Synaptophysin Expression in Rat Retina Following Acute High Intraocular Pressure

In response to injury, synapse alteration may occur earlier than the changes in the cell body of neurons. Although retinal ganglion cell death and thinning of the inner part of retina were found after acute high intraocular pressure (HIOP), the structural and functional changes of synapses in the retina remain unknown. In the present study, we investigated the protein and mRNA expression of synaptophysin (SYN), an important molecule closely related to synaptic activities, synaptogenesis and synaptic plasticity. In addition, we also studied the ultrastructural changes of the retinal synapses. We found that (1) synaptophysin was upregulated transiently at both protein and mRNA level following HIOP; (2) broadened distribution of synaptophysin protein was present within the outer nuclear layer at the early stage following HIOP; (3) in the outer nuclear layer bouton-like vesicle-containing structures were observed by electron microscopy. This data suggested that, besides degeneration, synapses in rat retina may undergo regenerative events following HIOP.     

Synapsin I expression in the rat retina during postnatal development

Summary The expression of the synapsin I gene was studied during postnatal development of the rat retina at the mRNA and protein levels. In situ hybridization histochemistry showed that synapsin I mRNA was expressed already in nerve cells in the ganglion cell layer of the neonatal retina, while it appeared in neurons of the inner nuclear layer from postnatal day 4 onward. Maximal expression of synapsin I mRNA was observed at P12 in ganglion cells and in neurons of the inner nuclear layer followed by moderate expression in the adult. At the protein level a shift of synapsin I appearance was observed from cytoplasmic to terminal localization during retinal development by immunohistochemistry. In early stages (P4 and P8), synapsin I was seen in neurons of the ganglion cell layer and in neurons of the developing inner nuclear layer as well as in the developing inner plexiform layer. In the developing outer plexiform layer synapsin I was localized only in horizontal cells and in their processes. Its early appearance at P4 indicated the early maturation of this cell type. A shift and strong increase of labelling to the plexiform layers at P12 indicated the localization of synapsin I in synaptic terminals. The inner plexiform layer exhibited a characteristic stratified pattern. Photoreceptor cells never exhibited synapsin I mRNA or synapsin I protein throughout development.Abbreviations GCL ganglion cell layer - INB inner neuroblast layer - INL inner nuclear layer - IPL inner plexiform layer - ONB outer neuroblast layer - ONL outer nuclear layer - OPL outer plexiform layer     

Histology of the ferret retina

The retina of the adult ferret, Mustelo furo, was studied with light and transmission electron microscopy to provide an anatomical basis for use of the ferret as a model for retinal research. The pigment epithelium is a simple cuboidal layer of cells characterized by a zone of basal folds, apical microvilli, and pigment granules at various stages of maturation. The distinction between rod and cone photoreceptor cells is based on their location, morphology, heterochromatin pattern and the electron density of their inner segments. The round, light-staining cone cell nuclei occupy the layer of perikarya along the apical border of the outer nuclear layer. The remainder of the outer nuclear layer consists of oblong, deeply-stained rod cell nuclei. Ribbon type synaptic complexes involving photoreceptor cell axons, horizontal cell processes, and bipolar cell dendrites characterize the outer plexiform layer. The inner nuclear layer is comprised of horizontal, bipolar, and amacrine cell perikarya as well as the perikarya of the Müller cells. The light-staining horizontal cell nuclei are prominent along the apical border of the inner nuclear layer. The light-staining amacrine cell nuclei form a more or less continuous layer along the basal border of the inner nuclear layer. Both conventional and ribbon-type synapses characterize the inner plexiform layer. The ganglion cells form a single cell layer. The optic fiber layer contains bundles of axons surrounded by Müller cell processes. Small blood vessels and capillaries are present in the basal portion of the retina throughout the region extending from the internal limiting membrane to the outer plexiform layer. The adult one-year-old retina is compared with the retina at the time of eye opening.     

