Localization of CD15 immunoreactivity in the rat retina

Using immunocytochemistry, we have investigated the localization of CD15 in the rat retina. In the present study, two types of amacrine cell in the inner nuclear layer (INL) and some cells in the ganglion cell layer were labeled with anti-CD15 antisera. Type 1 amacrine cells have large somata located in the INL, with long and branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). Type 2 cells have a smaller soma and processes branching in stratum 1 of the IPL. A third population showing CD15 immunoreactivity was a class of displaced amacrine cells in the ganglion cell layer. The densities of type 1 and type 2 amacrine cells were 166/mm(2) and 190/mm(2) in the central retina, respectively. The density of displaced amacrine cells was 195/mm(2). Colocalization experiments demonstrated that these CD15-immunoreactive cells exhibit gamma-aminobutyric acid and neuronal nitric oxide synthase (nNOS) immunoreactivities. Thus, the same cells of the rat retina are labeled by anti-CD15 and anti-nNOS antisera and these cells constitute a subpopulation of GABAergic amacrine cells.     

Morphology and distribution of Müller cells in the retina of the toad Bufo marinus

We have previously shown that an antibody against neuron-specific enolase (NSE) selectively labels Müller cells (MCs) in the anuran retina (Wilhelm et al. 1992). In the present study the light- and electron-microscopic morphology of MCs and their distribution were described in the retina of the toad, Bufo marinus, using the above antibody. The somata of MCs were located in the proximal part of the inner nuclear layer and were interconnected with each other by their processes. The MCs were uniformly distributed across the retina with an average density of 1500 cells/mm². Processes of MCs encircled the somata of photoreceptor cells isolating them from each other by glial sheath, except for those of the double cones. Some of the photoreceptor pedicles remained free of glial sheath. Electron-microscopic observations confirmed that MC processes provide an extensive scaffolding across the neural retina. At the outer border of the ganglion cell layer these processes formed a non-continuous sheath. The MC processes traversed through the ganglion cell layer and spread beneath it between the neuronal somata and the underlying optic axons. These processes formed a continuous inner limiting membrane separating the optic fibre layer from the vitreous tissue. Neither astrocytic nor oligodendrocytic elements were found in the optic fibre layer. The significance of the uniform MC distribution and the functional implications of the observed pattern of MC scaffolding are discussed.     

人胎视网膜神经肽Y免疫反应神经元的发育

本文用免疫细胞化学ＡＢＣ法,研究１５—３８周龄人胎视网膜神经肽Ｙ免疫反应（ＮｅｕｒｏｐｅｐｔｉｄｅＹｉｍｍｕｎｏｒｅｃｔｉｖｅ,ＮＰＹ－ＩＲ）神经元（以下称ＮＰＹ－ＩＲ细胞）的发育。结果表明：①胎龄１５周视网膜中央部已出现不同类型的ＮＰＹ－ＩＲ细胞：位于黄斑及其周围外核层的为ＮＰＹ－ＩＲ视锥细胞;位于内核层最内一列的为ＮＰＹ－ＩＲ无长突细胞位于节细胞层的可能为ＮＰＹ－ＩＲ移位无长突细胞或节细胞;内核层和节细胞层的ＮＰＹ－ＩＲ细胞的突起均分布在内网层的第１亚层。②胎龄２４周后,ＮＰＹ－ＩＲ视锥细胞完全消失。③随着视网膜的发育,内核层和节细胞层的ＮＰＹ－ＩＲ细胞数量增多,突起增粗增长,胞体分布由中央部扩展到周边部,其中内核层ＮＰＹ－ＩＲ细胞的密度呈现从中央部向周边部逐渐降低的分布方式,节细胞层ＮＰＹ－ＩＲ细胞则多数集中分布在视网膜的边缘和黄斑之间,形成较高密度的环状区。     

Amacrine cells in the retina of a cyprinid fish: functional characterization and intracellular labelling with horseradish peroxidase

