真鲷视网膜结构及其视觉特性研究：Ⅱ视网膜光感受细胞超微结构研究

对真鲷光感受细胞的超微结构进行观察,结果表明：视杆外段膜盘为游离膜盘,视锥外段膜盘则为连续的膜结构,视锥和视杆均含有连接纤毛和辅助外段。花萼状突起起源于内段。椭体内充满线粒体,无球状小体。双锥椭圆体并生膜为六层,视锥内段无鳍状突起,视锥突触带,在明适应视网膜中数量增多,在暗适应视网中数量减少,视杆突触带在这两种适应网膜中数量不变,每一杆小球只有一个突触带,而锥小足有４－６个突触带。     

Ultrastructure and serotonin immunocytochemistry of the parietal-pineal complex in the Japanese grass lizard, Takydromus tachydromoides.

The fine structure and immunocytochemical localization of serotonin in the cells of the receptor line were studied in the parietal eye and pineal organ proper of the Japanese grass lizard, Takydromus tachydromoides. Typical photoreceptor cells (PC) were the predominant cell type in the receptor line of the parietal eye, the outer segments of which had regular stacks of numerous disks similar to those of cones. The pineal organ contained relatively few PCs, which showed less well-developed outer segments than those of the parietal eye. In contrast, secretory rudimentary photoreceptor cells (SRPC) accounted for the majority of receptor cells in the pineal organ. These cells were structurally characterized by whorl-like lamellar outer segments and numerous dense-cored vesicles (80-280 nm in diameter). A small number of SRPC were also found in the parietal retina, which were similar to those in the pineal organ. In the parietal-pineal complex, numerous mitochondria located in the PC were larger and rounder than those in the SRPC. In the PC, basal processes prossessed only synaptic ribbons, whereas in the SRPC some of these processes contained synaptic ribbons and others contained dense-cored vesicles, rarely having both. Serotonin-immunoreactive cells were found not only in the pineal organ but also in the parietal eye, which closely resembled the cells of the receptor line in their size and shape. Furthermore, on immunoelectron microscopy for serotonin using the protein A-gold technique, gold particles indicating serotonin-immunoreactive sites were restricted in the core of dense-cored vesicles in the SRPC of the pineal organ. Regional differences in the distributions of the PC, SRPC and serotonin-immunoreactivity were found in the parietal-pineal complex.     

Light dependence of osmium reactivity in mouse photoreceptor cells.

Mouse photoreceptor cells exhibit local accumulations of osmium deposits after prolonged osmic staining at slightly elevated temperatures. Deposits were evident along the membranes of outer segment lamellae, Golgi cisternae and vesicles, nuclear envelopes, and synaptic vesicles. Other membranes within the photoreceptor cells were unreactive. No osmium reactivity was seen in other cells of the retina except for osmiophilic outer segment material which had been phagocytized by the pigment epithelium. In the outer segments, inner segments, and synaptic regions of the photoreceptor cells, the amount of osmium reactivity was increased by light stimulation and decreased following extended dark adaptation. The possible significance of the localized osmium reactivity is discussed.     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.     

Embryonic appearance of rod opsin in the urodele amphibian eye

Notophthalmus (Triturus) viridescens, a urodele amphibian (newt) common to the Eastern United States, is a promising subject for developmental and regeneration studies. We have available a monoclonal antibody shown to be specific in many vertebrates for rod opsin, the membrane apoprotein of the visual pigment rhodopsin. This antibody to an N-terminal epitope, by rigorous biochemical and immunological criteria, recognizes only rod photoreceptor cells of the retina in light-and electron-microscopic immunocytochemistry. To determine the ontogeny and localization of rhodopsin in developing rods as an indicator of function in the embryonic urodele retina, we have utilized this antibody in the immunofluorescence technique on sections of developing N. viridescens. It was applied to serial sections of the eye region of Harrison stage 28 (optic vesicle) through stage 43 (most adult retina histology complete) embryos, and subsequently visualized with biotinylated species antibody followed by extravidin fluorescein isothiocyanate. The first positive reaction to rhodopsin could be detected in two to four cells (total) of the stage 37 embryonic eye, in the region of the central retinal primordium where the photoreceptors will be found. Some indications of retinal outer nuclear and inner plexiform layers could be seen at this time. Later embryonic stages demonstrated increasing numbers of positive cells in the future photoreceptor outer nuclear layer and outer and inner segments, spreading even to the peripheral retina. Nevertheless, by stale 43, no positive cells could be found at the dorsal or ventral retinal margins. Thus, biochemical differentiation of a photoreceptor population in the urodele retina occurs at a stage before retinal histogenesis is complete. The total maturation of retinal rods occurs topographically over a long period until the adult distribution is achieved. Correspondence to: D.S. McDevitt     

An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.     

