The occurrence of three isoenzymes of protein kinase C (alpha, beta and gamma) in retinas of different species.

The localisation and immunochemical identification of 3 different forms of protein kinase C (PKC-alpha, PKC-beta and PKC-gamma) in retinas of different species were analysed by immunohistochemistry and SDS-PAGE-Western blotting, respectively. Only in some cases was there a correlation between the findings from each procedure. One reason for the lack of correlation could be the small amounts of PKC present in some retinas, which made detection possible only by first concentrating the antigen by SDS-PAGE and then carrying out Western blotting. Another possible reason is that an antibody recognises unknown antigens immunohistochemically, but, because of their specific characteristics, they are denatured when subjected to SDS-PAGE and Western blotting and therefore remain undetected. PKC-beta immunoreactivity is present in rabbit, frog and goldfish retinas but absent from the rat retina. However, SDS-PAGE and Western blotting experiments showed that the PKC-beta isoenzyme is absent from the fish retina but present in the rat retina. PKC-beta immunoreactivity in rabbit retina is present in ganglion and/or amacrine cells; in the frog retina the enzyme is associated with some bipolar cells. In the goldfish retina, PKC-beta is associated with a large population of cells in the ganglion cell layer as well as with some amacrine cell bodies. PKC-alpha is present primarily in bipolar cells of rat, fish and rabbit retinas and was not detected by immunohistochemistry or blotting experiments in the frog retina. SDS-PAGE and Western blotting of retinal extracts from different species showed that PKC-gamma occurs in the rabbit where it was associated with ganglion and/or amacrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate receptor agonists release [3H]GABA preferentially from horizontal cells

A total of 5-6 different cell types in vertebrate retinas accumulate [3H]gamma-aminobutyric acid (GABA). In frog retina, specific populations of cells in the horizontal, amacrine and ganglion cell layers are labeled autoradiographically after a 15-min in vitro incubation with [3H]GABA. Cells which may be bipolar or interplexiform cells are also labeled. Similar autoradiographic patterns are observed in chick retina except for the absence of labeled bipolar or interplexiform cells. In rat retinas, [3H]GABA uptake is limited primarily to Muller and amacrine cells. Depolarizing glutamate receptor agonists (glutamate, aspartate and kainic acid) applied in an in vitro perfusion system, stimulated massive release of [3H]GABA from frog and chick retina but not from rat retina. Under these conditions, autoradiographic labeling of horizontal cells was virtually depleted, while labeling of other cell types remained robust. In contrast, potassium caused release of the label from all 3 types of retina, and loss of autoradiographic labeling occurred uniformly in all cell types. We conclude that [3H]GABA-accumulating horizontal cells possess depolarizing glutamate receptors and that activation of these receptors leads to a release of GABA stores. On the other hand, Muller cells and the various subclasses of [3H]GABA-accumulating amacrine, bipolar and/or interplexiform cells, do not release GABA in response to glutamate receptor stimulation and thus appear to be relatively insensitive to excitatory amino acids.     

Patterns of glutamate immunoreactivity in the goldfish retina

Postembedding silver-intensified immunogold procedures reveal high levels of glutamate immunoreactivity in "vertical" elements of the goldfish retina: (1) Red-sensitive and green-sensitive cones display strong glutamate immunoreactivity, especially in their synaptic terminals, but blue-sensitive cones are poorly immunoreactive. (2) All type Mb (on-center) and Ma (off-center) mixed rod-cone bipolar cells and all identifiable cone bipolar cells are highly glutamate immunoreactive. We find no evidence for bipolar cells that lack glutamate immunoreactivity. (3) The majority of the somas in the ganglion cell layer and certain large cells of the amacrine cell layer resembling displaced ganglion cells are strongly glutamate immunoreactive. (4) Despite their high affinity symport of acidic amino acids, the endogenous levels of glutamate in Müller's cells are among the lowest in the retina. (5) GABAergic neurons possess intermediate levels of glutamate immunoreactivity. Quantitative immunocytochemistry coupled with digital image analysis allows estimates of intracellular glutamate levels. Photoreceptors and bipolar and ganglion cells contain from 1 to 10 mM glutamate. The bipolar and ganglion cell populations maintain high intracellular glutamate concentrations, averaging about 5 mM, whereas red-sensitive and green-sensitive cones apparently maintain lower levels. Importantly, photoreceptor glutamate levels are extremely volatile, and in vitro maintenance is required to preserve cone glutamate immunoreactivity in the goldfish. GABAergic horizontal and amacrine cells contain about 0.3-0.7 mM glutamate, which matches the values predicted from the Km of glutamic acid decarboxylase. Müller's cells and non-GABAergic amacrine cells contain less than 0.1 mM glutamate. Though Müller's cells are known to possess potent glutamate symport, they clearly possess equally potent mechanisms for maintaining low intracellular glutamate concentrations.     