外源性BDNF对急性高眼压后大鼠视网膜ERK1/2磷酸化的影响

为研究脑源性神经生长因子(BDNF)干预对急性高眼压后大鼠视网膜磷酸化的细胞外信号调节激酶(p-ERK1/2)表达变化的影响,本实验将成年大鼠随机分为单纯高眼压组、BDNF预处理高眼压组和溶媒预处理高眼压组,BDNF预处理高眼压组和溶媒预处理高眼压组动物左眼于加压前2d分别给予BDNF或溶媒预处理,右眼设为正常对照。各组动物左眼眼压升高至闪光视网膜电图b波消失的临界眼压并维持60min,动物分别存活1、3、7、14d后处死,冰冻切片行p-ERK的免疫组织化学染色。结果显示与正常对照相比,单纯高眼压组急性高眼压后p-ERK表达下调(P<0.05),1、3、7d组内核层出现p-ERK阳性细胞;溶媒预处理高眼压组实验结果与单纯急性高眼压组相似;BDNF预处理高眼压组急性高眼压后1、3、7d时p-ERK的表达与正常对照组相似,7d和14d出现了重新分布。此结果提示外源性BDNF可能通过促进急性高眼压后视网膜ERK1/2的活化对受损的视网膜起保护作用。     

Neuronal expression of P2X3 purinoceptors in the rat retina

P2X3 purinoceptors are involved in fast, excitatory neurotransmission in the nervous system, and are expressed predominantly within sensory neurons. In this study, we examined the cellular and synaptic localization of the P2X3 receptor subunit in the retina of the rat using immunofluorescence immunohistochemistry and pre-embedding immunoelectron microscopy. In addition, we investigated the activity of ecto-ATPases in the inner retina using an enzyme cytochemical method. The P2X3 receptor subunit was expressed in the soma of a subset of GABA immunoreactive amacrine cells, some of which also expressed protein kinase C-alpha. In addition, punctate immunoreactivity was observed within both the inner and outer plexiform layers of the retina. Double labeling studies showed that P2X3 receptor puncta were associated with both rod and cone bipolar cell axon terminals in the inner plexiform layer. Ultrastructural studies indicated that P2X3 receptor subunits were expressed on putative A17 amacrine cells at sites of reciprocal synaptic input to the rod bipolar cell axon terminal. Moreover, we observed P2X3 immunolabeling on amacrine cell processes that were associated with cone bipolar cell axon terminals and other conventional synapses. In the outer retina, P2X3 immunoreactivity was observed on specialized junctions made by putative interplexiform cells. Ecto-ATPase activity was localized to the inner plexiform layer on the extracellular side of all plasma membranes, but was not apparent in the ganglion cell layer or the inner nuclear layer, suggesting that ATP dephosphorylation occurs exclusively in synaptic regions of the inner retina. These data provide further evidence that purines participate in retinal transmission, particularly within the rod pathway.     

高血压大鼠视网膜溶酶体酶组织化学研究

目的：探讨溶酶体酶在高血压视网膜网变发生过程中的作用。方法：应用光镜定量酶组织化学方法对ＷＫＹ大鼠和自发性高血压大鼠视网膜原酸性磷酸的分布和活性变化进行定量观察。结果：视网膜各层酸性磷酸酶活性岂强到弱依次是（Ｆ检验，Ｐ〈０．０５）；（１）色素上皮层；（２）视杆维层内节和外网层（两层间活性无显著性差异）；（３）内网层；（４）节细胞层和神经纤维层，（５）外核层和内核层（两层间活性无显著性差异）杆锥层外     

Electron microscopy of glutamate decarboxylase (GAD) immunoreactivity in the inner plexiform layer of the rhesus monkey retina

Summary With indirect immunofluorescence, glutamate decarboxylase (GAD), the GABA synthesizing enzyme, was localized to cell bodies in the inner half of the inner nuclear layer and a few in the outer tier of the ganglion cell layer in the rhesus monkey retina. In the inner plexiform layer there were three strongly GAD-immunoreactive laminae separated by two less immunoreactive laminae. Electron microscopy demonstrated that the GAD was contained in amacrine cells and these GAD-immunoreactive amacrines were primarily pre- and postsynaptic to biopolar cell axon terminals. The GAD-containing processes possessed small synaptic vesicles and formed synapses that could be characterized as symmetrical. Large, dense-cored vesicles were often found in the cell bodies and synaptic processes of the GAD-immunoreactive amacrine cells. As the vast majority of the synaptic input and output of the GAD-containing amacrine cells was to and from bipolar cells and the strongest GAD-immunoreactivity correlated with the endings of bipolar cells that connect with a single cone, the functional effects of GABA in the primate retina are likely to be found in the responses of single cone pathways in the inner plexiform layer.     

