Localization of enkephalin-like immunoreactive amacrine cells in the larval tiger salamander retina: a light and electron microscopic study.

Immunohistochemistry was utilized to examine the light and electron microscopic localization of enkephalin-like (enk) immunoreactive amacrine cells in the larval tiger salamander retina. The vast majority of enk-immunoreactive cells were typical amacrine cells whose round or oval cell bodies (14-16 microns) were situated in the innermost cell row of the inner nuclear layer. A relatively small number of enk-stained oval cell bodies (14-22 microns) were located in the ganglion cell layer and were designated as those of displaced amacrine cells. Enkephalin immunostaining was observed in the inner plexiform layer as a fine plexus in sublamina 1 and as a dense network of fibers in sublamina 5. In both the center and periphery of the retina the density of typical enk-amacrine cells was determined to be 250 +/- 16.36 cells per mm2 surface area of the retina. At the ultrastructural level typical enk-stained amacrine cells possessed a round, indented nuclear membrane. Enk-immunoreactive processes sometimes contained dense-core vesicles (60-115 nm) in addition to a rather homogeneous population of small, round, agranular synaptic vesicles (25-35 nm). In sublamina 1 the processes of enk-amacrine cells were presynaptic to amacrine and bipolar cells. They also contacted processes devoid of synaptic vesicles which possibly arise from ganglion cells. As the postsynaptic element in sublamina 1, they received synaptic input from amacrine cells. In sublamina 5 the processes of enk-amacrine cells were presynaptic to amacrine cells, bipolar cells, and the somas of cells situated in the ganglion cell layer.(ABSTRACT TRUNCATED AT 250 WORDS)

A peptide histidine isoleucine/peptide histidine methionine-like peptide in the rabbit retina: colocalization with vasoactive intestinal peptide, synaptic relationships and activation of adenylate cyclase activity.

Antisera against peptide histidine isoleucine and peptide histidine methionine were found to label a subpopulation of amacrine and displaced amacrine cells in the rabbit retina with processes ramifying in sublaminas 1, 3 and 5 of the inner plexiform layer. Preadsorption controls demonstrated that this immunoreactivity was specific for a peptide histidine isoleucine- or peptide histidine methionine-like (peptide histidine isoleucine/peptide histidine methionine-like) peptide, and was not caused by cross-reactivity of the peptide histidine isoleucine or peptide histidine methionine antibodies with vasoactive intestinal peptide vasoactive intestinal peptide. In double-label studies, vasoactive intestinal peptide and peptide histidine isoleucine/peptide histidine methionine-like immunoreactivity were colocalized in the same population of retinal neurons. Electron microscopic analysis revealed that the peptide histidine isoleucine/peptide histidine methionine-labelled cells interacted with processes of bipolar cells, amacrine cells and ganglion cells. Peptide histidine methionine and peptide histidine isoleucine were slightly less potent than vasoactive intestinal peptide in stimulating adenylate cyclase activity in the rabbit retina, while the related peptides secretin, glucagon, and the C-terminal vasoactive intestinal peptide fragment, vasoactive intestinal peptide (10-28), showed little or no stimulatory activity. Stimulation of adenylate cyclase by high concentrations of vasoactive intestinal peptide and peptide histidine methionine were non-additive. These results suggest that a peptide histidine isoleucine/peptide histidine methionine-like peptide may function as a neuroactive peptide in the mammalian retina, and that this peptide appears to be cosynthesized and colocalized with vasoactive intestinal peptide and to mimic the activity of vasoactive intestinal peptide through interaction with vasoactive intestinal peptide receptor-adenylate cyclase complexes.

An ethanolic phosphotungstic acid (EPTA) analysis of photoreceptor and synaptic ultrastructure in the guinea pig retina.

Ethanolic phosphotungstic acid (EPTA) has been used to elucidate the structure of certain organelles contained within retinal cells not clearly discernible using conventional preparations. Both synaptic and nonsynaptic components of the guinea pig neural retina have been analyzed. Within the photoreceptor (PR) cell EPTA-stained components include the connecting cilia, their basal bodies, and the root filament system. Cross-striated fibrillar organelles, similar in appearance to the root filaments, are also observed in the nuclear region, the synaptic terminal and other parts of the PR cell. The possible structural continuity and significance of these structures are discussed. Within retinal synapses of both the inner and outer plexiform layers, ribbons and associated paramembranous specializations are stained. The photoreceptor ribbons have a trialaminar structure with filamentous, tufted borders. Synaptic cleft material and postsynaptic densities are also stained. Bipolar cell synapses in the inner plexiform layer contain stained short ribbons as well as closely associated peg-like densities extending towards the presynaptic membrane.

