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.     

The ganglion cell layer of the retina of the rat: a Golgi study.

In whole-mounts of Golgi stained rat retinae four cell types are described in the ganglion cell layer. Three of these cell types are considered to be analogous to the alpha, delta and gamma cells described in the cat retina by Boycott & W?ssle (1974). The fourth cell type is thoughtt to be a displaced amacrine cell. All the cell types described are present in all parts of the retina. There is no evidence for an increase in dendritic field size with increasing distance from the optic disk.     

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.     

Exogenous growth factors stimulate the regeneration of ganglion cells in the chicken retina

Recent reports have found that the posthatch chicken retina has the capacity for neuronal regeneration. The purpose of this study was to test whether the types of cells destroyed by neurotoxic lesions influence the types of cells that are regenerated, and whether exogenous growth factors stimulate neural regeneration in the chicken retina. N-methyl-D-aspartate (NMDA) was used to destroy amacrine and bipolar cells; kainate was used to destroy bipolar, amacrine, and ganglion cells; colchicine was used to selectively destroy ganglion cells. Following toxin-induced damage, bromo-deoxyuridine was used to label proliferating cells. In some animals, growth factors were injected into the vitreous chamber of the eye. We found that the proliferation of cells within the retina was stimulated by toxin-induced cell loss, and by insulin and FGF2. After either kainate- or colchicine-induced retinal damage, some of the newly generated cells expressed markers and had the morphology of ganglion cells. The combination of insulin and FGF2 stimulated the regeneration of ganglion cells in kainate- and colchicine-treated retinas. We conclude that exogenous growth factors can be used to stimulate neural regeneration in the retina. We propose that the type of neuron destroyed in the retina may allow or promote the regeneration of that neuronal type.     

'Coronate' amacrine cells in the rabbit retina have the 'starburst' dendritic morphology

Acetylcholine-synthesizing cells in the rabbit retina are symmetrically distributed about the inner plexiform layer: one population of cholinergic amacrines has cell bodies in the inner nuclear layer and an equivalent population of displaced amacrines has cell bodies in the ganglion cell layer. It has been suggested that the morphological correlates of the acetylcholine-synthesizing cells are either coronate amacrine cells or starburst amacrine cells. Coronate cells have a characteristic nuclear morphology and can be selectively labelled by neurofibrillar methods or with the fluorescent dye4',6-diamidino-2-phenyl-indole (DAPI). Starburst cells have a characteristic dendritic morphology but have only been described from Golgi-stained retinae. This paper bridges the gap between the previous studies. DAPI-labelled coronate cells were impaled with a micropipette under microscopic control and filled with Lucifer yellow by iontophoresis. The results show that the coronate amacrines in the ganglion cell layer are type b starburst cells, and that those DAPI-labelled neurones in the inner nuclear layer with a coronate-like nuclear morphology are type a starburst cells. At a given eccentricity the dendritic field diameter of type a starburst cells is about 1.13 times larger than that of type b starburst cells. The dendritic field coverage of coronate (type b starburst) cells increases linearly with decreasing coronate cell density and ranges from 25 on the peak visual streak to 70+ in the superior periphery.     

The ramification pattern of amacrine cells within the inner plexiform layer of the carp retina

Summary The morphology of amacrine cells in the retina of the carp is described using the Golgi technique. The ramification pattern of these cells was analyzed in flat-mounts of retinas. Based on these observations classification into five groups was made. Cells possessing one principal process leaving the soma were subdivided into starburst A-neurons and radiate neurons. Cells having two or more principal processes were subdivided into starburst B-neurons and spindle-shaped soma neurons. Small, diffuse amacrine cells form the fifth group. With respect to the shape of the field of arborization, the following cell types could be distinguished: (i) uniform cells, (ii) cells with a preferential direction, and (iii) cells with a marked edge, i.e., cells that lack processes in one direction. The latter form rarely occurs among starburst neurons; most of the spindle-shaped soma cells possess processes with a preferred direction, and cells with a marked edge are mainly found among the radiate neurons.All five cell types are found throughout the retina. The size of the cells varies within each group, and there is no correlation between size and distance from the optic nerve.The radial arborization pattern of each cell was examined in serial transverse sections. Starburst A-neurons ramify in the middle of the inner plexiform layer (IPL), radiate neurons in the inner half, and spindle-shaped soma neurons without overlapping processes (type B) as well as starburst B-neurons in the outer half. The ramification can be monostratified (narrow or broad), bistratified or multistratified. Small, diffuse amacrine cells and spindle-shaped soma neurons with overlapping processes (type A) ramify throughout the entire IPL.This work was supported by the Deutsche Forschungsgemeinschaft     