Summary Forty amacrine cells in retinae of a cyprinid fish, the roach, were intracellularly labelled with horseradish peroxidase following electrophysiological identification as sustained depolarizing, sustained hyperpolarizing or transient units. Labelled cells were analysed by light microscopy and compared with a catalogue of amacrine cells established in a previous Golgi study on the same species. About 30% of the cell types characterized by the Golgi method were encountered in the present study. When intracellularly labelled cells were differentiated on the basis of their dendritic organization in the plane of the retina, a given electrophysiological response pattern was found to be generated by different morphological types, and vice versa. However, examination of the ramification patterns of the dendrites within the inner plexiform layer (i.e. in the radial dimension of the retina), showed that this morphological parameter of a given amacrine cell could be correlated with its light-evoked response. Several amacrine cell types were found to possess special distal dendrites which arose from the main dendritic branches and extended well over a mm in the retina. Distal dendrites were oriented tangentially with respect to the optic nerve papilla, but did not appear to be involved in any synaptic connectivity. It is concluded that the Golgi-based classification is a valuable tool for identifying intracellularly labelled amacrine cells. However, although the correlation between layering of dendrites in the inner plexiform layer and electrophysiology was generally good, additional physiological parameters would be required to determine whether more extensive parallels exist between structural and functional characteristics of amacrine cells. Alternatively, the considerable morphological diversity of amacrine cells may be of limited physiological significance.A preliminary account of the present findings was presented to the Physiological Society (Djamgoz et al. 1984)     

Morphological analysis of CD15-immunoreactive neurons in the guinea pig retina

Using immunocytochemistry, morphometry and electron microscopy, we have investigated the distribution and characteristics of CD15-immunoreactive (IR) neurons in the guinea pig retina. In the present study, two types of amacrine cells, including interplexiform cells in the inner nuclear layer (INL) and some cells in the ganglion cell layer (GCL), were labeled with anti-CD15 antisera. Type 1 amacrine cells had large somata located in the INL, with long and branched processes ramifying mainly in strata 4 and 5 of the inner plexiform layer (IPL). Somata of type 2 cells had smaller diameters, and were also located in the INL. Their processes stratified in stratum 1. The densities of type I and type 2 amacrine cells increased from 152.8+/-36.7/mm2 and 160.6+/-61.7/mm2 in the peripheral retina, to 404.3+/-41.5/mm2 and 552.2+/-72.2/mm2 in the central retina, respectively. Cells in the GCL exhibiting CD15 immunoreactivity were rarely observed. Colocalization experiments, using consecutive semi-thin sections, demonstrated that these CD15-IR amacrine cells exhibited gamma-aminobutyric acid (GABA) immunoreactivity. In addition, the processes of the type 1 cells formed one member of the postsynaptic dyads that are formed in the axon terminals of rod bipolar cells. Most of these processes made reciprocal synapses back to the axon terminals of the rod bipolar cells. Thus, CD15-IR amacrine cells constitute a subpopulation of GABAergic amacrine cells in the guinea pig retina, and the type 1 cells among them provide the inhibitory input to rod bipolar cells.     

Localization of glutamate in the human retina during early prenatal development

In this study we have localized glutamate (GLU) in fetal (14–25 weeks gestation, Wg) human retinas by immunohistochemistry. At 14 Wg, GLU-immunoreactivity (IR) was localized only in the central part of retina, showing a prominently labelled nerve fiber layero A few ganglion cells and displaced amacrine cells were very weakly labelled. At 17 Wg, GLU was localized conspicuously in many ganglion cells, displaced amacrine cells, some amacrine cells and the prospective photoreceptor cell bodies in the neuroepithelial layero With progressive development at 20 and 25 Wg, the IR for GLU was found additionally in the Müller cell endfeet, some bipolar cells as well as in the horizontal cells that were aligned in a row along the outer border of the inner nuclear layer of the central retinao The photoreceptor cell bodies in the outer nuclear layer were also prominently immunopositive for GLU. The developmental distribution of GLU in the human retina tends to indicate that it plays an important role in the differentiation and maturation of retinal neurons.     