Immunocytochemical localization of LANT-6-like immunoreactivity within neurons in the inner nuclear and ganglion cell layers in vertebrate retinas

LANT-6 is a hexapeptide (H-Lys-Asn-Pro-Tyr-Ile-Leu-OH) isolated from chicken small intestine, which resembles the COOH-terminal half of neurotensin, except for the amino acid substitutions Lys/Arg and Asn/Arg. The present report concerns the immunocytochemical staining of vertebrate retinas using an antiserum directed against LANT-6. In the retinas from goldfish, bird and turtle, cells in both the inner nuclear and ganglion cell layers were labeled, but in the frog cells were labeled specifically and in the rat only cells in the ganglion cell layer were labeled. Labeled cell bodies in the inner nuclear layer gave rise to processes which were seen primarily within the following laminas of the inner plexiform layer (IPL): in the goldfish, lamina 3; chicken, laminae 1,3 and 4; and turtle, laminae 3,4 and 5. The cell bodies of the labeled neurons in the ganglion cell layer gave rise to dendrites which entered the IPL and axons which descended to the optic fiber layer. The cells with LANT-6-like immunoreactivity were distributed in both the central and peripheral parts of the retina in all the species examined except frog. Measured by radioimmunoassay, the levels of LANT-6-like-immunoreactivity in extracts of turtle, chicken, and goldfish retinas were 5–30 times those for neurotensin-like immunoreactivity, however no LANT-6-like immunoreactivity was detected in frog. Multiple chromatographic analyses indicated that while the LANT-6-like immunoreactivity in chicken retina was indistinguishable from synthetic LANT-6, LANT-6 like immunoreactivity in turle and goldfish retinas was primarily associated with large molecular forms. Treatment of turtle LANT-6-like immunoreactivity with pepsin, an enzyme known to mimic processing for neurotensin precursors, yielded 3 major peptides, one of which co-chromatographed with synthetic LANT-6. The present immunocytochemical localization of LLI within cells in the inner nuclear and ganglion cell layers, coupled with the biochemical characterization of LANT-6 in the vertebrate retinas and brains, suggests that neuropeptides such as LANT-6 may play a role in visual processing both within the retina and within the visual pathways to the brain.     

Distribution of protein gene product 9.5 (PGP 9.5) in the vertebrate retina: evidence that immunoreactivity is restricted to mammalian horizontal and ganglion cells.

Using light microscopic immunocytochemistry, we have studied the distribution of protein gene product 9.5 (PGP 9.5), a neuron-specific protein first extracted from human brain (Doran et al., '83:J. Neurochem. 40:1542-1547), in the vertebrate retina. Retinas were obtained from frog, chicken, rat, rabbit, cow, cat, dog, and human. No immunoreactivity was observed in frog and only a faint staining was present in chicken. In mammalian retinas, a strong positive reaction was restricted to horizontal and ganglion cells, with minor interspecies variations. Immunostaining was present throughout the cell body and the dendritic tree in horizontal cells. At the level of retinal ganglion cells, immunolabel was particularly abundant in cell bodies and axons forming the optic nerve. Only the main dendrites were stained, the remainder of the dendritic tree giving rise to a diffuse punctate reaction in the inner plexiform layer. In rats, displaced amacrine cells, which are known to contribute largely (40-50%) to the total neuronal population within the ganglion cell layer (Perry, '81: Neuroscience 6:931-944) were not immunoreactive, as demonstrated from (i) analysis of the morphology, cell size and cell density of immunoreactive neurons in wholemounts; (ii) colocalization of retrograde label and PGP 9.5 immunoreactivity in about 80% of ganglion cells after injection of peroxidase into the optic nerve; and (iii) reduction of immunoreactivity in the inner plexiform and ganglion cell layers following optic nerve transection. Western blot analysis of extracts from rabbit retinas indicated that the immunoreactive species is PGP 9.5 or a closely related molecule. Recent studies have demonstrated that PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., '89:Science 246:670-673). The present results, therefore, suggest that differences in the ubiquitination process exist between retinal neurons.     