Rhodopsin transport in the membrane of the connecting cilium of mammalian photoreceptor cells

The transport of the photopigment rhodopsin from the inner segment to the photosensitive outer segment of vertebrate photoreceptor cells has been one of the main remaining mysteries in photoreceptor cell biology. Because of the lack of any direct evidence for the pathway through the photoreceptor cilium, alternative extracellular pathways have been proposed. Our primary aim in the present study was to resolve rhodopsin trafficking from the inner to the outer segment. We demonstrate, predominantly by high-sensitive immunoelectron microscopy, that rhodopsin is also densely packed in the membrane of the photoreceptor connecting cilium. Present prominent labeling of rhodopsin in the ciliary membrane provides the first striking evidence that rhodopsin is translocated from the inner segment to the outer segment of wild type photoreceptors via the ciliary membrane. At the ciliary membrane rhodopsin co-localizes with the unconventional myosin VIIa, the product of human Usher syndrome 1B gene. Furthermore, axonemal actin was identified in the photoreceptor cilium, which is spatially co-localized with myosin VIIa and opsin. This actin cytoskeleton of the cilium may provide the structural bases for myosin VIIa-linked ciliary trafficking of membrane components, including rhodopsin.     

Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.     

Ultrastructural localization of endogenous calcium in the teleost retina

Summary The ultrastructural localization of endogenous calcium in the retina of adult cichlid fishOreochromis mossambicus (Teleostei) was studied using the cytochemical osmiate-bichromate method of Probst (1986). The specificity of this method for calcium localization was proven by means of EGTA treatment of ultrathin sections and electronspectroscopic-imaging technique (ESI) with an energy-filtering transmission electron microscope (CEM 902, Zeiss). Large amounts of electron-dense calcium containing deposits were found in the outer segments of rods, in the synaptic vesicles of receptor terminals and bipolar cells, in the perinuclear space of photoreceptors and in the endoplasmic reticulum of different cell types, especially in the inner segment and fibres of photoreceptor cells. In the inner plexiform layer calcium was detected in the extracellular space with greater accumulations in the synaptic cleft. Principal differences in the localization of calcium between rods and cones and between several types of synapses and vesicles are shown. The possible role of calcium in the subcellular structures of retinal cells is discussed.     

Ultrastructural study of the cellular types in the pineal organ of Gambusia affinis (teleost)

The pineal organ of Gambusia affinis was studied via light and electron microscopy. The cell types studied included photoreceptor cells, supporting cells, and a third cell type. The photoreceptor cells, which appear to form clusters, are divided into four regions: outer segment, inner segment, cell soma, and synaptic pedicle. Synaptic ribbons are commonly observed in the synaptic pedicle. The supporting cells separate the photoreceptor cells from the thick basal lamina that surrounds the entire pineal organ. The supporting cells show highly organized membrane formations, some lipid-like inclusions, and a diplosome. One of the centrioles gives rise to an invaginated cilium. The third cell type is observed infrequently and appears to be located mainly in the vicinity of the outer segments. The morphological characteristics of this cell type are similar to those of phagocytic cells. The ultrastructural features of the pineal organ of G. affinis are compared with those of other teleosts.     

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles.     

An ultrastructural study of embryonic chick retinal neurons in culture

Summary The differentiation of cells and synapses in explants of 9-day-old chick embryo retina has been studied by light and electron microscopy over a period of 35 days in vitro, and samples of retina from the 9-day chick foetus were directly fixed and prepared for study.At the time of explantation the retinae were poorly differentiated and no lamination was apparent. From day 14 onwards, (i) outer and inner nuclear layers (ONL, INL) separated by a layer of neuropil corresponding to the outer plexiform layer (OPL) and (ii) a layer of scattered large ganglion cells separated from the INL by a zone of neuropil resembling the inner plexiform layer (IPL) were apparent, and (iii) a well-differentiated outer limiting membrane was established close to the surface of the explants. In the oldest cultures some development of photoreceptor outer segments occurred but a distinct optic nerve fibre layer did not form.Although cell identification presented problems even in the oldest cultures, the major retinal cell types described in vivo could be identified. Photoreceptor cells developed pedicles in the OPL which became filled with synaptic vesicles and synaptic ribbons and established ribbon synapses (including triads) with and were commonly invaginated by processes from horizontal and bipolar cells. Processes of bipolar cells in the IPL formed simple and dyad synapses. At least two types of presynaptic amacrine cells were also identified in the INL, one of which contained large numbers of dense-core vesicles. The ganglion cells, though sparse, were large and well differentiated.These findings show that all the major neuronal types of the retina are capable of developing and differentiating in vitro, lagging behind the time-table of development and differentiation in vivo by approximately 7 days, but resulting in a histotypically organised retina with synaptic neuropil showing many similarities to the corresponding neuropil in vivo.     

