of relatively sharp lamellar reflections of small mosaic spread. The unit cell repeat was reversibly varied over the range of 143 to 183 A. The diffraction patterns changed dramatically at 150 A; consequently, the low (less than 150 A) and high (greater than 150 A) periodicity data were independently analyzed via a swelling algorithm. The high periodicity data yielded two statistically equivalent phase choices corresponding to two symmetric, but different membrane profiles. The low periodicity data yielded essentially one, characteristically asymmetric profile. These profiles have been modeled with regard to the separate profiles of rhodopsin, lipid and water, subject to the known composition of the isolated disk membranes.

The structure of the photoreceptors of the vervet monkey (Cercopithecus aethiops) has been investigated by light and electron microscopy. In this species the photoreceptors can be readily differentiated and adequately described by the classical terminology of rods and cones, with rods being the more numerous. Rods are long, slender cells while cones are shorter and stouter. Both rods and cones are highly differentiated cells and consist of an outer segment, a connecting cilium, and inner segment, a nuclear region and a synaptic process leading to a synaptic ending. Morphological differences are noted between rods and cones for the various regions of these cells.

To investigate the hypothesis that sugar molecules might act as markers for ROS disc phagocytosis, frog eyecups were incubated in a normally permissive medium for ROS disc shedding with and without added simple sugars. L-fucose, alpha-methyl-D-mannopyranoside and D-mannose all significantly reduced the numbers of packets of ROS discs found in the retinal pigment epithelium. D-fucose, L-mannose, D-fructose, D-galactose, D-glucose and sucrose were without significant effect at the same concentration. Ultrastructural examination of the retinas indicates that the sugars were effective on the disc shedding process rather than on phagocytosis of already shed disc packets.

The rod-outer-segment disk membranes were studied in cattle retinas by electron microscope observation of ultrathin resin sections and complementary freeze replicas. In sections, the membranes are characterized by discontinuous electron densities along the unit membrane structure, in which electron-less-dense globular substructures, forming an intermediate layer, are enhanced. Such substructures are estimated as 60 A in diameter. In freeze replicas, the membranous particles of 77 A in diameter are distributed quite densely in the P-face and scattered in the E-face. Like a number of preceding investigators who have failed to obtain convincing findings to correlate similar profiles with the visual pigment molecules, we should also raise a question about preparation involving chemical and thermal procedures affecting the dimensions of the final images. Our constant results obtained from the two methods, however, will suggest that the thin-section substructures and freeze-replica particles represent two different phases respectively of the same molecular existence rather than a complete profile of the visual pigment. It is also uncertain yet whether one membranous particle should be in fact associated to one molecular pigment or an aggregation of several molecules. The last problem will be resolved to some extent by topographical analyses of the complementary replicas in progress in our laboratory.

Retinal neurons from rats acutely intoxicated with trimethyltin (TMT) were examined by light and electron microscopy to determine if there is a relationship between the subcellular response of a neuron to TMT and its morphologic subspecialization. Subcellular pathologic alterations were present in neurons from all three cellular layers of the sensory retina. However, the type and degree of subcellular response varied among the highly subspecialized neurons of the different retinal layers. Clusters of dense-cored vesicles and tubules were mainly limited to neurons of the ganglion-cell layer, large accumulations of dense bodies were mainly limited to neurons of the inner nuclear layer, and neuronal necrosis was mainly limited to the photoreceptor cells. The inner segment of the photoreceptor cell shared with the perikaryon of more conventional neurons a special vulnerability to TMT cytotoxicity. Our results suggest that the morphologic subspecialization of neurons affects the type and the degree of subcellular response to TMT.

An animal model has been developed in this study to produce commotio retinae (Berlin's edema) by means of standardized, non-penetrating B.B. pistol injury to the cornea. Parameters have been established to ensure uniform blunt injury, enabling study of the retina and blood-retinal barrier by light- and electron microscopy, including use of horseradish peroxidase as a vascular tracer. Retinal whitening and swelling were found in the peripapillary and central regions, as occurs in the human. The earliest damage involved breakage of the connecting cilia of rods and cones, with rapid disorganization of the outer segments. Later changes included swelling of photoreceptor inner segments and breakdown of the outer blood-retinal barrier at the level of the retinal pigment epithelium. The outer blood-retinal barrier was re-established between 7 and 14 days, and at the longest survival (56 days), incompletely regenerated outer segments were present. This model for commotio retinae provides a new approach for the study of outer-segment regeneration in rods and cones after mechanical injury, as well as mechanisms that underlie re-establishment of the blood-retinal barrier following non-penetrating trauma to the eye.