Early development of GABAergic cells of the retina in sharks: an immunohistochemical study with GABA and GAD antibodies

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27-29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.     

Synaptic organization of type 2 catecholamine amacrine cells in the rhesus monkey retina

Summary Two types of amacrine cell immunoreactive for tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthetic pathway, are present in the retina of the rhesus monkey,Macaca mulatta. The well-known dopaminergic, or type 1 catecholamine amacrine cells have relatively large cell bodies almost exclusively in the inner nuclear layer with processes that densely arborize in the outermost stratum of the inner plexiform layer and fine, radially-oriented fibres in the inner nuclear layer. Type 2 catecholamine amacrine cells, in contrast, have smaller cell bodies in the inner nuclear layer, the inner plexiform layer and the ganglion cell layer, and have sparsely-branching processes ramifying in the centre of the inner plexiform layer. Although type 2 catecholamine cells are more numerous than type 1 catecholamine amacrines, type 2 cells contain less than one-third the amount of tyrosine hydrolase as the type 1 cells. Electron microscopy of retinal tissue immunoreacted for tyrosine hydrolase by the peroxidase-antiperoxidase method revealed synaptic input from amacrine cells at conventional synapses, and bipolar cells at ribbon synapses onto the type 2 catecholamine amacrine cells. Curiously, although the synaptic input is comparatively easily found, the output synapses, or synapses of the type 2 catecholamine amacrine cells onto other neuronal elements, are rarely found. Some synapses of the type 2 catecholamine cells onto non-immunoreactive amacrine cells have been identified, however. This unusual pattern of synaptic organization, with many identifiable input synapses but few morphologically characterizable output synapses, suggests a paracrine function for the dopamine released by the type 2 catecholamine amacrine cells in the primate retina.     

Ultramicroscopical immunolocalization of PAX6 in the adult chicken retina

Cell type-specific PAX6 protein expression was examined in all retinal layers of the normal chicken retina. The most intense PAX6 immunostaining was found in the ganglion cell and inner nuclear layers, and in lower amounts in the optic nerve fiber, the inner plexiform and the photoreceptor layers. PAX6 immunostaining was variable in terms of its subcellular localization, even within one cell. PAX6 immunostaining was mainly localized in nuclear heterochromatin of the ganglion cell and inner nuclear layers whereas in the outer nuclear layer, PAX6 immunostaining was only observed in the intercellular space and the cytoplasm. In photoreceptors, the myoid portion of the inner segment showed PAX6 immunostaining, but the ellipsoid portion and the outer segment did not. The ultrastructural distribution pattern of PAX6 in the adult chicken retina suggests that normal expression of PAX6 is variable even in subcellular structures in the same cell type.     

Differential localization of rat Eag1 and Eag2 potassium channels in the retina

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K⁺ channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K⁺ channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K⁺ channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.     

Patterns of cell proliferation and rod photoreceptor differentiation in shark retinas

We studied the pattern of cell proliferation and its relation with photoreceptor differentiation in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). Cell proliferation was studied with antibodies raised against proliferating cell nuclear antigen (PCNA) and phospho-histone-H3, and early photoreceptor differentiation with an antibody raised against rod opsin. As regards the spatiotemporal distribution of PCNA-immunoreactive cells, our results reveal a gradual loss of PCNA that coincides in a spatiotemporal sequence with the gradient of layer maturation. The presence of a peripheral growth zone containing pure-proliferating retinal progenitors (the ciliary marginal zone) in the adult retina matches with the general pattern observed in other groups of gnathostomous fishes. However, in the shark retina the generation of new cells is not restricted to the ciliary marginal zone but also occurs in retinal areas that contain differentiated cells: (1) in a transition zone that lies between the pure-proliferating ciliary marginal zone and the central (layered) retina; (2) in the differentiating central area up to prehatching embryos where large amounts of PCNA-positive cells were observed even in the inner and outer nuclear layers; (3) and in the retinal pigment epithelium of prehatching embryos. Rod opsin immunoreactivity was observed in both species when the outer plexiform layer begins to be recognized in the central retina and, as we previously observed in trout, coincided temporally with the weakening in PCNA labelling.     