Synaptic plasticity and functionality at the cone terminal of the developing zebrafish retina

Previous studies have analyzed photoreceptor development, some inner retina cell types, and specific neurotransmitters in the zebrafish retina. However, only minor attention has been paid to the morphology of the synaptic connection between photoreceptors and second order neurons even though it represents the transition from the light sensitive receptor to the neuronal network of the visual system. Here, we describe the appearance and differentiation of pre- and postsynaptic elements at cone synapses in the developing zebrafish retina together with the maturation of the directly connecting second order neurons and a dopaminergic third order feedback-neuron from the inner retina. Zebrafish larvae were examined at developmental stages from 2 to 7dpf (days postfertilization) and in the adult. Synaptic maturation at the photoreceptor terminals was examined with antibodies against synapse associated proteins. The appearance of synaptic plasticity at the so-called spinule-type synapses between cones and horizontal cells was assessed by electron microscopy, and the maturation of photoreceptor downstream connection was identified by immunocytochemistry for GluR4 (AMPA-type glutamate receptor subunit), protein kinase beta(1) (mixed rod-cone bipolar cells), and tyrosine hydroxylase (dopaminergic interplexiform cells). We found that developing zebrafish retinas possess first synaptic structures at the cone terminal as early as 3.5dpf. Morphological maturation of these synapses at 3.5-4dpf, together with the presence of synapse associated proteins at 2.5dpf and the maturation of second order neurons by 5dpf, indicate functional synaptic connectivity and plasticity between the cones and their second order neurons already at 5dpf. However, the mere number of spinules and ribbons at 7dpf still remains below the adult values, indicating that synaptic functionality of the zebrafish retina is not entirely completed at this stage of development.     

Polarized sorting of rhodopsin on post-Golgi membranes in frog retinal photoreceptor cells

We have isolated a subcellular fraction of small vesicles (mean diameter, 300 nm) from frog photoreceptors, that accumulate newly synthesized rhodopsin with kinetics paralleling its appearance in post-Golgi membranes in vivo. This fraction is separated from other subcellular organelles including Golgi and plasma membranes and synaptic vesicles that are sorted to the opposite end of the photoreceptor cell. The vesicles have very low buoyant density in sucrose gradients (rho = 1.09 g/ml), a relatively simple protein content and an orientation of rhodopsin expected of transport membranes. Reversible inhibition of transport by brefeldin A provides evidence that these vesicles are exocytic carriers. Specific immunoadsorption bound vesicles whose protein composition was indistinguishable from the membranes sedimented from the subcellular fraction. Some of these proteins may be cotransported with rhodopsin to the rod outer segment; others may be involved in vectorial transport.     

Immunocytochemical study on photoreceptor cell differentiation in the cultured retina of the chick

Photoreceptor cell differentiation was investigated in a dissociated monolayer culture of chick embryonic retinas with electron microscopic immunohistochemistry. The antibody was raised against bovine rhodopsin purified on SDS-polyacrylamide gel electrophoresis. In the developing retina, immunoreactivity was first recognized on the 14th day of incubation and was localized on the plasma membrane of the growing inner segment. On the 16th day, immunoreactivity was observed on some differentiating outer segments but not on inner segments. In the culture from 6 1/2-day-old embryonic retinas, immunoreactivity was found on the 7th day of culturing on the plasma membrane of large-sized neurons. Electron microscopic observations confirmed that such stained cells showed reaction product on the plasma membrane, and that they displayed fine structures characteristic of intact photoreceptor cells. They had a number of microvillous processes and often one thick process, both of which were intensely stained. Outer segment formation, however, was not observed in the present monolayer culture. These results indicate that opsin synthesis and its transport to the plasma membrane begins prior to and probably independently of outer segment formation.     

Histochemical localization of retinochrome and rhodopsin studied by fluorescence microscopy

Retinochrome is readily reduced by sodium borohydride into an N-retinyl protein that emits visible fluorescence upon irradiation with near-ultraviolet light. Rhodopsin is also converted to a similar fluorescent product, but only when denatured with formaldehyde before reduction. Based upon this difference, retinochrome was discriminated from rhodopsin on frozen sections. The distribution of these two photopigments in various photosensitive tissues was examined by means of epifluorescence microscopy. In the octopus retina (Octopus vulgaris), the yellow-green fluorescence of reduced retinochrome was observed in both the basal regions of the outer segments and throughout the inner segments of the visual cells, while the fluorescence of reduced rhodopsin was restricted to within the rhabdomal layer of the outer segments. In the squid parolfactory vesicles (Todarodes pacificus), rhodopsin was present in the central lumen, which contains the distal processes of the photoreceptor cells, while retinochrome was detected in the myeloid bodies scattered within the vesicular wall. In the slug retina (Limax flavus), rhodopsin was found in the microvilli, and retinochrome appeared to be concentrated in the photic vesicles of the visual cells.     