Immunolocalization of cellular retinoic acid binding protein to Müller cells and/or a subpopulation of GABA-positive amacrine cells in retinas of different species

Retinoic acid (RA) and its specific binding protein, cellular RA binding protein (CRABP), are found in relative abundance in bovine and rat retinas. Since RA does not participate in the visual cycle, the presence of RA and its binding protein in retina suggests that they may be involved in other aspects of retinoid action. As an initial step in identifying the role of RA and its binding protein in retina, monoclonal antibodies were prepared against CRABP purified from bovine retina and used to localize this antigen by immunocytochemistry in retinas of different species. Human and monkey retinas showed specific cytoplasmic labeling of Müller cells. Cat, bovine, rabbit, rat, turtle, and chick retinas showed specific cytoplasmic labeling of some somata in the inner nuclear and ganglion cell layers and characteristic strata in the inner plexiform layer. Cat and bovine retinas also showed cytoplasmic labeling of Müller cells. Immunoreactivity in these species was absent with nonimmune serum or abolished when the antibodies were preabsorbed with purified antigen. Chameleon, goldfish, and frog retinas were nonreactive. We used double-labeling immunofluorescence experiments to determine if the CRABP-positive cells were also positive for known neurotransmitters or associated enzymes. CRABP-positive amacrine cells of cat, cow, rabbit, rat, and chick represented a subset of the more numerous gamma-aminobutyric acid (GABA)-positive amacrine cells. However, turtle CRABP-positive amacrine cells were negative for GABA despite the fact that turtle retina contains many GABA positive cells. CRABP-positive amacrine cells in rat retinas were not immunoreactive for glycine, choline acetyltransferase, somatostatin, or tyrosine hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical study of calretinin and calbindin D-28K expression in the retina of three cartilaginous fishes and a cladistian (Polypterus)

The distribution of two calcium-binding proteins, calbindin D-28K (CB) and calretinin (CR) was studied in the retina of a cladistian, Polypterus senegalus, and three cartilaginous fishes (Scyliorhinus canicula, Raja undulata and Torpedo marmorata). Western blot analysis of brain extracts revealed the lack of cross-reactivity of the used antibodies. In Polypterus, CB and CR immunoreactivities were observed in some amacrine and ganglion cells, but scarce cells showed CR/CB colocalization. Furthermore, CR immunoreactivity was present in a number of displaced bipolar cells and in some putative displaced ganglion cells, whereas CB immunoreactivity was found in some cones. No positive retinal structure was observed with the CB antibody used in cartilaginous fishes. Instead, CR was expressed in some amacrine, horizontal and ganglion cells of the dogfish and skate and, in some ganglion cells of the electric ray. The comparative analysis suggests, (1) the presence of CB-positive photoreceptor cells in the retina of cladistians seems to be apomorphic (in jawed fishes) in contrast with the plesiomorphic condition of this character in land vertebrates; (2) the presence of CR in amacrine and ganglion cells is a conserved feature along vertebrate phylogeny, whereas its variable expression in bipolar and horizontal cells represents a derived character; (3) the absence of CB in horizontal cells in cladistians could represent a derived character; and (4) the presence of CR displaced bipolar and putative displaced ganglion cells in Polypterus is shared with basal groups of actinopterygians.     

Immunocytochemical study of calretinin and calbindin D-28K expression in the retina of three cartilaginous fishes and a cladistian (Polypterus)

The distribution of two calcium-binding proteins, calbindin D-28K (CB) and calretinin (CR) was studied in the retina of a cladistian, Polypterus senegalus, and three cartilaginous fishes (Scyliorhinus canicula, Raja undulata and Torpedo marmorata). Western blot analysis of brain extracts revealed the lack of cross-reactivity of the used antibodies. In Polypterus, CB and CR immunoreactivities were observed in some amacrine and ganglion cells, but scarce cells showed CR/CB colocalization. Furthermore, CR immunoreactivity was present in a number of displaced bipolar cells and in some putative displaced ganglion cells, whereas CB immunoreactivity was found in some cones. No positive retinal structure was observed with the CB antibody used in cartilaginous fishes. Instead, CR was expressed in some amacrine, horizontal and ganglion cells of the dogfish and skate and, in some ganglion cells of the electric ray. The comparative analysis suggests, (1) the presence of CB-positive photoreceptor cells in the retina of cladistians seems to be apomorphic (in jawed fishes) in contrast with the plesiomorphic condition of this character in land vertebrates; (2) the presence of CR in amacrine and ganglion cells is a conserved feature along vertebrate phylogeny, whereas its variable expression in bipolar and horizontal cells represents a derived character; (3) the absence of CB in horizontal cells in cladistians could represent a derived character; and (4) the presence of CR displaced bipolar and putative displaced ganglion cells in Polypterus is shared with basal groups of actinopterygians.     