Morphology and synaptic connectivity of nitric oxide synthase-immunoreactive neurons in the guinea pig retina

Immunocytochemical methods with an antiserum against neuronal nitric oxide synthase (NOS) were applied to identify the morphology and synaptic connectivity of NOS-like immunoreactive neurons in the guinea pig retina. In the present study, two types of amacrine cells were labeled with anti-NOS antisera. Type 1 cells had large somata located in the inner nuclear layer (INL) with long, sparsely branched processes ramifying mainly in stratum 3 of the inner plexiform layer (IPL). The somata of type 2 cells (smaller diameters) were located in the INL. Some displaced amacrine cells in the ganglion cell layer were labeled. The soma size of the displaced amacrine cells was similar to that of the type 2 amacrine cells. However, processes originating from type 2 amacrine cells and displaced amacrine cells stratified mainly in strata 1 and 5, respectively. Some cone bipolar cells were weakly NOS-immunoreactive. The synaptic connectivity of NOS-like immunoreactive amacrine cells was identified in the IPL by electron microscopy. NOS-labeled amacrine cell processes received synaptic input from other amacrine cell processes and bipolar cell axon terminals in all strata of the IPL. The most frequent postsynaptic targets of NOS-immunoreactive amacrine cells were other amacrine cell processes. Cone bipolar cells were postsynaptic to NOS-labeled amacrine cells in all strata of the IPL. Labeled amacrine cells synapsing onto ganglion cells were found only in sublamina b. A few synaptic contacts were observed between labeled cell processes. In the outer plexiform layer, dendrites of labeled bipolar cells made basal contact with cone pedicles or formed a synaptic triad opposed to a synaptic ribbon of cone pedicles.     

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.     

Neurotransmitter candidates in the retina of the mudpuppy,Necturus maculosus

Summary Neurons accumulating (³H)-glycine and (³H) GABA were demonstrated with the use of autoradiography. Both were accumulated by different types of amacrine cells, similar those of goldfish. (³H)-GABA was also accumulated by horizontal cells, again similar to the goldfish. These results and physiological studies from other laboratories suggest that GABA and glycine are neurotransmitter candidates in amacrine cells of the mudpuppy.Immunoreactive neuropeptide Y (NPY), glucagon, vasoactive intestinal peptide (VIP), somatostatin, substance P, and neurotensin were found in different types of stratified amacrine cells. Weakly immunoreactive enkephalin and bombesin processes were also seen in the inner plexiform layer. Gastrin-immunoreactive neurons were not detectable.Endogenous 5-hydroxytryptamine was visualized immunohistochemically in a population of diffuse amacrine cells and some cells in the ganglion cell layer. This suggests that 5-hydroxytryptamine may be a neurotransmitter in the retina of the mudpuppy.     

Brain-derived neurotrophic factor modulates the dopaminergic network in the rat retina after axotomy

Dopaminergic cells in the retina express the receptor for brain-derived neurotrophic factor (BDNF), which is the neurotrophic factor that influences the plasticity of synapses in the central nervous system. We sought to determine whether BDNF influences the network of dopaminergic amacrine cells in the axotomized rat retina, by immunocytochemistry with an anti-tyrosine hydroxylase (TH) antiserum. In the control retina, we found two types of TH-immunoreactive amacrine cells, type I and type II, in the inner nuclear layer adjacent to the inner plexiform layer (IPL). The type I amacrine cell varicosities formed ring-like structures in contact with AII amacrine cell somata in stratum 1 of the IPL. In the axotomized retinas, TH-labeled processes formed loose networks of fibers, unlike the dense networks in the control retina, and the ring-like structures were disrupted. In the axotomized retinas treated with BDNF, strong TH-immunoreactive varicosities were present in stratum 1 of the IPL and formed ring-like structures. Our data suggest that BDNF affects the expression of TH immunoreactivity in the axotomized rat retina and may therefore influence the retinal dopaminergic system. E.-J. Lee and M.-C. Song contributed equally to this work. This work was supported by Korea Research Foundation (grant no. E00004, 2004).     

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.     