Rabbit retinal ganglion cells survive optic nerve transection and entubulation repair with type I collagen nerve guide tubes.

The major objective of the experiments reported in this paper was to qualitatively test the hypothesis that rabbit retinal ganglion cells survive optic nerve transection and entubulation repair of the proximal optic nerve stump. The optic nerve of rabbits was transected immediately behind the globe, and a 1-cm length of a Type I collagen nerve guide tube was sutured onto the short proximal stump. The nerve guide was either left empty or was filled with a Type I collagen gel (Vitrogen, Collagen Corp.). Following 8-12 weeks survival time, the animals were sacrificed and the retinae were prepared as whole mounts and processed for immunocytochemistry using an antibody which selectively labels the retinal ganglion cells. Although no formal cell counts were carried out, the animals which received Vitrogen within the nerve guide showed a qualitative enhancement of retinal ganglion cell survival compared to the group with the nerve guide alone. The results suggest that specific manipulations of the central nervous system microenvironment may enhance neuronal survival following axonal transection.

GABAergic ganglion cells in the rabbit retina.

The ganglion cells are the output neurons of the retina. There is, however, relatively little known about the neurotransmitters used by these cells. In the present study, ganglion cells identified with a ganglion cell-specific monoclonal antibody (AB5) are shown in separate double-label experiments to be gamma-aminobutyric acid (GABA)-like immunoreactive and to possess a high-affinity uptake mechanism for [3H]GABA accumulation. The localization of these markers of GABA activity to AB5-labelled ganglion cells provides the first definitive evidence for the presence of a classical transmitter in retinal ganglion cells and suggests that GABA may perform a role as a neurotransmitter in these cells.

Ganglion cell density in albino and pigmented rabbit retinas labeled with a ganglion cell-specific monoclonal antibody.

Retinas from two rabbits, one normally pigmented and one albino, were labeled with monoclonal antibody AB5, which has been shown to be a specific marker for ganglion cells. This method obviates criteria for distinguishing among ganglion cells, displaced amacrine cells, and glia. Labeled cells were counted within small fields at some 2000 regularly spaced points on each retina. These counts were transformed to maps of ganglion cell density. In general, the density map for the pigmented retina was similar to those obtained by earlier studies with non-specific stains, thereby confirming the basic validity of most previous studies and demonstrating the applicability of AB5 labeling to work of this type. The ganglion cell density map of the albino retina was abnormal, showing a clear deficit of ganglion cells in the nasal portion of the visual streak. This result not only indicates that the albino anomaly has retinal effects, but also suggests a major impact on ganglion cells whose projections (in normal animals) are contralateral.

A monoclonal antibody specific for retinal ganglion cells of mammals.

The development of specific markers for retinal ganglion cells is an area of great interest in retinal research. In this study we report on a monoclonal antibody (AB5) which specifically labels ganglion cells in rabbit, cat and monkey, as well as a variety of other mammalian species. Labelling of ganglion cells was also observed in isolated cell preparations of rabbit retina.

Postnatal development of ganglion cells in the rabbit retina: characterizations with AB5 and GABA antibodies.

The use of cell-specific monoclonal antibodies provides a means by which the emergence, differentiation and maturation of retinal neurons can be studied. The present study investigates the labelling of ganglion cells in the developing rabbit retina by a ganglion cell-specific monoclonal antibody, AB5(12,13). AB5 labelling of ganglion cells was observed as early as day postnatal. By 6-8 days postnatal, AB5-labelled ganglion cells had begun differentiating into the various ganglion cell subtypes observed in the adult retina. This differentiation process appeared to continue throughout the first 3 weeks postnatal. The AB5 monoclonal antibody was also used in a double-label paradigm with an anti-gamma-aminobutyric acid (GABA) polyclonal antibody to differentiate the GABAergic ganglion cells from other GABAergic elements in the retina and to study their development. GABAergic ganglion cells were first observed at 3 days postnatal and by 6 days postnatal, it was possible to observe a wide variety of GABAergic ganglion cells ranging from small cells to large alpha-type cells. The appearance of AB5 labelling in ganglion cells at relatively early stages of development suggests that the AB5 monoclonal antibody may be a useful tool for studying the development of ganglion cell structure, distribution, synaptic relationships and neurochemical specificity.