Displaced amacrine cells of the mouse retina

The aim of this study was to characterize and classify the displaced amacrine cells in the mouse retina. Amacrine cells in the ganglion cell layer were injected with fluorescent dyes in flat-mounted retinas. Dye-filled displaced amacrine cells were classified according to dendritic field size, horizontal and vertical stratification patterns, and general morphology. We identified 10 different morphological types of displaced amacrine cell. Six of the cell types identified here are novel cell types that have not been described previously in the mouse retina, to the best of our knowledge. The displaced amacrine cells included four types of medium-field cells, with dendritic field diameters of 200-500 microm, and six types of wide-field cells, with dendritic fields extending over 500 microm. Narrow-field displaced amacrine cells, with dendritic field diameters smaller than 200 microm, were not encountered. The most frequently labeled displaced amacrine cell type was the starburst amacrine cell. At least three cell types identified here have nondisplaced counterparts in the inner nuclear layer as well. Displaced amacrine cells display a rich variety of stratification and branching patterns, which surely reflect the wide range of their functional roles in the processing of visual signals in the inner retina.     

Cellular localization of cyclooxygenase-1 and cyclooxygenase-2 in the normal mouse,rat, and human retina

Prostaglandins, synthesized by cyclooxygenase (COX), regulate diverse neurophysiological actions such as regulation of autonomic responses, transmission of pain, generation of fever, control of sleep-wake cycle, synaptic signaling, and cross-talk between neurons and glia in the central nervous system. Although prostaglandins have been widely studied in the anterior segment tissues of the eye, relatively little is known about prostaglandins in the neural retina. By using immunohistochemistry, we have compared the cellular expression and localization of COX-1 and COX-2 in the normal mouse, rat, and human retina. In the normal mouse retina, COX-1 immunoreactivity is present in the outer segments of photoreceptor cells, horizontal cells, microglia, retinal ganglion cells, and displaced amacrine cells. In the normal rat retina, COX-1 immunoreactivity is present in microglia, retinal ganglion cells, and displaced amacrine cells. In the normal human retina, COX-1 immunoreactivity is present in microglia, astrocytes, retinal ganglion cells, and displaced amacrine cells. In the normal mouse and rat retina, COX-2 immunoreactivity is present in processes of the outer plexiform layer and in certain amacrine cells and retinal ganglion cells. In the normal human retina, COX-2 immunoreactivity is only present in processes of the outer plexiform layer. These results suggest that prostaglandins, synthesized by COX-1 or COX-2, may contribute to normal physiological and homeostatic functions in the retina.     

Choline acetyltransferase immunoreactivity suggests that ganglion cells in the goldfish retina are not cholinergic

Published evidence that ganglion cells in the retinae of nonmammalian species are cholinergic is strong but indirect. In this paper we report results of attempts to demonstrate choline acetyltransferase immunoreactivity in ganglion cells of goldfish retina using two different antisera against choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme. We obtained ChAT-immunoreactive staining of amacrine and displaced amacrine cells in the retina and type XIV cells in the tectum, but we obtained no direct immunocytochemical evidence that ganglion cells in the goldfish retina are cholinergic. Thus, ganglion cells identified by retrograde transport of propidium iodide were never ChAT-immunoreactive. Intraocular injections of colchicine did not result in the appearance of a population of ChAT-immunoreactive neurons in the ganglion cell layer. ChAT-immunoreactive axons were not observed in intact, ligated, or transected optic nerves. And finally, the ChAT immunoreactivity of cells and fibers in the optic tectum was unaffected by deafferentation. These experiments provide no positive evidence that any ganglion cells in goldfish retina contain the acetylcholine-synthesizing enzyme, ChAT. While it is possible that our method is too insensitive to detect the enzyme in ganglion cell somata or too specific to recognize the form of ChAT present in these cells, the fact that we can stain putatively cholinergic retinal amacrine cells and tectal neurons makes these alternative explanations improbable. We conclude that it is unlikely that any of the ganglion cells in the retina are cholinergic and that alternative explanations should be sought for previously published results that suggest that they are.     

