Morphology of calretinin and tyrosine hydroxylase-immunoreactive neurons in the pig retina

The morphology of calretinin- and tyrosine hydroxylase-immunoreactive (IR) neurons in adult pig retina was studied. These neurons were identified using antibody immunocytochemistry. Calretinin immunoreactivity was found in numerous cell bodies in the ganglion cell layer. Large ganglion cells, however, were not labeled. In the inner nuclear layer, the regular distribution of calretinin-IR neurons, the inner marginal location of their cell bodies in the inner nuclear layer, and the distinctive bilaminar morphologies of their dendritic arbors in the inner plexiform layer suggested that these calretinin-IR cells were AII amacrine cells. Calretinin immunoreactivity was observed in both A-and B-type horizontal cells. Neurons in the photoreceptor cell layer were not labeled by this antibody. The great majority of tyrosine hydroxylase-IR neurons were located at the innermost border of the inner nuclear layer (conventional amacrines). The processes were monostratified and ran laterally within layer 1 of the inner plexiform layer. Some of the tyrosine hydroxylase-IR neurons were located in the ganglion cell layer (displaced amacrines). The processes of displaced tyrosine hydroxylase-IR amacrine cells were also located within layer 1 of the inner plexiform layer. Some processes of a few neurons were located in the outer plexiform layer. A very low density of neurons had additional bands of tyrosine hydroxylase-IR processes in the middle and deep layers of the inner plexiform layer. The processes of tyrosine hydroxylase-IR neurons extended radially over a wide area and formed large, moderately branched dendritic fields. These processes occasionally had varicosities and formed "dendritic rings". These results indicate that calretinin- and tyrosine hydroxylase-IR neurons represent specific neuronal cell types in the pig retina.     

An electron microscopic study of neuronal degeneration and glial cell reaction in the retina of glaucomatous rats

The present investigation was focused on the ultrastructural changes in the neurons and glial cells in the retina of rats with experimentally-induced glaucoma. An experimental glaucoma model was created by limbal-derived vein cauterization. Animals were sacrificed at 1, 3 weeks and 3 months post-operation. Retinae were dissected and processed for electron microscopy. Neuronal degeneration was observed in all the different layers of the retina at both 1 and 3 weeks post-operation. Some degenerating neurons were found in the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL). And the dying neurons presented apoptotic-like more than necrotic neurons. Many degenerating axons and axon terminals were observed between neurons in the GCL, inner plexiform layer (IPL), INL, and outer plexiform layer (OPL). Activated astrocytes and microglial cells were present in close association with degenerating neurons and axons. The Müller cells in the INL also presented longer and darker processes with more microfilaments than in normal cells. Degenerating neuronal debris, degenerating axonal profiles and electron-dense bodies were often found in the cytoplasm of macrophages. The results suggest that both microglial cells and astrocytes are activated in the process of neuronal degeneration in the retina of experimentally-induced glaucomatous rats. It is hypothesized that they may play a protective role in removing degenerating neuronal elements in the retina after the onset of glaucoma.     

Neurofibrillar long-range amacrine cells in mammalian retinae

A distinct population of wide-field, unistratified amacrine cells are shown to be selectively stained by using neurofibrillar methods in rabbit and cat retinae. Their cell bodies may be located in the inner nuclear, inner plexiform or ganglion cell layers and they branch predominantly in stratum 2 of the inner plexiform layer. Characteristically, each cell has two or more long-range distal processes which extend for 2-3 mm beyond a more symmetrical, proximal dendritic field of 0.6-0.8 mm diameter. Although the neurofibrillar long-range amacrines account for less than 1 amacrine in 500, they achieve effective coverage of the retina by both the proximal and distal dendrites.     

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.     

