Arnold G. Hyndman

Excitatory Amino Acid‐Induced Toxicity in Chick Retina: Amino Acid Release, Histology, and Effects of Chloride Channel Blockers

Abstract: Acute excitotoxicity in embryonic chick retina and the ability of C1− channel blockers to prevent toxicity were evaluated by measurement of endogenous amino acid release and histology. Treatment of retina with kainate, quisqualate, or N‐methyl‐D‐aspartate resulted in a large dose‐dependent release of γ‐aminobutyric acid and taurine, moderate release of glutamine and alanine, and no measurable release of glu‐tamate or aspartate. Concentrations inducing maximal γ‐aminobutyric acid release were 50 μM quisqualate, 100 μM kainate, and 100 μM N‐methyl‐D‐aspartate. Treatment with 1 mM glutamate resulted in significant γ‐aminobutyric acid release, as well as an elevation in medium aspartate levels. Typical excitotoxic retinal lesions were produced by the agonists and, at the lower concentrations tested, revealed a regional sensitivity. There was a positive correlation between the amount of γ‐aminobutyric acid release and the extent of tissue swelling, suggesting that release may be secondary to toxic cellular events. Omission of C1− completely blocked cytotoxic effects due to kainate or glutamate. Likewise, addition of the C1−/bicarbonate anion channel blocker 4,4′‐di‐isothiocyanatostilbene‐2,2′‐disulfonate at 600 μM protected retina from cytotoxic damage from all excitotoxic analogs and restored amino acid levels to baseline values. Furosemide. which blocks Na+/K+/2C1− cotransport, was only minimally effective in reducing amino acid release induced by the agonists. Consistent with the latter, histological examination showed the continued presence of the lesion but with general reduction of cellular edema. These results indicate that although influx of C1− is a central component of the acute excitotoxic phenomenon, mechanisms other than passive Cl−flux may be involved.

Origins of the Extracellular Glutamate Released During Total Metabolic Blockade in the Immature Retina

Abstract: Previous studies have shown that complete blockade of metabolism in embryonic chick retina causes a time‐dependent increase in the release of glutamate into the extracellular space. The present study examined the cellular source of this glutamate, i.e., neuronal and/or glial. Pure cultures of retinal neurons or glia were labeled for 10 min at 37°C with [3H]acetate. Retinal glia, but not retinal neurons, were found to selectively and preferentially metabolize acetate, thus producing 3H‐labeled amino acids in the glial compartment. This finding provides direct evidence to substantiate findings from several other laboratories that have indirectly determined the preferential metabolism of acetate by glia by using mixed neuronal/glial populations. To study the cellular source of glutamate released during total metabolic blockade, whole retina were prelabeled with [3H]acetate plus [U‐14C]glucose (to label the neuronal compartment). Total metabolic blockade was instituted with a combination of iodoacetate (IOA) plus KCN, and the release of glutamate into the medium was followed at 5, 15, and 30 min. During total energy blockade, net extracellular glutamate was not elevated at 5 min [0.17 ± 0.02 vs. 0.12 ± 0.01 µM for treated vs. control retina (means ± SEM), respectively], but was increased significantly at 15 (1.2 ± 0.26 µM) and 30 min (2.6 ± 0.22 µM). Total [3H]glutamate in the medium during IOA/KCN treatment was unchanged at 5 min, but was increased 1.5‐ and threefold above basal levels at 15 and 30 min, respectively. During the time when extracellular glutamate increased, the specific activity of [3H]glutamate remained fairly constant, 731 ± 134 and 517 ± 82 dpm/nmol (means ± SEM) at 15 and 30 min, respectively. In contrast, 14C‐labeled glutamate in the medium did not increase during IOA/KCN treatment and paralleled basal levels. Thus, the specific activity of 14C‐labeled extracellular glutamate decreased from 309 ± 87 dpm/nmol at 15 min to 42 ± 8 dpm/nmol at 30 min. Prior loading of the tissue with 0.5 mM trans‐pyrrolidine‐2,4‐dicarboxylate (t‐PDC), a glutamate transport inhibitor, blocked 57% of the glutamate released at 30 min of IOA/KCN exposure, suggesting that reversal of an Na+‐dependent glutamate transporter was a key contributor to the appearance of extracellular glutamate during energy deprivation. The increase in extracellular [3H]glutamate, constancy of the specific activity of extracellular [3H]glutamate, decrease in the specific activity of extracellular [14C]glutamate, and attenuation of release by prior loading with t‐PDC indicate that glial pools of glutamate released via reversal of the transporter contribute significantly to the rise in extracellular glutamate after metabolic inhibition in this preparation.