Age-dependent remodelling of retinal circuitry

We have investigated morphological changes in second-order neurons of the mouse retina during aging by using immunohistochemistry and electron microscopy. We observed sprouting of rod bipolar cells dendrites and horizontal cells arborizations: neuronal processes of both neuronal types showed irregular extensions beyond the outer plexiform layer, toward the outer limiting membrane, as well as into the outer nuclear layer (ONL). These processes were first observed in animals of 12 months of age and increased in numbers steadily until 24 months, which represent the last age examined. The ectopic processes are decorated by puncta immunoreactive for pre-synaptic markers typical of photoreceptor terminals juxtaposed to post-synaptic neurotransmitter receptors, demonstrating the presence of the entire molecular machinery of functional synapses. Electron microscopy confirmed that ectopic processes receive synapses from photoreceptor terminals. We conclude that during the second year of life retinal rod bipolar and horizontal cells undergo sprouting and form ectopic synapses in the ONL.     

Differential localization of rat Eag1 and Eag2 potassium channels in the retina

Despite of their wide expression in the brain, the precise neurophysiological role of rat Eag1 (rEag1) and Eag2 (rEag2) K(+) channels remains elusive. Our previous studies in hippocampal pyramidal neurons demonstrate a somatodendritic localization of rEag1 and rEag2 channels, suggesting that the two channel isoforms may contribute to setting the membrane excitability of somas and dendrites. Here, we aim to further characterize the cellular and subcellular localization patterns of rEag1 and rEag2 proteins by studying their laminar distribution in the retina. Confocal microscopic analyses of immunofluorescence data revealed that rEag1 and rEag2 K(+) channels exhibit distinct cellular expression pattern in the retina. rEag1 immunoreactivity was most prominent in the outer half of the inner plexiform layer, whereas strong rEag2 immunostain was found in the outer and inner segments of photoreceptor cells, the outer plexiform layer, and the inner nuclear layer. These results suggest that rEag1 and rEag2 K(+) channels may play a significant role in the transmission of electrical signals along the retinal neuronal circuits. We also performed double-labeling experiments to demonstrate that rEag1 and rEag2 are predominantly expressed in the somatodendritic compartment of retinal neurons. In addition, we presented evidence suggesting that rEag1 channels may be expressed in the GABAergic amacrine cell. Finally, based on their different immunostaining patterns over the inner region of the retina, we propose that compared to rEag2, rEag1 expression encompasses a significantly broader range of the somatodendritic compartment of the retinal ganglion cell.     

On the synaptogenic sequence in the chick retina

Study of the developing chick retina with the electron microscope revealed that dyad ribbon synapses begin to form in the inner plexiform layer before synaptic ribbons begin to appear in photoreceptor terminals of the outer plexiform layer. This centrifugal (inner to outer) sequence of synaptogenesis in the predominantly cone retina of the chick differs from the centripetal sequence that has been reported for the predominantly rod retinas of the mouse and rat. This difference does not favor the hypothesis, suggested by others, that the photo-receptor may influence the maturation of inner retinal elements. The different patterns of synaptogenesis are discussed briefly with reference to anatomical differences between the retinas of different species.     

The absence of the clathrin-dependent endocytosis in rod bipolar cells of the FVB/N mouse retina

The high rate of exocytosis at the ribbon synapses is balanced by following compensatory endocytosis. Unlike conventional synaptic terminals where clathrin-mediated endocytosis (CME) is a predominant mechanism for membrane retrieval, CME is thought to be only a minor mechanism of endocytosis at the retinal ribbon synapses, but CME is present there and it works. We examined the clathrin expression in the FVB/N rd1 mouse, which is an animal model of retinitis pigmentosa. The broadly distributed pattern of clathrin immunoreactivity in the inner plexiform layer was similar in both the control and FVB/N mouse retinas, but the immunoreactive punta within the rod bipolar axon terminals located in the proximal IPL were decreased in number and reduced in size at postnatal days 14 and they came to disappear at postnatal days 21. This preferential decrease of the clathrin expression at ribbon synapses in the rod bipolar cell axon terminals of the FVB/N mouse retina demonstrates another plastic change after photoreceptor degeneration and this suggests that clathrin may be important for normal synaptic function at the rod bipolar ribbon synapses in the mammalian retina.     