Class 5 transmembrane semaphorins control selective Mammalian retinal lamination and function

In the vertebrate retina, neurites from distinct neuronal cell types are constrained within the plexiform layers, allowing for establishment of retinal lamination. However, the mechanisms by which retinal neurites are segregated within the inner or outer plexiform layers are not known. We find that the transmembrane semaphorins Sema5A and Sema5B constrain neurites from multiple retinal neuron subtypes within the inner plexiform layer (IPL). In Sema5A?/?; Sema5B?/? mice, retinal ganglion cells (RGCs) and amacrine and bipolar cells exhibit severe defects leading to neurite mistargeting into the outer portions of the retina. These targeting abnormalities are more prominent in the outer (OFF) layers of the IPL and result in functional defects in select RGC response properties. Sema5A and Sema5B inhibit retinal neurite outgrowth through PlexinA1 and PlexinA3 receptors both in vitro and in vivo. These findings define a set of ligands and receptors required for the establishment of inner retinal lamination and function.     

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.     

Adrenergic retinal neurons

Summary Adrenergic retinal neurons have been studied in cynomolgus monkeys, cats, rabbits, guinea-pigs, rats, and mice with the fluorescence technique of Falck and Hillarp. With some species variations, three adrenergic fibre layers have been observed: an outer adrenergic fibre layer (all species) at the border between the inner nuclear and inner plexiform layers, a middle adrenergic fibre layer (rabbits, guinea-pigs, rats, and mice) in the middle of the inner plexiform layer, and an inner adrenergic fibre layer (rabbits) at the border between the inner plexiform layer and the ganglion cell layer. Similarly, three kinds of adrenergic nerve cells have been found: a somewhat heterogenous group of outer adrenergic cells (all species) situated in the innermost cell rows of the inner nuclear layer, eremite cells (rabbits, guinea-pigs, rats, and mice) within the inner plexiform layer and alloganglionic cells (all species) with a position and appearance resembling some of the ordinary non-adrenergic cells of the ganglion cell layer. All the adrenergic cells are star-shaped with slender branching processes running to the different adrenergic layers.The research reported in this document has been sponsored by the Air Force Office of Scientific Research under grant AF EOAR 66-14 through the European Office of Aerospace Research (OAR), United States Air Force, by the United States Public Health Service (grant no. NB 05236-02), by the Swedish Medical Research Council (grant no. B 66-320), and by the Faculty of Medicine, University of Lund, Sweden.     

THE DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE ACTIVITY IN VERTEBRATE RETINA1

Abstract— Choline acetyltransferase (ChAc) activity was determined in retinal layers from 10 vertebrates. In all animals, the highest activity was in the inner plexiform layer, intermediate activity in the inner nuclear and ganglion cell layers, and very low activity in the photoreceptor and outer plexiform layers and optic nerve. The pattern of distribution of enzyme activity within the inner nuclear layer corresponds quantitatively to the distribution of amacrine cells within that layer. A species difference of almost 90-fold was found between the lowest and highest values for ChAc activity in inner plexiform layer. The variation in enzyme activity found among homeotherms in inner nuclear and inner plexiform layers is related to the number of amacrine cell synapses in the inner plexiform layer. But the differences in enzyme activity are generally greater than those which have been found in numbers of amacrine cell synapses between species. The data suggest that cholinergic neurons in retina are to be found predominantly among the amacrine cell types and that not all amacrine cells will be found to be cholinergic.     

Microscopic investigation of the comparative morphology of the retina

Morphological differences in the architectonics (the relations and composition of the layers and sublayers) of the retina are described in various vertebrates: pike, frog, and cat. These differences apply to both cellular and plexiform layers. The differences are particularly marked in the composition of the sublayers of the inner nuclear layer. In the frog the greatest degree of subdivision into layers of processes of the ganglion and amacrine cells is observed to correspond to the particularly complex differentiation of the inner plexiform layer of the retina (about 10 sublayers). In all the animals studied the ganglion cells can be divided into two principal types: symmetrical and asymmetrical, with many varieties. Asymmetrical amacrine cells are found in the pike and frog retina. The presence of vertical processes branching in the outer plexiform layer is confirmed for amacrine cells in the cat retina. The structural features of the retina are discussed in connection with physiological findings.     

