The Fine Structure of the Retina Studied with the Electron Microscope : IV. Morphogenesis of Outer Segments of Retinal Rods

The morphogenesis of the outer segments of retinal rods was studied mainly in the kitten before the opening of the eye, and the probable sequence of the morphogenetic stages is deduced. Since the development of retinal rods is not synchronous, the deductions were based on observations of many single and serial sections. One centriole extends ciliary tubules of about 0.5 µ long, in the growing primitive cilium. Beyond this length, each ciliary tubule becomes a row of small vesicles (called "ciliary vesicles" in this paper), which penetrate into the distal region of the cilium. Where the ciliary vesicles establish contact with the plasma membrane of the distal region of the cilium, more or less deep infoldings of the plasma membrane are observed. In the distal region can be seen rows of tubular or vesicular structures. A few of these membranous structures are continuous with the bottoms of the infoldings. At the following stage, the infoldings disappear and the ciliary vesicles lose contact with the distal plasma membrane. Nonetheless, the formation of the tubular structures continues in the distal region of the primitive outer segment. The tubular structures appear to be transformed into the primitive rod sacs by sidewise enlargement. At a subsequent time, presumably, these primitive rod sacs flatten and are rearranged into a position perpendicular to the long axis of the outer segment. The detailed structure of the basal body of the connecting cilium was also studied by means of serial sections.     

The Fine Structure of Some Retinal Photoreceptors

An electron microscope study has been made of octopus and amphibian photoreceptors, after fixing with KMnO₄ and embedding in araldite. What has previously been seen as a single dense stratum bounding the tubular compartments (octopus) or the double membrane discs (rods and cones), now shows a double structure. We interpret this as showing that these tubules and discs have similar bounding surfaces, which are probably directly related to the cell membrane. This is confirmed by the finding that the tubules and discs are (at least occasionally) continuous with the cell membrane.     

Fine Structure and Morphogenesis of Borna Disease Virus

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.     

Studies on the Fine Structure of Microorganisms : V. Morphogenesis of Nuclear and Membrane Structures during Ascospore Formation in Yeast

The fine structure of cells of Saccharomyces cerevisiae engaged in the formation of ascospores was studied in electron micrographs of ultrathin sections. Although the mode of the first reduction division could not be clearly determined, the second nuclear division appeared to proceed in a manner similar to that observed previously during vegetative division. That is, division by constriction of the existing nucleus occurs without dissolution of the nuclear membrane and without involvement of discrete chromosomes. Variously shaped areas of low electron density were discerned within the nucleoplasm; these had not been previously seen in the vegetative nucleus. The significance of this nuclear differentiation and its possible similarity to nuclear structures reported in bacteria and an imperfect fungus are discussed. The cytoplasmic membrane appears first in the developing ascospore. The formation of an outer coat and an inner coat then follows. The cytoplasmic vacuole was observed not to be incorporated into the spore. An unusual intracytoplasmic membrane was observed in the spore and appeared to be at least temporarily continuous with the nuclear membrane.     

The Fine Structure of the Rod-Bipolar Cell Synapse in the Retina of the Albino Rat

The fine structure of the rod-bipolar synapse is described and illustrated. Each rod spherule possesses a large, single, oval or elongate mitochondrion approximately 0.5 x 2.0 microns. Surrounding the mitochondrion are elements of agranular endoplasmic reticulum. The bipolar dendrite projects into the lower pole of the spherule and usually terminates in two lobes separated by a cleft. The plasma membranes appear dense and thicker in the region of the synapse. In the rod spherule cytoplasm, contiguous with the plasma membrane is a dense, slightly concave arciform structure, the rod arciform density, extending from the base of the bipolar bifid process through the cleft to an equivalent point on the opposite side. Also within the spherule, and external (towards the sclera) to the rod arciform density, is a parallel, dense, thin lamella, the rod synaptic lamella. This is approximately 25 mµ in thickness and 400 mµ in width at its widest extent. This halfmoon-shaped plate straddles the cleft between the two lobes of the bipolar process. The lamella appears to consist of short regular rodlets or cylinders 5 to 7 mµ in diameter, oriented with their long axes perpendicular to the plane of the lamella. Minute cytoplasmic vesicles found in the cytoplasm of both the rod spherule and the bipolar terminal are most abundant near the rod synaptic lamella.     

