Immunologic detection of retina cognin on the surface of embryonic cells

The retina cell-aggregating glycoprotein, referred to as the retina cognin, has been demonstrated to be located at the surface of embryonic neural retina cells. The term cognin is used to indicate its postulated role in the mechanism of mutual recognition and morphogenetic association of embryonic cells. Antiserum was prepared to the highly purified retina cognin derived from isolated cell membranes of chick embryo retina, and it was used to detect the cognin on cells from chick embryos by means of complement-mediated cell lysis. Retina cells (from 10-day embryos) freshly dissociated with trypsin showed little—if any—lysis by the cognin antiserum; this is consistent with the sensitivity of the cognin to trypsin. However, the cells became susceptible to immunolysis after a period of incubation at 37 °C, which indicates regeneration of the cognin at the cell surface during the recovery period. This regeneration required protein synthesis. Immunofluorescence tests showed binding of the antiserum to the surface of the recovered cells, thereby further demonstrating the surface location of the cognin. The presence, availability or ability to regenerate the cognin, as assayed here, declined sharply with the embryonic age of the cells. Addition of exogenous cognin to freshly trypsin-dissociated retina cells (from 10-day embryos) markedly increased their susceptibility to immunolysis by the cognin antiserum, which indicates that the added cognin becomes associated with the surface of these cells. In contrast, addition of retina cognin to cells freshly trypsinized from 10-day embryo optic tectum and cerebrum, or from 14-day retina did not increase their susceptibility to immunolysis by the cognin antiserum. These results are consistent with earlier findings that enhancement of cell aggregation by the retina cognin is tissue-specific and stage-specific. Cells from non-neural tissues of the chick embryo were not lysed by the retina cognin antiserum. However, neural tissues, such as optic tectum, were found to contain cells which showed surface cross-reaction with the retina cognin antiserum.     

Retina cognin does not bind to itself during membrane interaction in vitro

Retina cognin (R-cognin) is an intrinsic membrane protein of vertebrate retinal cells which supports tissue-specific cell adhesion and mediates cell type-specific associations during development. As a first step in understanding how R-cognin mediates specific adhesion of retinal cell membranes, we asked if cognin bound to another cognin molecule or to a different macromolecule, a possible cognin-binding protein. To do this, we constructed an affinity column with retinal cell membrane proteins (enriched for cognin) bound to the matrix. Proteins in a detergent extract of retinal cell membranes were exposed to this matrix and those which bound specifically eluted and identified by immunoelectrophoresis. Most prominent among these was a protein with an apparent mass of 64 kDa. The binding of this material to the column was blocked by cognin antibody. To eliminate possible artifacts of molecular interactions in vitro, we sought independent confirmation that 64 kDa protein actually bound R-cognin. Using a modified retina membrane vesicle system, we asked what proteins could be photoaffinity cross-linked to cognin during vesicle aggregation. Cross-linking produced a 114 kDa complex on gels which could be resolved into a 50 kDa (cognin) and a 64 kDa band under reducing conditions. Identification of a 64 kDa protein by independent techniques suggests that cognin promotes association of embryonic chick neural retina cells by binding to this macromolecule or these molecules. Identification of a second component in the mechanism should allow elucidation of cognin's role in mediating cell-cell interactions in developing neural retina.     

Possible Roles for Cognin in Chick Embryo Neural Retina

In this review, we discuss current information about a cell-cellrecognition protein present in chick embryo neural retina. Thisprotein, retina cognin, has cell adhesion or aggregation promotingpropertiesin vitro. We discuss five questions. First, what isretina cognin (R-cognin)? Second, what do we know about cogninin chick retina? We discuss its histological distribution inretina and how that distribution changes during embryonic andearly post-hatching development. Third, where is cognin withincells? We review light microscopy evidence for its localizationin plasma membranes of somas and neurites of selected retinalneurons as an intrinsic membrane protein. Fourth, how is cognindistributed in membranes? We summarize evidence that cogninmight not be uniformly distributed over cellsurfaces and thatit might bind to specific proteins on the surfaces of otherretina cells. From the available information, we ask what wecan deduce about cognin's biological role in the neural retina.     

Electron microscopic observation of pituitary adenylate cyclase-activating polypeptide (PACAP)-containing neurons in the rat retina

The distribution and localization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the rat retina were studied by immunocytochemistry with both light and electron microscopy. PACAP-like immunoreactivity (PACAP-LI) was detected in the amacrine and horizontal cells as well as in the inner plexiform layer, the ganglion cell layer and the nerve fiber layer. PACAP-LI seemed to be concentrated predominantly in the neuronal perikarya and their processes, but not in other cells in the retina. At the ultrastructural level, PACAP-LI was visible in the plasma membranes, rough endoplasmic reticulum, and cytoplasmic matrix in the PACAP-positive neurons in the inner nuclear layer. In the inner plexiform layer, PACAP-positive amacrine cell processes made synaptic contact with immunonegative amacrine cell processes, bipolar cell processes, and ganglion cell terminals. These findings suggest that PACAP may function as a neurotransmitter and/or neuromodulator.     