胭脂鱼眼早期形态发生研究

采用组织学方法观察了胭脂鱼(Myxocyprinus asiaticus) 眼的发生过程, 结果显示: 胭脂鱼眼的发育经历了眼原基形成期、眼囊形成期、视杯形成期、晶体板形成期、晶体囊形成期、角膜原基形成期、角膜上皮形成期、视网膜细胞增殖期、晶状体成熟期、眼色素形成期以及眼成型期等11个时期。视网膜发育最早, 起始于眼原基的形成, 直至眼成型期分化完成, 形成了厚度不一的8层细胞, 由内向外依次为神经纤维层、神经细胞层、内网层、内核层、外网层、外核层、视杆视锥层和色素上皮层, 且发育历时最长, 约264h。晶状体的发育在视网膜之后, 始于晶体板的形成, 于出膜前期成熟, 发育历时最短, 约74h。角膜发育最晚, 始于角膜原基的形成, 出膜1 d分化为透明的成熟角膜, 发育历时约96h。出膜4 d仔鱼眼色素沉积明显, 视网膜各层分化明显, 晶状体内部完全纤维化, 眼的形态结构基本发育完全。     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Summary Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

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.     

Neuropeptide Y (NPY) immunoreactive neurons in the retina of different species

Neurons displaying Neuropeptide Y (NPY) immunoreactivity were found among amacrine cells in the retina of baboon, pig, cat, pigeon, chicken, frog, trout, carp and goldfish. The immunoreactive cell bodies were located in the middle and the innermost cell rows of the inner nuclear layer with processes forming one, two or three more or less well-defined sublayers in the inner plexiform layer. The location and the density of the sublayers varied with the species investigated. In the frog retina, bipolar-like cell bodies were found in the middle of the inner nuclear layer as well as sparsely occurring ovoid cell bodies in the ganglion cell layer. Like the amacrine cells, these cells emitted processes ramifying in three sublayers in the inner plexiform layer.     

Microtubule-associated proteins characteristic of embryonic brain are found in the adult mammalian retina

We have used monoclonal antibodies against each of the major mammalian brain microtubule-associated proteins (MAPs), MAP1, MAP2, MAP3, MAP5, and tau, to study the timing of appearance and the cytological distribution of these proteins during the development of the rat retina. Western blots of adult rat retina reveal MAPs that are characteristic of embryonic brain, i.e., MAP5 and the low-molecular-weight forms of MAP2 (MAP2c) and tau (juvenile tau). At the onset of neuronal differentiation within the embryonic retina, MAP5, MAP3, MAP2c, and tau are found in the perikarya or extending axons of ganglion cells. High-molecular-weight MAP2, a dendrite marker, does not appear in the retina until the second day of postnatal development, when ganglion cell dendrites ramify within the inner plexiform layer. MAP1, which is characteristic of adult brain, does not appear in the retina until 1 week after birth, and is limited to ganglion cells and their processes. In the adult retina, MAP5 and MAP2c are concentrated within the inner segments and cell bodies of photosensitive cells, whereas tau is found in horizontal cells and more internal cell layers. Since photosensitive cells are unique among retinal neurons in their constant regeneration of their primary processes, the photoreceptive outer segments, both MAP5 and MAP2c appear not only to be involved in events associated with the embryonic differentiation and growth of neurites, but also in process regeneration in adult neurons that maintain some embryonic characteristics.     

IMMUNOHISTOCHEMICAL LOCALIZATION OF GABA IN CHAMELEON RETINA (CHAMALEO CHAMALEO)

We used a policlonal antiserum against GABA and demonstated GABA-immunoreactivity (GABA-IR) in several populations of amacrine cells in the inner nuclear layer (INL), and other cells in the inner plexiform layer (IPL) of the central and peripheral retina of the chameleon. Horizontal cells do not contain GABA-IR and the chameleon retina is therefore an exception among non-mammals. GABA-IR was not seen in cell bodies in the position of photoreceptor, bipolar and interplexiform cells suggesting that GABA is not involved in synaptic transmission in the outer plexiform layer of chameleon retina.     