GABA-like immunoreactive cells containing nicotinic acetylcholine receptors in the chick retina.

The possibility that GABA-like immunoreactive cells of the chick retina also contain neuronal nicotinic acetylcholine receptors was investigated by means of immunohistochemical techniques. Double-labeled cell bodies containing GABA-like immunoreactivity and nicotinic receptor-like immunoreactivity were seen in the inner third of the inner nuclear layer and were presumably amacrine cells. Approximately 29-36% of the GABA-positive cells in the inner nuclear layer contained nicotinic receptor immunoreactivity. Their soma sizes ranged from 5-12 microns. Some double-labeled cells ranging from 7-21 microns were observed in the ganglion cell layer as well. Between 9-37% of the GABA-positive cells in this layer contained nicotinic receptor-like immunoreactivity. Following injection of a retrograde tracer into the optic tectum, some of the retrogradely labeled cells were also double labeled with antibodies against GABA and nicotinic receptors. This indicates that at least some of the GABA-positive cells containing nicotinic acetylcholine receptors in the ganglion cell layer are indeed ganglion cells. The present data appear to represent the first demonstration of the presence of acetylcholine receptors in GABA-containing cells in the retina, thus providing a basis for a possible influence of acetylcholine upon those presumptive GABAergic cells.     

Neurotrophin receptors (Trk A, Trk B, and Trk C) in the developing and adult human retina

In this study, the ontogeny and distribution patterns of three neurotrophin receptors (Trk A, Trk B, and Trk C) were examined in the human retinas. Immunohistochemistry was performed on sections of retina and optic nerve from fetuses (11-24 weeks of gestation, wg), one infant (4-month-old) and two adult (35- and 65-years-old) subjects. At 11 wg, Trk A was expressed in the nerve fiber and inner plexiform layers, while Trk B and Trk C were expressed in many neuroblastic cells. By 16-17 wg, the photoreceptors showed immunoreactivity for all three receptors. The ganglion cell layer and amacrine cells were conspicuously immunoreactive for Trk A and Trk C, but labeled diffusely for Trk B. The horizontal cells were labeled for Trk A and Trk B. The pattern was same in the retinas at midgestation (20-21 wg). Shortly after this period, there was an apparent decrease in receptor immunoreactivity in the fetal retinas. In the infant retina, Trk A immunoreactivity was absent from horizontal cells. The photoreceptors were immunopositive for Trk B and Trk C, in infant and adult retinas. In the adults, few cells of the ganglion cell layer and inner nuclear layer were clearly labeled for Trk A and Trk C, and diffusely for Trk B. The glial cells of the retina and optic nerve immunoreacted for Trk A only, right from fetal 16 wg. The early expression of Trk B and Trk C on neuroblastic cells suggests that both play a role in cell proliferation. The developmental distribution pattern of Trk A, on the other hand, provides evidence for its involvement in differentiation of the inner plexiform layer, horizontal cells and neuroglia. The results strongly suggest that photoreceptor development is mediated by Trk receptors. The novel localization of Trk B and Trk C on adult photoreceptors points to a possible therapeutic potential for BDNF and NT-3, respectively, in photoreceptor diseases.     

Biphasic retinal neurogenesis in the brush-tailed possum, Trichosurus vulpecula: further evidence for the mechanisms involved in formation of ganglion cell density gradients.