Morphology of the retina in deep‐water fish Nezumia sclerorhynchus (Valenciennes, 1838) (Gadiformes: Macrouridae)

Using light microscopy, we examined the retina of benthopelagic fish Nezumia sclerorhynchus. Although the retina is typical of other vertebrates, having three nuclear and two synaptic layers, it presents some features associated with the animal's deep‐sea habitat. A stratum argenteum containing iridescent crystals is located in the choroid. The pigment cell layer shows bulky cells filled with melanin granules but without the typical apical processes. The visual cells, consisting of a big population of rods, are arranged in several banks. No cones were observed. The outer segments are very long and cylindrical, and the inner segments are constituted by a small ellipsoid at the proximal end. The outer nuclear layer contains several rows of oval nuclei, and the spherules in the outer plexiform layer have less regular outlines than nuclei. The inner retina is characterized by very large horizontal cells, and presumable bipolar and amacrine cells separated by large spaces that are occupied by neuronal processes. Finally, the low density of ganglion cells produces a thin nerve fibre layer. The results of this study suggest that the retina of Nezumia sclerorhyncus exhibits high visual sensitivity and that vision is a sense that plays an important role in its behaviour.     

SOMATOSTATIN-LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA

Immunocytochemical techniques were employed to locate somatostatin (SS)-containing cells in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS-like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS-positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.     

Distribution of calcitonin gene-related peptide immunoreactivity in the central nervous system of the forg,Rana esculenta

Summary Tyrosine hydroxylase (TH) immunocytochemistry was utilized to quantify dopaminergic synapses in the inner plexiform layer of the retina of Bufo marinus. Since dopaminergic cells have bistratified dendritic arborisation in the inner plexiform layer, attention was given to the segregation of synapses between the scleral and the vitreal sublaminae. Light-microscopically, a more elaborate dendritic branching was observed in the scleral than in the vitreal sublamina. In contrast, about 55% of synapses occurred in the vitreal one fifth of the inner plexiform layer, 30% in the scleral fifth, and 15% in the intermediate laminae. Input sources and output targets showed only minor quantitative differences between sublaminae 1 and 5. TH-immunoreactive processes were found in presynaptic (62.8%) and postsynaptic (37.2%) positions. Synapses to the stained dendrites derived from bipolar (40.4%) and amacrine (59.6%) cells, whereas outputs from the TH-positive processes were directed to amacrine cells (56.8%) and to small and medium-sized dendrites (35.4%); at least some of these can be considered as ganglion cell dendrites. TH-positive profiles neither formed synapses with each other nor were presynaptic to bipolar cell terminals. Junctional appositions of the immunoreactive profiles were occasionally seen on non-stained amacrine and ganglion cell dendrites in the scleral sublamina of the inner plexiform layer and on optic axons in the optic fibre layer. Although dopaminergic cells are mainly involved in amacrine-amacrine interactions, inputs from bipolar terminals and outputs to ganglion cell dendrites were also substantial, suggestive of a role also in vertical information processing.     

Calbindin and calretinin immunoreactivities in the retina of a chondrostean,Acipenser baeri

The distribution of calbindin and calretinin in the retina of the sturgeon Acipenser baeri was studied with immunocytochemistry. Western blot analysis of brain extracts, together with immunocytochemical results in the retina and brain, indicated the presence of the two calcium-binding proteins in sturgeon. Calbindin immunocytochemistry revealed only a large displaced bipolar cell type with narrowly stratified axons, similar to some mixed rod and cones bipolar cells described in teleosts. The plexus formed by the axons of these cells in the inner plexiform sublayer was similar to that formed by calbindin-immunoreactive diffuse bipolar cells of some mammals. Calretinin immunocytochemistry also stained these displaced bipolar cells, most ganglion cells including displaced ganglion cells (Dogiel cells), and some amacrine cells of the inner nuclear layer. The distribution of calbindin and calretinin immunoreactivities in the retina of a primitive bony fish indicates that these proteins are highly specific to the cell type.     