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.     

Interactions leading to colour opponency in ganglion cells of the turtle retina

In the turtle retina, colour-dependent photoresponses could be recorded intracellularly from ganglion cells receiving only bipolar cell input. Thus, the mechanism for colour discrimination by these ganglion cells (type A) is contained in the outer plexiform layer of the retina and depends on interaction between horizontal and cone cells. Ganglion cells receiving an additional amacrine input (type B) are not influenced by colour, and have about 0.7 logarithmic unit lower absolute sensitivity to peak wavelength than have type A ganglion cells.     

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.     

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.     

Immunocytochemical and autoradiographic localization of GABA system in the vertebrate retina

Summary The localization of -aminobutyric acid (GABA) neurons in the goldfish and the rabbit retina has been studied by immunocytochemical localization of the GABA-synthesizing enzyme L-glutamate decarboxylase (GAD, L-glutamate 1-carboxy-lase, EC 4.1.1.15) and by [³H] GABA uptake autoradiography. In the goldfish retina, GAD is localized in some horizontal cells (H1 type), a few amacrine cells and sublamina b of the inner plexiform layer. Results from immunocytochemical studies of GAD-containing neurons and autoradiographic studies of GABA uptake reveals a marked similarity in the labeling pattern suggesting that in goldfish retina, the neurons which possess a high-affinity system for GABA uptake also contain significant levels of GAD. In the rabbit retina, when Triton X-100 was included in immunocytochemical incubations with a modified protein A-peroxidase-antiperoxidase method, reaction product was found in four broad, evenly spaced laminae within the inner plexiform layer. In the absence of the detergent, these laminae were seen to be composed of small, punctate deposits. When colchicine was injected intravitreally before glutamate decarboxylase staining, cell bodies with the characteristic shape and location of amacrine cells were found to be immunochemically labeled. Electron microscopic examination showed that these processes were presynaptic to ganglion cell dendrites (infrequently), amacrine cell telodendrons, and bipolar cell terminals. Often, bipolar cell terminals were found which were densely innervated by several GAD-positive processes. No definite synapses were observed in which a GAD-positive process represented the postsynaptic element. In autoradiographic studies by intravitreal injection of [³H] GABA a diffuse labeling of the inner plexiform layer and a dense labeling of certain amacrine cell bodies in the inner nuclear layer was observed. Both immunocytochemical and autoradiographic results support the notion that certain, if not all, amacrine cells use GABA as their neurotransmitter.     

Distribution of R-cognin and choline acetyltransferase in the ganglion cell layer of developing chick neural retina

Retina cognin, a cell membrane glycoprotein which mediates cell-cell recognition and adhesion in vitro, is initially present throughout the retina and becomes confined to the ganglion cell layer at 14–15 days of embryogenesis. Within this layer it is found on membranes of virtually all ganglion and displaced amacrine cells, but not on membranes of retinal glial cells (Müller fibers) which traverse this layer. The distribution of cognin as determined by immunocytochemistry is described and compared with that of choline acetyltransferase. The significance of cognin as a possible address marker during development of neural retina is discussed.     

Transient requirement for ganglion cells during assembly of retinal synaptic layers

The inner plexiform layer (IPL) of the vertebrate retina comprises functionally specialized sublaminae, representing connections between bipolar, amacrine and ganglion cells with distinct visual functions. Developmental mechanisms that target neurites to the correct synaptic sublaminae are largely unknown. Using transgenic zebrafish expressing GFP in subsets of amacrine cells, we imaged IPL formation and sublamination in vivo and asked whether the major postsynaptic cells in this circuit, the ganglion cells, organize the presynaptic inputs. We found that in the lak/ath5 mutant retina, where ganglion cells are never born, formation of the IPL is delayed, with initial neurite outgrowth ectopically located and grossly disorganized. Over time, the majority of early neurite projection errors are corrected, and major ON and OFF sublaminae do form. However, focal regions of disarray persist where sublaminae do not form properly. Bipolar axons, which arrive later, are targeted correctly, except at places where amacrine stratification is disrupted. The lak mutant phenotype reveals that ganglion cells have a transient role organizing the earliest amacrine projections to the IPL. However, it also suggests that amacrine cells interact with each other during IPL formation; these interactions alone appear sufficient to form the IPL. Furthermore, our results suggest that amacrines may guide IPL sublamination by providing stratification cues for other cell types.