We investigated cell generation in the retina of the brush-tailed possum (Trichosurus vulpecula) by using tritiated (3H)-thymidine labelling of newly generated cells. Animals aged between postnatal day (P) 5 and 85 each received a single injection of 3H-thymidine. Following autoradiographic processing, maps of labelled cells were constructed from retinal sections. Retinal cell generation takes place in two phases, the first is concluding in the retinal periphery at P53 as the second is seen to commence in midtemporal retina. In the first phase, cells in central retina are generated earlier than those in peripheral regions. In the second phase, cells complete their final division in midtemporal retina first and in the periphery last. Cells generated in the first phase comprise virtually all cells in the ganglion cell layer, amacrine cells, horizontal cells, and cones. Ganglion cells are produced at a slightly earlier stage than displaced amacrine cells, horizontal cells, or cones. Amacrine cells in the inner nuclear layer are the final cells produced in the first phase. When ganglion cells and amacrine cells are pooled, their combined rate of production matches that of the other cell types. These data indicate that the ratio of displaced amacrine cells: horizontal cells: cones: combined ganglion cells and amacrine cells does not change throughout development. However, the ratio of ganglion cells:macrines changes steadily as development proceeds to favour amacrine cells. In the second phase, sparse numbers of nonganglion cells in the ganglion cell layer and large numbers of bipolar and Müller cells are produced along with all rods. The two phases in the possum are similar to those seen in the wallaby, the quokka. However, fewer cells are added in central retina in the possum than in the quokka and cell addition continues for a more extended period in the periphery in the possum. We suggest that this difference in cell addition could account for the development of a more pronounced visual streak of retinal ganglion cells in the possum than in the quokka. A comparison of possum retinal cell generation with that of other marsupials adds support for the "homochrony theory."     

Localization of G protein-coupled receptor kinases (GRKs) in the avian retina

G protein-coupled receptor kinases (GRKs) are enzymes involved in agonist-dependent regulation of G protein-coupled receptors. In the present work, we characterized, by immunohistochemistry, the presence of GRKs 2, 3 and 5 in the chick retina, a tissue whose structure and neurochemistry are well known. These enzymes are expressed in specific cell types and regions of the retina. Immunoreactivity for GRK2 was found over photoreceptor inner segments, cell bodies of horizontal, amacrine and ganglion cells. Labeling for this enzyme was also observed over the two plexiform layers. Immunoreactivity for GRK3 was found in cell bodies of amacrine and ganglion cells. In plexiform layers, specific GRK3 immunoreactivity was observed only at the inner plexiform layer, where three bands of high labeling were detected. In contrast to GRK2 and 3, intense immunoreactivity for GRK5 was observed only over Müller cells. Occasionally, labeled amacrine cell bodies were also observed. These results suggest that GRKs 2, 3 and 5 are expressed and involved in the physiology of specific cells types of the retina. They also suggest that receptor-GRK specificity may be determined by the co-expression of the receptor and the kinase within individual cell populations in this tissue.     

GABA-like immunoreactivity in the macaque monkey retina: a light and electron microscopic study

The distribution of GABA-like immunoreactivity in the macaque monkey retina was studied by using postembedding techniques on semithin and ultrathin sections. At the light microscopic level, both inner and outer plexiform layers showed strong GABA-like immunoreactivity in the central retina. All the horizontal cells, some bipolar cells, 30-40% of amacrine cells, occasional interplexiform cells, and practically all displaced amacrine cells were labeled. In the peripheral retina (beyond 5 mm eccentricity), the outer plexiform layer and the horizontal cells were not labeled, but all other cell types showed the same labeling pattern as in the central retina. Synapses of the inner plexiform layer involving a pre- or postsynaptic GABA-labeled process were studied electron microscopically. Synapses involving a GABA-labeled presynaptic amacrine cell process made up 80% of the synapses observed. These GABA-labeled amacrine processes synapsed onto amacrine, bipolar, and ganglion cell processes as well as onto amacrine and ganglion cell bodies. Synapses involving a postsynaptic GABA-labeled process made up 20% of the synapses studied. The GABA-like immunoreactive processes were postsynaptic to bipolar cells at the dyads and to amacrine cells at conventional synapses.     

Neuronal nitric oxide synthase is expressed in the axotomized ganglion cells of the rat retina

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity.     

Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina

Postembedding immunocytochemistry was used to determine the cellular localization of the amino acid neurotransmitters glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine in the avian retina. The through retinal pathway was glutamatergic, with all photoreceptors, bipolar cells, and ganglion cells being immunoreactive for glutamate. Bipolar cells displayed the highest level of glutamate immunoreactivity, with the cell bodies terminating just below the middle of the inner nuclear layer. All lateral elements, horizontal cells, amacrine cells, and interplexiform cells were immunoreactive for glycine or GABA. The GABAergic neurons consisted of two classes of horizontal cells and amacrine cells located in the lower part of the inner nuclear layer. GABA was also localized in displaced amacrine cells in the ganglion cell layer, and a population of ganglion cells that co-localize glutamate and GABA. Both the horizontal cells and GABAergic amacrine cells had high levels of glutamate immunoreactivity, which probably reflects a metabolic pool. At least two types of horizontal cells in the avian retina could be discriminated on the basis of the presence of aspartate immunoreactivity in the H2 horizontal cells. Glycine was contained in a subclass of amacrine cells, with their cell bodies located between the bipolar cells and GABAergic amacrine cells, two subclasses of bipolar cells, displaced amacrine cells in the ganglion cell layer, and ganglion cells that colocalize glutamate and glycine. Glycinergic amacrine cells had low levels of glutamate. We have also identified a new class of glycinergic interplexiform cell, with its stellate cell body located in the middle of the inner nuclear layer among the cell bodies of bipolar cells. Neurochemical signatures obtained by analyzing data from serial sections allowed the classification of subclasses of horizontal cells, bipolar cells, amacrine cells, and ganglion cells. © 1993 Wiley-Liss, Inc.     

Localization of aspartate-like immunoreactivity in the retina of the turtle (Pseudemys scripta).

Aspartate has been reported to be a putative excitatory neurotransmitter in the retina, but little detailed information is available concerning its anatomical distribution. We used an antiserum directed against an aspartate-albumin conjugate to analyze the anatomy, dendritic stratification, and regional distribution of cell types with aspartate-like immunoreactivity in the turtle retina. The results showed dramatic differences in immunoreactivity in the peripheral versus the central retina. Strong aspartate-like immunoreactivity was shown in the peripheral retina, with many well-labeled processes in the inner plexiform layer. Many bipolar, horizontal, amacrine, and ganglion cells, some photoreceptors, and some unidentified cells were strongly immunoreactive in the peripheral retina. In contrast, although the central retina showed well-labeled horizontal cells, there was only light labeling in the inner plexiform layer with weakly immunoreactive amacrine and ganglion cells and no labeled bipolar cells. There were several strongly immunoreactive efferent nerve fibers which left the optic nerve head and arborized extensively in the retina. At the electron microscopic level, electron-dense reaction product was associated with synaptic vesicles at bipolar and amacrine cell synapses in the inner plexiform layer. These results suggest that aspartate may be involved in many diverse synaptic interactions in both the outer plexiform layer and the inner plexiform layer of the turtle retina.     

Spatial density and immunoreactivity of bipolar cells in the macaque monkey retina.

The anatomical substrates of spatial and color vision in the primate retina are investigated by measuring the immunoreactivity and spatial density of bipolar, amacrine and horizontal cells in the inner nuclear layer of the macaque monkey retina. Bipolar cells can be distinguished from amacrine and horizontal cells by their differential immunoreactivity to antisera against glutamate, glycine, GABA, parvalbumin, calbindin (CaBP D-28K), and the L7 protein from mouse cerebellum. The spatial density of bipolar cells is compared to the densities of photoreceptors and ganglion cells at different retinal eccentricities. In the centralmost 2 mm, cone bipolar cells outnumber ganglion cells by about 1.4:1. The density of cone bipolar cells is thus high enough to allow for input to different (parasol and midget) ganglion cell classes by different (diffuse and midget) bipolar cell classes. The density gradient of cone bipolar cells follows closely that of ganglion cells in central retina but falls less steeply in peripheral retina. This suggests that the convergence of cone signals to the receptive fields of ganglion cells in the peripheral retina occurs in the inner plexiform layer. The density of cone bipolar cells is 2.5-4 times that of cones at all eccentricities studied, implying that cone connectivity to bipolar cells remains constant throughout the retina. Different subgroups of bipolar cells are distinguished by their relative immunoreactivity to the different antisera. All rod and cone bipolar cells show moderate to strong glutamate-like immunoreactivity. The bipolar cells that show weak to moderate GABA-like immunoreactivity are also labeled with the antiserum to the L7 protein and are thus identified as rod bipolar cells. Nearly half of all cone bipolar cells showed glycine-like immunoreactivity. The results suggest that the inhibitory neurotransmitter candidates GABA and glycine are segregated respectively in rod and cone bipolar cell pathways. A diffuse, cone bipolar cell type can be identified by the anti-parvalbumin and the anti-calbindin antisera. All horizontal cells show parvalbumin-like immunoreactivity. Nearly all amacrine cells show GABA-like or glycine-like immunoreactivity; a variety of subpopulations also show immunoreactivity to one or more of the other markers used.