Cellular and subcellular localization of heme oxygenase-2 in monkey retina

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.     

Retinal neurogenesis: the formation of the initial central patch of postmitotic cells

We have investigated the relationship between the birthdate and the onset of differentiation of neurons in the embryonic zebrafish neural retina. Birthdates were established by a single injection of bromodeoxyuridine into embryos of closely spaced ages. Differentiation was revealed in the same embryos with a neuron-specific antibody, zn12. The first bromodeoxyuridine-negative (postmitotic) cells occupied the ganglion cell layer of ventronasal retina, where they formed a small cluster of 10 cells or less that included the first zn12-positive cells (neurons). New cells were recruited to both populations (bromodeoxyuridine-negative and zn12-positive) along the same front, similar to the unfolding of a fan, to produce a circular central patch of hundreds of cells in the ganglion cell layer about 9 h later. Thus the formation of this central patch, previously considered as the start of retinal neurogenesis, was actually a secondary event, with a developmental history of its own. The first neurons outside the ganglion cell layer also appeared in ventronasal retina, indicating that the ventronasal region was the site of initiation of all retinal neurogenesis. Within a column (a small cluster of neuroepithelial cells), postmitotic cells appeared first in the ganglion cell layer, then the inner nuclear layer, and then the outer nuclear layer, so cell birthday and cell fate were correlated within a column. The terminal mitoses occurred in three bursts separated by two 10-h intervals during which proliferation continued without terminal mitoses.     

Organotypic culture of neural retina as a research model of neurodegeneration of ganglion cells

Organotypic models deserve special attention among the large variety of methods of vertebrate retina cultivation. The purpose of this study was to make a detailed qualitative and quantitative characterization of a model employing roller organotypic cultivation of the neural retina of rat eye posterior segment, with special attention to morphological and functional characteristics of retinal ganglion cells. The study included morphological analysis of retina histological preparations as well as estimation of RNA synthesis and evaluation of neuron survival by the Brachet and TUNEL methods, respectively. Retina has been shown to display normal morphofunctional characteristics for the first 12 h of cultivation. After 24 h, a substantial number of ganglion cells underwent pyknosis and stopped RNA synthesis. Almost all the cells of the retinal ganglion layer became apoptotic by 3–4 days in vitro. In the course of cultivation, neural retina is detached from the underlying layers of the posterior eye segment and undergoes significant cytoarchitectonic changes. The causes of ganglion cell death during organotypic cultivation of eye posterior segment are discussed. This method can serve as a suitable model for the screening of new retinoprotectors and for research on ganglion cell death resulting from retina degenerative diseases, e.g. glaucoma.     

Developmental study of the expression of B50/GAP-43 in rat retina

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons–bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule. © 1993 John Wiley & Sons, Inc.     

Antibodies against neurofilament subunits label retinal ganglion cells but not displaced amacrine cells of hamsters.

Although neurofilament (NF) antibodies have been used to visualize ganglion cells and their axons in the retina, it is not known, however, how many ganglion cells contain NF, and how the various NF subunits are distributed in the ganglion cells. Moreover, it is not known whether displaced amacrine cells in the ganglion cell layer are also labelled. In order to see whether NF antibodies can be used as a specific marker for ganglion cells, antibodies raised against the low (NF-L), middle (NF-M) and high (NF-H) molecular weight subunits of NF were employed to stain retinal whole-mounts of adult hamsters after pre-labelling the ganglion cells with Granular Blue. It was found that NF-L and NF-H antibodies labelled 38,777 and 17,750 cells in the ganglion cell layer respectively. By co-localization with GB-labelled cells, 88% of NF-L positive cells and 91% of NF-H positive cells were found to be ganglion cells. In contrast, the NF-M antibody labelled only very few ganglion cells (418 per retina) although robust staining of axonal bundles was observed. Thus, NF antibodies may prove useful in studying this population of ganglion cells.     