The rabbit retina: a long-term model system for aluminum-induced neurofibrillary degeneration.

Aluminum (Al) was injected into the rabbit eye as a potential long-term model system for Al-induced neurofibrillary degeneration (NFD). Neurofibrillary tangles made up of 10 nm phosphorylated neurofilaments were observed in a subpopulation of retinal ganglion cells, located primarily in the peripheral retina. The distribution of affected cells suggested a differential susceptibility of ganglion cells to Al intoxication. Importantly, none of the animals demonstrated any of the central neurological dysfunctions characteristic of previous Al intoxication models. The retinal model should allow for long-term studies of Al intoxication and its potential relationship to neurofibrillary degenerative disorders such as Alzheimer's disease.

Specific labelling of ganglion cells in the cat retina by a monoclonal antibody.

The introduction of monoclonal antibody technology has made it possible to produce specific markers for various retinal cell types. We report here on a monoclonal antibody, AB5, which specifically labels the retinal ganglion cells of the cat. The labelled cells could be categorized as either alpha, beta or gamma subtype based on morphological criteria. This antibody will be useful for studies of the morphology, localization and synaptic connectivity of ganglion cells in the cat retina.

Enkephalin in the goldfish retina.

Enkephalin-like immunoreactive amacrine cells were visualized using the highly sensitive avidin-biotin method. The somas of these cells were situated in the inner nuclear and ganglion cell layers. Enkephalin-stained processes were observed in layers 1, 3, and 5 of the inner plexiform layer. The biosynthesis of sulfur-containing compounds in the goldfish retina was studied by means of a pulse-chase incubation with 35S-methionine. A 35S-labeled compound, which comigrated with authentic Met5-enkephalin on high-performance liquid chromatography (HPLC), was synthesized and was bound competitively by antibodies to enkephalin and by opiate receptors. This compound was tentatively identified as "Met5-enkephalin." The newly synthesized 35S-Met5-enkephalin was released upon depolarization of the retina with a high K+ concentration. This K+-stimulated release was greatly suppressed by 5 mM Co2+, suggesting that the release was Ca2+ dependent. Using a double-label technique, enkephalin immunoreactivity and gamma-aminobutyric acid (GABA) uptake were colocalized to some amacrine cells, whereas others labeled only for enkephalin or GABA. The possible significance of enkephalin-GABA interactions is also discussed.

An immunocytochemical marker for hamster retinal ganglion cells.

We examined the specificity and developmental time course of the labelling of retinal ganglion cells in Syrian hamsters by a monoclonal antibody AB5. In adult hamsters, AB5 selectively labelled somata in the ganglion cell layer, dendrites in the inner plexiform layer and axons in the nerve fibre layer. When retinal ganglion cells were retrogradely labelled with DiI prior to AB5 immunocytochemistry, all of the retrogradely labelled retinal ganglion cells in the ganglion cell layer were AB5 immunoreactive, indicating that AB5 labels all classes of ganglion cell in that layer. In retinae depleted of retinal ganglion cells by neonatal optic nerve transections, AB5 did not label any somata or processes, indicating that AB5 specifically labels retinal ganglion cells. During development, AB5 labelling first appeared as a weak staining of cell bodies in the ganglion cell layer on postnatal day 12 (P12; PO = first 24 h following birth) and acquired the staining pattern seen in the adult by postnatal day 14. From the onset of AB5 immunoreactivity, AB5-labelled somata of varying sizes were present across the entire retinal surface. Although AB5 labelled retinal ganglion cell axons in the nerve fibre layer of the retina it did not label the optic nerve or retinal ganglion cell axons in the brain at any age examined. AB5 labelling was also found to be compatible with bromodeoxyuridine immunocytochemistry and, therefore, useful for determining the time of generation of hamster retinal ganglion cells.