Kinetics of the Photocurrent of Retinal Rods

The shapes of the photocurrent responses of rat rods, recorded with microelectrodes from the receptor layer of small pieces of isolated retinas, have been investigated as a function of temperature and of stimulus energy. Between 27 and 37°C the responses to short flashes can be described formally as the output of a chain of at least four linear low-pass filters with time constants in the range 50-100 msec. The output of the filter chain is then distorted by a nonlinear amplitude-limiting process with a hyperbolic saturation characteristic. Flashes producing ~30 photons absorbed per rod yield responses of half-maximal size independently of temperature. The maximum response amplitude is that just sufficient to cancel the dark current. The rate of rise of a response is proportional to flash energy up to the level of 10⁵ photons absorbed per rod, where hyperbolic rate saturation ensues. The responses continue to increase in duration with even more intense flashes until, at the level of 10⁷ photons absorbed per rod, they last longer than 50 min. The time-courses of the photocurrent and of the excitatory disturbance in the rod system are very similar. The stimulus intensity at which amplitude saturation of the photocurrent responses begins is near that where psychophysical “rod saturation” is seen. An analysis of these properties leads to the following conclusions about the mechanism of rod excitation. (a) The kinetics of the photocurrent bear no simple relation to the formation or decay of any of the spectroscopic intermediates so far detected during the photolysis of rhodopsin. (b) The forms of both the amplitude- and rate-limiting processes are not compatible with organization of rhodopsin into “photoreceptive units” containing more than 300 chromophores. Even at high stimulus intensities most rhodopsin chromophores remain connected to the excitatory apparatus of rods. (c) The maximum rate of rise of the photocurrent is too fast to be consistent with the infolded disks of a rod outer segment being attached to the overlying plasma membrane. Most of the disks behave electrically as if isolated within the cell. (d) Control of the photocurrent at the outer segment membrane is not achieved by segregation of the charge carriers of the current within the rod disks. Instead, it is likely to depend on control of the plasma membrane permeability by an agent released from the disks.     

Control of Retinal Sensitivity : I. Light and Dark Adaptation of Vertebrate Rods and Cones

Rods and cones in Necturus respond with graded hyperpolarization to test flashes spanning about 3.5 log units of intensity. Steady background levels hyperpolarize the rods, and the rod responses become progressively smaller as background level is increased. In cones, higher background levels reduce the effectiveness of test flashes, so higher ranges of test intensities are required to elicit the full range of graded responses. When backgrounds are terminated, cones return rapidly, but rods return slowly to the dark potential level. The effects of backgrounds on both rods and cones can be observed at intensities that cause negligible bleaching as determined by retinal densitometry. During dark adaptation, changes are observed in the rods and cones that are similar to those produced by backgrounds. Receptor sensitivities, derived from these results, show that rods saturate, cones obey Weber's law, and sensitization during dark adaptation follows a two-phase time-course.     

Single-Cell Transcriptomic Comparison of Human Fetal Retina,hPSC-Derived Retinal Organoids,and Long-Term Retinal Cultures

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Fine Structure of the Retinal Pigment Epithelium of the River Lamprey (Lampetra fluviatilis,Cyclostomi)

The retinal pigment epithelium of Lampetra fluviatilis was studied by electron microscopy. The epithelial cells differ in many details from those of gnathostomes. The lateral cell membranes are difficult to distinguish. The smooth endoplasmic reticulum is well developed; in some animals undulated membrane complexes, comprising systems of tightly fused double membrane plates, are related to the endoplasmic reticulum. Myeloid bodies are common and well developed, but pigment granules are comparatively sparse. The intercellular space between pigment epithelium and photoreceptors is rather wide. There are only a few inclusion bodies with membranous contents. The importance of the pigment epithelium in the retinal metabolic exchange is discussed in view of the fine structure of the cells. Compared with that of hagfishes, the lamprey retina is well developed. However, any comparison must be made against the background of a diphyletic development of the two groups.     

The Structure and Morphogenesis of the Ventral Ciliature in Paraurostyla hymenophora*

SYNOPSIS. The structure and morphogenesis of the ventral ciliature of Paraurostyla hymenophora (Stokes) are described. The oral primordium apparently originates in association with transverse cirrus #6, from which it migrates anteriorly simultaneous with kinetosomal proliferation. The primordium eventually forms an elongate ciliary field from which the future opisthe's fronto-ventro-transverse (FVT) and undulating membrane primordial fields arise. Concomitantly, the future proter's FVT primordial field is initiated by the disaggregation of frontal cirri #4, #5, and #6. Primordia then develop simultaneously within marginal and ventral cirral rows by a disaggregation of cirri within the respective rows, and do not give rise to new cirri until the FVT fields complete segregation into discrete cirri. Near the completion of cirral production from the FVT primordia, each ventral cirral primordium (VCP) forms the 2 rightmost transverse cirri. Segregation of new cirri within the marginal cirral primordia and VCP then occurs, eventually replacing all old cirri within their respective marginal and ventral cirral rows. At the end of cortical morphogenesis, all old ciliary organelles, with the exception of the adoral zone of membranelles, are either reorganized or replaced. These results suggest an evolutionary affinity between the ventral and marginal cirral rows and raise questions about the control of the developmental competence of individual primordia.     