Nitric oxide synthase immunoreactivity in the developing and adult human retina

Nitric oxide synthase (NOS) catalyzes the formation of nitric oxide (NO) from L-arginine. In this study, the cellular localization of neuronal NOS (nNOS) activity in the human retina since fetal development was examined by immunohistochemistry. No detectable staining in the fetal retina was present at 14 weeks of gestation (wg), the earliest age group examined. A centro-peripheral gradient of development of nNOS immunoreactivity was evident at 16–17 wg, with the midperipheral retina showing nNOS immunoreactivity in most of the cell types and the inner plexiform layer while the peripheral part demonstrated moderate immunoreactivity only in the ganglion cell layer and photoreceptor precursors. A transient increase in nNOS immunoreactivity in the ganglion cells and Müller cell endfeet between 18–19 and 24–25 wg was observed at the time when programmed cell death in the ganglion cell layer, loss of optic nerve fibres as well as increase in glutamate immunoreactivity and parvalbumin (a calcium binding protein) immunoreactivity in the ganglion cells was reported. These observations indicate that programmed cell death of ganglion cells in the retina may be linked to glutamate toxicity and NO activity, as also suggested by others in the retina and cerebral cortex. The presence of nNOS immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions, besides their involvement in photoreceptor function of transduction and information processing.     

Localization of the Thy-1 antigen to the surfaces of rat retinal ganglion cells

Indirect immunofluorescence has been used to localize the Thy-1 antigen to ganglion cell axons, the ganglion cell layer and the inner plexiform layer in cryostat sections of adult and neonatal rat retina. In similar immunofluorescence experiments monoclonal antibodies raised against the 200,000 molecular weight neurofilament polypeptide bound only to ganglion cell axons and processes in the outer plexiform layer.Less than 1% of cells dissociated from 8 day postnatal rat retina had superficial Thy-1 antigen demonstrable by immunofluorescence; these cells were generally large and their size spectrum was similar to that of ganglion cells $\text{in}$$\text{situ}$. After culture for 1 day many of these Thy-1 positive cells had generated neurofilament antigen.We conclude that Thy-1 is found chiefly or exclusively on ganglion cells of eight day retina, and may be useful in the identification and isolation of these cells by immunoselection procedures.     

Morphology of congenital microphthalmia in chicks (Gallus gallus)

This study examines the morphology of sporadic congenital microphthalmia in 1-day-old chicks, with particular emphasis on the neural retina. On the basis of the size of the eyeball it is possible to classify microphthalmia into two groups, severe and mild. In severe microphthalmia (less than 5 mm in equatorial diameter), the eyeball is severely malformed, but in most cases it shows evidence of an organized neural retina. Although ganglion cells and an optic nerve head are present in a small proportion of these retinae, we could not trace an optic nerve projection to the brain. These results indicate that some ganglion cells are able to be sustained after the period of naturally occurring cell death, suggesting either that those ganglion cells have established some contact with the central nervous system or that the presence of their axons in a rudimentary optic nerve is adequate for survival. In mild microphthalmia (greater than 5 mm in equatorial diameter), the most consistent abnormality is a defect in the pecten, which together with other abnormalities such as orbital cysts and colobomas indicates that the major abnormality occurs in the region of the choroid fissure. Associated with these defects are abnormalities within the ganglion cell layer. In some cases the number of ganglion cells was reduced, and in others the numbers of both ganglion and displaced amacrine cells were reduced. Unexpectedly, there were localized regions completely devoid of cells in the ganglion cell layer. The timing of the congenital defect may provide some clue as to the presence of a critical period in which displaced amacrine cells are formed or are sensitive to events related to ganglion cell loss.     

Sonic hedgehog, secreted by amacrine cells, acts as a short-range signal to direct differentiation and lamination in the zebrafish retina

Neurogenesis in the zebrafish retina occurs in several waves of differentiation. The first neurogenic wave generates ganglion cells and depends on hedgehog (hh) signaling activity. Using transgenic zebrafish embryos that express GFP under the control of the sonic hedgehog (shh) promoter, we imaged the differentiation wave in the retina and show that, in addition to the wave in the ganglion cell layer, shh expression also spreads in the inner nuclear layer. This second wave generates amacrine cells expressing shh, and although it overlaps temporally with the first wave, it does not depend on it, as it occurs in the absence of ganglion cells. We also show that differentiation of cell types found in the inner and outer nuclear layers, as well as lamination of the retina, depends on shh. By performing mosaic analysis, we demonstrate that Shh directs these events as a short-range signal within the neural retina.     

Amacrine cells, displaced amacrine cells and interplexiform cells in the retina of the rat

The amacrine cells in the retina of the rat are described in Golgi-stained whole-mounted retinae. Nine morphologically distinct types of cell were found: one type of diffuse cell, five types of unistratified cell, two types of bistratified cell, and one type of stratified diffuse cell. Measurements show that the largest unistratified cells have a dendritic field 2 mm across. One type of interplexiform cell is also described. Wide-field diffuse amacrine cells and unistratified amacrine cells were found with their somata located in either the inner nuclear layer or the ganglion cell layer. It is clear that there may be an amacrine cell system in the ganglion cell layer of the rat retina.     