Target cells of vitamin D in the vertebrate retina

Using PAP technique, cellular localization of vitamin D-dependent calcium-binding protein (D-CaBP) was investigated in vertebrate retina with monospecific antisera against chick duodenal D-CaBP. In the chick retina, the receptor cells were positive. In the inner nuclear layer, horizontal cells and some bipolar cells were also positive. Some amacrine cells as well as different levels of the inner plexiform layer were also positive for D-CaBP. A few interspersed ganglion cells were positive but their axons forming the optic tract were negative. Müller's cells were negative. In 1-day-old chicks and 4-week-old rachitic chicks there was paucity and absence, respectively, of D-CaBP staining in horizontal cells. In the mouse, rat, and rabbit the receptors had only trace amounts of reaction product in their outer segment and pedicle. Horizontal cells were densely positive throughout their cellular body and processes. Some amacrine cells in the inner nuclear layer were positive. In the mouse and rat three horizontal levels of the outer plexiform layer were very prominent because of their dense staining for D-CaBP. Many ganglion cells were also positive along with their axons forming the optic nerve. In the rabbit, no positive layers were seen in the inner plexiform layer, and ganglion cells with their fibers were negative. In the frog retina there were smaller amounts of D-CaBP in the receptor cells and horizontal cells than that of the chick retina. Also, the fibers of the ganglionic cells were positive for D-CaBP. In all species studied, some amacrine cells were stained for D-CaBP. Because of its possible roles in membrane calcium transport and intracellular Ca++ regulation, it has perhaps similar functions in these positive cells. The synthesis of D-CaBP is dependent upon vitamin D. These positive cells are thus target cells of vitamin D.     

Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers

In the developing mouse retina degenerating neurons can be observed initially in the ganglion cell layer followed by a phase of cell death in the inner nuclear layer. Using an immunohistochemical method to localize the mouse macrophage specific antigen F4/80, we show that macrophages migrate from the vascular supply overlying the developing retina and phagocytose the degenerating neurons. The macrophages subsequently differentiate to become the microglia of the retina and form a regularly spaced distribution across the retina in the inner and outer plexiform layers. These experiments provide strong evidence for the mesodermal origin of central nervous system microglia.     

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.     

NMDA-induced retinal injury is mediated by an endoplasmic reticulum stress-related protein, CHOP/GADD153

We investigated the role of an endoplasmic reticulum stress-associated protein, CHOP/GADD153, after NMDA-induced mouse retinal damage. After injection of NMDA into the vitreous, TUNEL-positive cells were detected in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 6 h after NMDA injection, and these gradually increased in number up to 24 h. Analysis by real-time RT-PCR revealed that CHOP mRNA was induced by about 3-fold, at 2 h after NMDA injection. Immunoreactivity for the CHOP protein was intense in cells of the GCL following NMDA treatment. Immunoblot analysis showed that NMDA injection increased the expression of CHOP protein in the retina. Compared with wild-type mice, CHOP/ mice were more resistant to NMDA-induced retinal cell death as determined by TUNEL assay. At 7 days after NMDA treatment, the thickness of the inner plexiform layer and INL were larger in CHOP/ mice than in wild-type mice. The number of residual cells in the GCL following NMDA treatment was significantly higher in CHOP/ mice than in wild-type mice. In conclusion, CHOP is induced in mouse retina by NMDA treatment, and CHOP/ mice are more resistant to NMDA-induced retinal damage, suggesting that CHOP plays an important role in NMDA-induced retinal cell death.     

Calmodulin gene expression in the neural retina of the adult rat

Calmodulin (CaM) mRNAs are expressed with low abundancy in the adult rat neural retina. However, when digoxigenin (DIG)-labeled cRNA probes specific for each CaM mRNA population were hybridized at slightly alkaline pH (pH 8.0), the widespread distribution of CaM mRNA-expressing cells was revealed, with similar abundance for all three CaM genes. The CaM genes displayed a uniquely similar, layer-specific expression throughout the retina, and no significant differences were found in the distribution patterns of the CaM mRNA populations or the labeled cell types. The strongest signal for all CaM mRNAs was demonstrated in the ganglion cell layer and the inner nuclear layer, where the highest signal intensity was found within the inner sublamina. Similarly intermediate signal intensities for all CaM genes were detected in the inner and outer plexiform layers, within the vicinity of the outer limiting membrane and in the retinal pigment epithelium. A very low specific signal was characteristic in the outer nuclear layer and the photoreceptor inner segment layer, while no specific hybridization signal was observed in the photoreceptor outer segment layer. In summary, all CaM genes exhibited a similar and a characteristically layer-specific expression pattern in the adult rat retina.