The Visual Cycle in the Inner Retina of Chicken and the Involvement of Retinal G-Protein-Coupled Receptor (RGR)

The vertebrate retina contains typical photoreceptor (PR) cones and rods responsible for day/night vision, respectively, and intrinsically photosensitive retinal ganglion cells (ipRGCs) involved in the regulation of non-image-forming tasks. Rhodopsin/cone opsin photopigments in visual PRs or melanopsin (Opn4) in ipRGCs utilizes retinaldehyde as a chromophore. The retinoid regeneration process denominated as “visual cycle” involves the retinal pigment epithelium (RPE) or Müller glial cells. Opn4, on the contrary, has been characterized as a bi/tristable photopigment, in which a photon of one wavelength isomerizes 11-cis to all-trans retinal (Ral), with a second photon re-isomerizing it back. However, it is unknown how the chromophore is further metabolized in the inner retina. Nor is it yet clear whether an alternative secondary cycle occurs involving players such as the retinal G-protein-coupled receptor (RGR), a putative photoisomerase of unidentified inner retinal activity. Here, we investigated the role of RGR in retinoid photoisomerization in Opn4x (Xenopus ortholog) (+) RGC primary cultures free of RPE and other cells from chicken embryonic retinas. Opn4x (+) RGCs display significant photic responses by calcium fluorescent imaging and photoisomerize exogenous all-trans to 11-cis Ral and other retinoids. RGR was found to be expressed in developing retina and in primary cultures; when its expression was knocked down, the levels of 11-cis, all-trans Ral, and all-trans retinol in cultures exposed to light were significantly higher and those in all-trans retinyl esters lower than in dark controls. The results support a novel role for RGR in ipRGCs to modulate retinaldehyde levels in light, keeping the balance of inner retinal retinoid pools.     

Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina

Hyperglycemia-induced retinal oxidative and nitrative stress can accelerate vascular cell aging, which may lead to vascular dysfunction as seen in diabetes. There is no information on whether this may contribute to the progression of diabetic retinopathy (DR). In this study, we have assessed the occurrence of senescence-associated markers in retinas of streptozotocin-induced diabetic rats at 8 and 12 weeks of hyperglycemia as compared to normoglycemic aging (12 and 14 months) and adult (4.5 months) rat retinas. We have found that in the diabetic retinas there was an up-regulation of senescence-associated markers SA-β-Gal, p16^INK4a and miR34a, which correlated with decreased expression of SIRT1, a target of miR34a. Expression of senescence-associated factors primarily found in retinal microvasculature of diabetic rats exceeded levels measured in adult and aging rat retinas. In aging rats, retinal expression of senescence associated-factors was mainly localized at the level of the retinal pigmented epithelium and only minimally in the retinal microvasculature. The expression of oxidative/nitrative stress markers such as 4-hydroxynonenal and nitrotyrosine was more pronounced in the retinal vasculature of diabetic rats as compared to normoglycemic aging and adult rat retinas. Treatments of STZ-rats with the anti-nitrating drug FeTPPS (10mg/Kg/day) significantly reduced the appearance of senescence markers in the retinal microvasculature. Our results demonstrate that hyperglycemia accelerates retinal microvascular cell aging whereas physiological aging affects primarily cells of the retinal pigmented epithelium. In conclusion, hyperglycemia-induced retinal vessel dysfunction and DR progression involve vascular cell senescence due to increased oxidative/nitrative stress.     

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.     

The Fine Structure of the Integument of Free-Living and Parasitic Copepods. A Review

A perusal of the literature on copepod cuticles has been made, and results of the investigation of six species made by the author are included in this review. The integument of copepods is of the arthropod type. Pore canals and other structures traversing the cuticle, common in most arthropods, are not always present in free-living and some parasitic copepods. In parasitic forms, with advanced morphological changes, the cuticle is generally very thin and the epicuticle in many species forms external microvilli-like structures. In the copepods hitherto investigated the epicuticle is probably the sole layer present in the cuticle. Some copepods show specialized regions of the cuticular surface, the function of which still remains obscure. Integumental organs and integumental structures are numerous and variable. The association of bacteria with the cuticle has been observed in many species. The structure of the integument of parasitic species lacking an alimentary tube and in close contact with the host tissue or hemocoelic cavity supports the idea that the integument could be the obligatory site of nutrient uptake. In spite of the relatively few species of copepods that have been investigated, a remarkable variation of cuticular fine structure has been revealed.