NMDA-receptor blockade enhances cell apoptosis in the developing retina of the postnatal rat

During visual system development, programmed cell death occurs in order to facilitate the establishment of correct connections and synapses. During this period, glutamate plays a very important role as an excitatory neurotransmitter. With a view to evaluating if NMDA glutamate receptors participate in the regulation of apoptosis which occurs during the development of the rat retina, we subcutaneously injected the NMDA receptor antagonist MK-801 into rats at different stages of early postnatal development (P2 to P9). Ensuing cell death in the retina and superior colliculus was analyzed by using the Feulgen method. MK-801 administration had no effect on the survival of photoreceptor cells. In contrast, the presence of this antagonist induced a significant increase in the number of apoptotic cells in the neuroblastic layer (P7 and P8) and ganglion cell layer (P6-P8), as well as in the superior colliculus which receives afferent contacts from retinal ganglion cells during P7-P9. We conclude that during development, specific types of cells in the mammalian retina are critically dependent for their survival on glutamate stimulation through NMDA receptors. These findings thus throw fresh light on the mechanisms of development of the rat visual system by identifying NMDA glutamate receptors as participants in the regulation of apoptotic processes which occur during the initial stages of development.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

Cytochemical localization of 5'-nucleotidase in the frog (Rana pipiens) retina. A histochemical and cytochemical study

Localization of 5'-nucleotidase in the frog retina was investigated using histochemical and cytochemical techniques. Light-microscopic observations revealed the presence of this enzyme in the inner retinal layers (the nerve fiber layer, ganglion cell layer, and inner plexiform layer). Ultrastructural investigations revealed that the enzyme activity is associated with the plasma membranes of the Müller cell processes, whereas the Müller cell processes present in the outer retinal layers did not demonstrate any detectable enzyme activity. This observation would appear to confirm our previous findings, that 5'-nucleotidase is an ectoenzyme, but its distribution in frog retina differs from that in rodents and it is only present in the inner layers of the retina. The prominent localization of 5'-nucleotidase on the glial plasma membrane may be viewed in the context of the widely accepted interaction between neurones and glial cells. Since nucleotides do not penetrate the plasma membrane, a mechanism to produce membrane-permeable adenosine, important for neuronal function, is postulated. It is known that 5'-nucleotidase produces adenosine by hydrolyzing adenosine 5'-monophosphate (5'-AMP). Therefore one would expect that the glial membrane-bound enzyme can accomplish the final step in this mechanism by producing the adenosine in the extracellular spaces.     

Early patterns of neuronal degeneration in the retina of the goldfish after optic nerve sectioning

The early patterns of retinal degeneration were studied in the goldfish after optic nerve sectioning by l.m. and e.m. Beginning on the 2nd postsurgical day there was an initial degeneration of neurons in the ganglion cell and inner nuclear layers of the central retina. Massive ganglion cell degeneration in the whole retina (60%) as well as degeneration of neurons in inner and outer nuclear layer of the peripheral retina was evident around the 7th postsurgical day. The early degenerating cells appeared to be cones and cone bipolars.     

人胎视网膜SOD免疫阳性细胞的发育与细胞凋亡

选择不同胎龄的人胎18例,用免疫细胞化学ABC法和TUNEL法观察人胎视网膜超氧化物歧化酶（SOD）免疫阳性细胞的发育和细胞凋亡。结果显示;（1）E15w节细胞层开始出现SOD免疫阳性细胞,E20w和E28wSOD免疫阳性细胞排列较整齐,分布于视网膜的外核层,内核层,节细胞层;E40wSOD免疫阳性细胞主要集中于视网膜的外核层,内核层,节细胞层,其数量增多,特别是内核层SOD免疫阳性细胞增多明显。（2）TUNEL法标记的视网膜凋亡细胞胞核具有指环状典型的凋亡特征,E12w人胎视网膜未见凋亡细胞,E15w,E17w凋亡细胞较多,大小不一,分布于视网膜的全层;E20w凋亡细胞主要集中在内核层,数量减少,E28w凋亡细胞仅见于内核层,胞核呈指环样外观,着色较深,但数量较E20w进一步减少;E40w视网膜全层未见凋亡细胞。结果提示,E28w视网膜SOD抗氧化酶系,光感受的基本结构初步发育成熟,其抗氧化保护作用可能主要来源于内核层的SOD免疫阳性细胞。