The fine structure of the pigment epithelium and photoreceptor cells of the newt,Triturus viridescens dorsalis (Rafinesque)

The morphology of the retinal pigment epithelium and photoreceptor cells has been studied in the common newt Triturus viridescens dorsalis by light, conventional transmission and scanning electron microscopy. The pigment epithelium is formed by a single layer of low rectangular cells, separated by a multilayered membrane (Bruch's membrane) from the vessels of the choriocapillaris. The scleral border of the pigment epithelium is highly infolded and each epithelial cell contains smooth endoplasmic reticulum, myeloid bodies, mitochondria, lysosomes, phagosomes and an oval nucleus. Inner, pigment laden, epithelial processes surround the photoreceptor outer and inner segments. The three retinal photoreceptor types, rods, single cones and double cones, differ in both external and internal appearance. The newt, rod, outer segments appear denser than the cones in both light and electron micrographs, due to a greater number of rod lamellae per unit distance of outer segment and to the presence of electron dense intralamellar bands. The rod outer segments possess deep incisures in the lamellae while the cone lamellae lack incisures. Both rod and cone outer segments are supported by a peripheral array of dendritic processes containing longitudinal filaments which originate in the inner segment. The inner segment mitochondria, forming the rod ellipsoid, arelong and narrow while those in the cone are spherical to oval in shape. The inner segments of all three receptor cell types also contain a glycogen-filled paraboloid and a myoid region, just outside the nucleus, rich in both rough and smooth endoplasmic reticulum. The elongate, cylindrical nuclei differ in density. The rod nuclei are denser than those of the cones, contain clumped chromatin and usually extend further vitreally. Similarly, the cytoplasm of the rod synaptic terminal is denser than its cone counterpart and contains synaptic vesicles almost twice as large as those of the cones. Photoreceptor synapses in rods and cones are established by both superficial and invaginated contacts with bipolar or horizontal cells.     

Stages of development of retinal photoreceptor cells in postnatal ontogenesis of the pied flycatcherFicedula hypoleuca

Electron microscopic study of the retina of the altricial pied flycatcherFicedula hypoleuca nestlings has permitted identification, in the course of development of photoreceptor cells, of several consecutive stages, each characterized by the appearance in the cell of some new stagespecific structures. At the 1st stage (the presumptive photoreceptor stage), the cell is composed of the nucleus surrounded by a small amount of the cytoplasm and two processes. The 2nd stage is characterized by formation of the inner segment in the apical cell region, while in the distal region, of the cleft (electric) contacts between the photoreceptor axon terminal and the processes of the second neurons. At the 3rd stage, a pile of membrane disks of the outer segment is formed, while in the synaptic region, the first band synapses considered as the chemical ones. At the 4th stage, a lipid droplet appears in a part of the cells, and at the 5th stage, a paraboloid. The photoreceptors of the 6th stage are young cells that continue growing but are already capable for specific functioning. Closely connected with development of the photoreceptor cells is maturation of the Müller’s glia and pigment epithelium, so that every identified stage of the photoreceptor cell development corresponds to a certain structural state of these elements. The possibility of involvement of the forming retinal cells in performance of early behavioral forms is discussed.     

Utilization of retinoids in the bullfrog retina

The capacity to generate 11-cis retinal from retinoids arising naturally in the eye was examined in the retina of the bullfrog, Rana catesbeiana. Retinoids, co-suspended with phosphatidylcholine, were applied topically to the photoreceptor surface of the isolated retina after substantial bleaching of the native visual pigment. The increase in photoreceptor sensitivity associated with the formation of rhodopsin, used as an assay for the appearance of 11-cis retinal in the receptors, was analyzed by extracellular measurement of the photoreceptor potential; in separate experiments using the isolated retina or receptor outer segment preparations, the formation of rhodopsin was measured spectrophotometrically. Treatments with the 11- cis isomers of retinal and retinol induced significant increases in both the rhodopsin content and photic sensitivity of previously bleached receptors. The all-trans isomers of retinyl palmitate, retinol, and retinal, as well as the 11-cis isomer of retinyl palmitate, were inactive by both the electrophysiological and spectrophotometric criteria for the generation of rhodopsin. Treatment with any one of the "inactive" retinoids did not abolish the capacity of subsequently applied 11-cis retinal or 11-cis retinol to promote the formation of rhodopsin. The data are discussed in relation to the interconversions of retinoids ("visual cycle of vitamin A") thought to mediate the regeneration of rhodopsin in vivo after extensive bleaching.