The influence of cyclic GMP on the structural state of photoreceptor membranes was studied. The spin probe technique was used to obtain the dependences of microviscosity of lipid domains of membranes on cGMP concentration. It was shown that microviscosity of 5-doxylsteraric acid environment in rod outer segment, disc and plasma mambranes changed at concentrations of 2.10(7); 5.10(-6) and 6.10(-6). Non-cyclic GMP was ineffective. The ultrasound treatment of membraneous samples resulted on disappearance of the structural changes.

A model for random cross-linking of identical monomers diffusing in a membrane was formulated to test whether rhodopsin's cross-linking behavior was quantitatively consistent with a monomeric structure. Cross-linking was performed on rhodopsin both in intact retinas and in isolated rod outer segment (ROS) membranes using the reagent glutaraldehyde. The distribution of covalent oligomers formed was analyzed by SDS-polyacrylamide gel electrophoresis and compared to predictions for the random model. A similar analysis was made for ROS membranes cross-linked by diisocyanatohexane and retinas cross-linked by cupric ion complexed with o-phenanthroline. Patterns of cross-linking produced by these three reagents are reasonably consistent with the monomer model. Glutaraldehyde was also used to cross-link the tetrameric protein aldolase in order to verify that cross-linking of a stable oligomer, under conditions comparable to those used for ROS, yielded the pattern predicted for a tetrameric protein having D2 symmetry. This pattern is markedly different from the one for a random-collision model. Moreover, a comparison of rates showed that aldolase cross-linking with glutaraldehyde is significantly faster than cross-linking of membrane-bound rhodopsin. It is concluded that rhodopsin is monomeric in dark-adapted photoreceptor membranes and that the observed cross-linking results from collisions between diffusing rhodopsin molecules.

A cytochemical method was developed for localization in isolated rod outer segments of manganese-dependent pyrimidine 5'-nucleotidase (MDPNase), an enzyme activity with possible relevance to shedding that we recently reported in photoreceptors and retinal pigment epithelial (RPE) cells in the intact rat retina. The purpose of this study was to eliminate the possibility that the previously observed cytochemical staining of the rods was due to diffusion of reaction product from the RPE cell lysosomes, which were also heavily stained. Rod outer segments (ROS) were isolated on continuous sucrose gradients from retinal homogenates prepared from rats raised in cyclic light (12 hr light:12 hr dark) and killed during the first 2 hr after light onset. ROS-containing bands were removed from the gradients and the isolated rods were fixed in 0.25% glutaraldehyde and pelleted. Chopped sections of the pellets were incubated in cytochemical medium for MDPNase activity and processed for light- and electron-microscopic localization of the enzyme activity. Two patterns of cytochemical staining were seen in ROS isolated from retinas obtained at this time of day. A few of the pellets contained clusters of ROS that were heavily coated along their surfaces and seemingly interconnected by thick strands of highly reactive extracellular material that displayed a punctate pattern of cytochemical staining. This material may have originated from the apical processes of the RPE cells, which were heavily stained in tissue fixed in situ around the time of light onset. The second staining pattern, visible only by electron microscopy, was more commonly observed. In the majority of the isolated ROS profiles, discrete streaks of cytochemical reaction product were seen in association with the internal aspects of the discs, at sites that seemed to correspond to the rims, and to narrow zones within the disc interiors. This distribution of reactive sites closely resembled that observed over most of the length of the ROS in the intact retina fixed at the same time of day. Occasionally, ROS profiles were encountered in which additional reactive sites were localized to the interdisc spaces between the plasma membrane and the rims of the discs. The latter pattern resembled the distribution of reaction product seen during this period over the tips of the ROS fixed in situ. As in the intact retinas, the cytochemical staining of the isolated ROS was inhibited by fluoride ions and strongly stimulated by manganese ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Photoreceptors can segregate inner segment proteins from outer segment proteins. It is uncertain whether such a sorting process occurs during disc morphogenesis in rods, thereby resulting in an outer segment plasma membrane which differs compositionally from the discs. In this study, the plasma membranes of intact, purified frog rod outer segments (ROS) have been labeled with a peanut lectin (PNA), either with or without prior neuraminidase treatment of the ROS. Neuraminidase removes terminal sialic acid residues from membrane-bound sialoglycoconjugates. PNA was demonstrated to bind to the plasma membrane of ROS only after neuraminidase treatment, as detected by fluorescence light microscopy and electron microscopy. In order to identify the sialoglycoproteins responsible for this labeling, ROS proteins were resolved by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper for incubation with PNA. A single, prominent band at 125 Kd bound PNA after neuraminidase treatment of ROS. This band represents a protein which is an integral component of the ROS plasma membrane and is not associated with the interphotoreceptor matrix or other potential contaminants. Selective degradation of ROS prior to neuraminidase treatment indicates that this sialoglycoprotein is absent from discs and, thus, can serve as a marker specific for ROS plasma membrane during fractionation studies.