Growth of postnatal rat retina in vitro. Development of neurotransmitter systems

In this study, we demonstrate that explanted neonatal rat retina can be maintained in culture for periods up to 3 weeks. The cultured retinas displayed a distinct layering that was almost identical to litter-matched retinas of the same age, but the majority of the ganglion cells did not survive and photoreceptor outer segments did not develop properly. Distinct synaptophysin immunoreactivity was expressed in both the inner and outer plexiform layers of cultured retina and the pattern mimicked that one observed in vivo. After 2-3 weeks in vitro, the inner retina expressed immunoreactivities to various components of the cholinergic and nitrergic transmitter systems, including nitric oxide activated cyclic GMP immunoreactivity. The investigated cell populations displayed similar distribution patterns as in situ, but morphological differences appeared in vitro. Such differences were mainly observed as irregularities in the arborization patterns in the inner part of the inner plexiform layer. We suggest that these discrepancies may arise as a result of reduced ganglion cell survival. Our observations demonstrate that some neurotransmitter systems develop in vitro and their neural circuitry appears similar to the in vivo situation. The presence of synapses, receptor proteins and transmitter substances implies that neural communication can occur in cultured retinas.     

高血压大鼠视网膜组织Ca^2＋酸性磷酸酶组织化学的研究

目的：应用光镜定量酶组织化学方法对正常京都种大鼠（ＷＫＹ）和自发性高血压大鼠（ＳＨＲ）视网膜组织的Ｃａ＾２＋－酸性磷酸酶的分布和活性进行定量观察，结果：Ｃａ＾２＋酸性磷酸酶在ＷＫＹ视网膜组织的活性由强到弱依次为（Ｆ检验，Ｐ〈０．０５）；（１）杆锥细胞内节和外核层；（２）节细胞层；（３）内核层；（４）内网层和外网层；（５）杆锥细胞外节阴性，在ＳＨＲ视网膜组织中，各层Ｃａｓ＾２＋酸性磷酸酶活性下降，以     

Expression profile of heat shock protein 108 during retinal development in the chick

In the developing chick retina, heat shock protein 108 (HSP108), which exhibits transferrin binding activity, has been demonstrated at the mRNA level, while transferrin shows two expression peaks. Here, we investigated the expression profile of HSP108 in the developing chick retina at the protein level. The localization of HSP108 in embryonic days 15 (E15), E18, and postnatal day 2 (P2) chick retina was examined immunohistochemically using monoclonal antibody 9G10 specific for chick HSP108, while the expression levels of HSP108 in developing chick retina from E12 to P2 and adult were measured by Western blot analysis. HSP108 was expressed in the ganglion cell layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, inner segments of photoreceptors and retinal pigment epithelium. Two peaks of HSP108 expression were found at around E13 and E18, respectively. Since the two HSP108 peaks appeared to be correlated with the transferrin expression peaks during retinal development, HSP108 may be associated with iron metabolism during the development of the retina.     

The development of the retina of the albino rat

Morphogenesis of the retina of the Sprague-Dawley albino rat was studied by light microscopy from day 11 of gestation until 225 days after birth. A quantitative analysis during development of retinal volume, thickness of the entire neural retina and thickness of each of the retinal layers, both posteriorly and peripherally, was made. The results indicate that initially a single neuroblastic layer forms and continually thickens by mitosis at its outer border. The retinal layers then form in sequence, moving from the inner retinal border outward and always beginning posteriorly and then spreading peripherally. The transient layer of Chievitz does not appear. All adult layers are present by eight days after birth and each layer thins after reaching its maximal thickness. Total thickness of the retina excluding pigmented epithelium, is greatest on postnatal day 5, but retinal volume only reaches a peak on postnatal days 7 to 12. The nerve fiber, inner plexiform, outer plexiform and bacillary layers all continue to increase in thickness after the ganglion cell and inner and outer nuclear layers reach their maximal width and are beginning to become thinner.