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.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Changes in retinal neuronal populations in the DBA/2J mouse

DBA/2J (D2) mice develop a form of progressive pigmentary glaucoma with increasing age. We have compared retinal cell populations of D2 mice with those in control C57BL/6J mice to provide information on retinal histopathology in the D2 mouse. The D2 mouse retina is characterized by a reduction in retinal thickness caused mainly by a thinning of the inner retinal layers. Immunocytochemical staining for specific inner retinal neuronal markers, viz., calbindin for horizontal cells; protein kinase C (PKC) and recoverin for bipolar cells, glycine, -aminobutyric acid (GABA), choline acetyltransferase (ChAT), and nitric oxide synthase (NOS) for amacrine cells, and osteopontin (OPN) for ganglion cells, was performed to detect preferentially affected neurons in the D2 mouse retina. Calbindin, PKC, and recoverin immunoreactivities were not significantly altered. Amacrine cells immunoreactive for GABA, ChAT, and OPN were markedly decreased in number, whereas NOS-immunoreactive amacrine cells increased in number. However, no changes were observed in the population of glycine-immunoreactive amacrine cells. These findings indicate a significant loss of retinal ganglion and some amacrine cells, whereas glycinergic amacrine cells, horizontal, and bipolar cells are almost unaffected in the D2 mouse. The reduction in amacrine cells appears to be attributable to a loss of GABAergic and particularly cholinergic amacrine cells. The increase in nitrergic neurons with the consequent increase in NOS and NO may be important in the changes in the retinal organization that lead to glaucomain D2 mice. Thus, the D2 mouse retina represents a useful model for studying the pathogenesis of glaucoma and mechanisms of retinal neuronal death and for evaluating neuroprotection strategies.Jung-Il Moon and In-Beom Kim contributed equally to this work.This work was supported by a Korea Research Foundation Grant (FP 0005) and by BK 21 in Korea.     

Autoradiographic Localization of <Superscript>3</Superscript>H-GABA in Rat Retina

γ-AMINOBUTYRIC acid (GABA) is present in all layers of vertebrate retinae^1–3: in the rabbit retina it seems to be most concentrated in the ganglion cell layers² while in the frog it is concentrated primarily in cell layers which are rich in amacrine cells¹. Recent autoradiographic studies of the distribution of ³H-GABA in rat brain slices after incubation in vitro suggest that the labelled amino-acid is selectively concentrated by certain neural elements^4,5. In a study of the distribution of ³H-GABA in rabbit retina after injection of the labelled amino-acid into the eye, Ehinger⁶ found that radioactivity was accumulated principally in the inner plexiform, inner nuclear and ganglion cell and nerve fibre layers. Labelling was also concentrated in some cells occupying the same position as amacrine cells and in some nerve cells of the ganglion cell layer.     

半滑舌鳎仔、稚鱼视网膜结构与视觉特性

对1-50d半滑舌鳎仔、稚鱼视网膜和全长50mm的半滑舌鳎幼鱼视网膜结构和视觉特性进行了研究。结果表明:(1)3d仔鱼色素层形成,15d仔鱼没有显著的视网膜运动反应,25d时具有正常感受自然光的明视功能,43d半滑舌鳎稚鱼适应自然光的功能丧失;(2)半滑舌鳎仔鱼阶段感受细胞主要为高密度的单锥,视杆细胞和双锥细胞出现的较晚;单锥融合成双锥时,由于半滑舌鳎视锥细胞椭圆体细长,融合程度较差,尽管在视网膜横切面上能够看到双锥,但在切向切面上仍呈现单锥排列方式;随其生长发育,视锥和神经节细胞密度降低,视杆细胞密度增加,31d后视杆细胞数量显著增加;同时,外核层细胞核与神经节细胞的比值增大,网络会聚程度提高;相关数据表明,20-31d是视网膜结构和视觉特性发生明显变化的过渡时期,这是与半滑舌鳎从浮游生活到底栖生活生态环境的变化相适应的;(3)半滑舌鳎内核层结构特殊,50mm时只有1层水平细胞,属感光系统不发达类型,双极细胞和无长突细胞共4-5层,但不可分辨;内核层细胞层数的减少,基本上没有分化的水平细胞、双极细胞和无长突细胞,说明半滑舌鳎视网膜的光敏感性不高;(4)半滑舌鳎仔鱼浮游生活阶段视敏度较高,视觉在捕食行为中具有重要意义;底栖生活后,视敏度和光敏感性都较差,视觉在捕食行为中不可能具有重要作用     

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina. (J Histochem Cytochem 58:377–389, 2010)     

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.