The hypothesis that melatonin regulates circadian rod outer segment disc shedding in mammals was tested by determining the effect of exogenous melatonin on histological phagosome counts. Melatonin was administered as a subcutaneous implant or injection to photoentrained albino rats. Retinas of treated rats and paired controls were obtained at various times including the time of expected disc shedding. Phagosome counts on electron micrographs were converted to size-frequency distributions. Injected melatonin did not induce abnormal circadian shedding. However, implanted melatonin increased the frequency of large phagosomes (P less than 0.01). These data further implicate melatonin in the control of disc shedding.

Albino rats were born and raised through 12 weeks of age in 12L:12D regimes of 5, 300- or 800-lx illuminance. Upon killing, the animals' retinas were examined for the following: (1) rhodopsin of whole retina and isolated rod outer-segment membrane; (2) retinal morphology, including outer segment length and outer nuclear layer area; and (3) outer-segment membrane lipid biochemistry. The three groups of animals show significant differences with respect to one another for nearly every parameter measured. Rod outer-segment membranes of rats raised in dim cyclic light (5 lx) have high rhodopsin packing densities, high levels of polyunsaturated fatty acids, and low cholesterol levels in comparison with animals raised in brighter illuminances (300- or 800 lx). The mole ratio of phospholipid to rhodopsin in the outer-segment membrane of rats raised in 5-lx cyclic light is only 43% of that of rats raised in 800-lx cyclic light. The difference between these two groups of animals for docosahexaenoic acid is greater than three times, with dim light-reared animals showing higher levels. These rats (5 lx-reared) have less cholesterol in their photoreceptor outer segments, 6.6 mol% compared with 19.7 mol% for animals from the 800-lx regime. In all cases, rats from the intermediate rearing illuminance (300 lx) exhibit intermediate membrane composition values. It is likely that these differences in membrane composition illustrate a profound effect of light history on photoreceptor function.

Albino rats were raised in cyclic lighting and subjected to either constant light inhibition of the shedding peak or a 10 hr phase advance in the lighting schedule, with lighting levels of varied intensity. All responses followed sigmoid curves of shedding vs. intensity. For constant light inhibition the curve had a shallow slope with a 50% peak at 0.85 ft-cd. Shedding after a phase shift showed a steep curve with a 50% peak at 0.3 ft-cd when animals were observed at 9:30 P. M. and a shallow slope with a 50% peak at 0.003 ft-cd when observed at 7:30 A. M. It is concluded that the mechanisms of shedding inhibition and phase shifts in the shedding rhythm differ with respect to the role of light.

A 31P-NMR study of magnetically oriented bovine rod outer segments is presented. We demonstrate that carefully isolated bovine rod outer segments retain the capacity to orient in a magnetic field. Maximal orientation (85-90%) is achieved at field strengths over 4.7 T in the NMR spectrometer. The lineshape of the 'oriented spectra' is totally different from the 'bilayer lineshape' of randomly oriented photoreceptor membranes. The oriented spectra consist of two phospholipid peaks, a major low-field peak (75-80% of the total intensity) near 30 ppm, and a minor high-field peak near - 14 ppm as well as two sharp metabolite peaks around 0 ppm. The phospholipid peaks are a composite of three narrower partially resolved resonances assigned to the individual phospholipid classes phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine. Based on the morphology and magnetic anisotropy of the rod outer segment, the major phospholipid peak is attributed to the flat part of the disk membranes while the phospholipids of the plasma membrane are thought to contribute only to the minor peak. Disk rim phospholipids and non-oriented material contribute to the minor peak and, in addition, contribute some intensity to the middle part of the spectrum. The phospholipid class composition of the major peak is estimated by spectral simulation and is consistent with the phospholipid class composition of rod outer segment membranes. Hence, 31P analysis of oriented rod outer segments resolves the main phospholipids in at least two different membrane pools in the rod outer segment and allows the differential investigation of these pools. Most of the mobile phosphate metabolite intensity resides in the Pi peak at 3.5 ppm. A slight shift in the Pi resonance position indicates a 0.2 pH unit acidification upon illumination of rhodopsin. The absence of detectable nucleotide resonances, when compared with chemical analysis, indicates that the majority of the nucleotide population present is rather immobile and probably bound to the membranes.