Transferrin binding protein is expressed by oligodendrocytes in the avian retina

It has been documented that some axons of ganglion cells in the nerve fiber layer of avian retina are wrapped in a myelin sheath. However, the identity of myelin-forming cells has not been established. In this study we demonstrated immunohistochemical evidence for the existence of a large population of oligodendrocytes in avian retina, using an antiserum against transferrin binding protein (TfBP), the avian homologue of the mammalian GRP 94 family of stress-regulated proteins. TfBP+ cells were mostly confined to the ganglion cell and optic nerve fiber layers of the retina, in which they were closely associated with the soma and axons of ganglion cells. The double-labeling experiments clearly show that TfBP is specific to oligodendrocytes. The morphology, distribution, and antigenic properties indicated by our findings suggest that TfBP+ cells are retinal oligodendrocytes that may be responsible for the myelination of ganglion cell axons in avian retina. A putative tropic role of TfBP+ oligodendrocytes to the ganglion cells is also discussed in conjunction with the physical properties of TfBP and avascular retinae of birds.

The ontogeny of transferrin receptors in the embryonic chick retina: an immunohistochemical study

Transferrin receptor (TfR) immunoreactivity in the developing chick retina was examined. Immunoreactivity was detectable in the ganglion cells of embryonic day (E) 4 retina. At E9, diffuse TfR immunoreactivity appeared in the outer portion of the inner nuclear layer. Amacrine cells were the most intensely TfR-positive cells in the inner nuclear layer. At E11, the inner segment of photoreceptor cells showed moderate immunoreactivity. With the appearance of the outer segments, positive immunoreactivity was observed in these structures. TfRs developmental distribution in the retina may reflect the developmental and physiological role of transferrin.

Transferrin in chick retina: distribution and location during development

Chick retinas from embryonic day 6 (E6) to 3 weeks post-hatching were examined for the presence and location of endogenous transferrin. Immunocytochemistry revealed that transferrin was differentially distributed in retinal layers. Furthermore, the pattern of transferrin distribution changed with developmental age. At day E6, transferrin was found in 2 distinct bands which were located in the area of the Müller cell end-feet. By day E9, additional regions of transferrin immunoreactivity could be found in the inner and outer plexiform layers (IPL, OPL) and the nerve fiber layer (NFL). These latter 3 bands (IPL, OPL and NFL) became more prominent from E9 until E17 as the synaptic layers and nerve fiber layer increased in density and maturation. Perikarya in the nuclear layers size, density and maturation. Perikarya in the nuclear layers were negative. At day E17 and later, the newly forming outer segments of photoreceptor cells were strongly reactive for transferrin while the somas of the photoreceptor cells, in the ONL, were negative. Retinas from chicks 1 day to 3 weeks post-hatching retained strong immunoreactivity for transferrin in the photoreceptor cell outer segments and OPL, lessened immunoreactivity in the IPL and loss of immunoreactivity in the NFL. Iron distribution in the retina for all ages examined showed only 2 bands that locally corresponded to the Müller cell end-feet. Iron stores were not found in the synaptic layers or photoreceptor cell outer segments. These studies suggest an iron storage function for retinal glia and a role for transferrin in neuronal development and differentiation.

Neurons and glia in purified retinal cultures identified by monoclonal antibodies to intermediate filaments.

Two monoclonal antibodies known to bind intermediate filaments were used to identify neurons and glia from embryonic chick neural retina. Neurofilament specific antibody RT-97-F1, bound neuroepithelial cells, migrating neurons, as well as the photoreceptor layer, plexiform layers and optic fiber layer. The other, 3A7, bound intermediate filaments of Müller cells. In purified neuronal cultures, RT-97-F1 bound 75, 83 and 98% of the cells present at 24, 48 and 72 h respectively, while 3A7 bound 26, 15 and 0.3% for the same times in vitro. In purified glial cultures, RT-97-F1 showed a weak perinuclear binding, while 3A7 strongly bound intermediate filaments of nearly 100% of the cells. These antibodies confirmed and quantitated the high purity of our cultures.

Identification of a population of amarcine cells rich in insulin receptors

Insulin receptor immunoreactivity in the developing chick retina was examined by immunocytochemistry. Insulin receptor immunoreactivity could be detected throughout the retina at all stages studied. Beginning at E12, a limited number of amacrine cells located in the inner level of the inner nuclear layer were strongly immunoreactive. By E19 there was a decrease in immunoreactivity throughout the retina, with the exception of the ganglion cell layer and a few amacrine cells and their process; this distribution was present in 3-day-old posthatched chick retina. The pattern of immunoreactivity of insulin receptors may indicate a unique role for insulin in the development and physiology of some amacrine cells.

Transferrin concentration and location during formation of chick retina: Developmental correlates

The amount of transferrin in chick retina was measured during development and compared to transferrin location seen immunocytochemically. Between embryonic day 6 (E6), and 5 days post hatching, two periods occur in which transferrin concentrations rise sharply and decline. During the first, transferrin concentration rises 5-fold between E6 and 10, then rapidly declines by E14. A second increase begins on E17 and peaks by E19-20. Immunocytochemical findings demonstrate that during the first rise in concentration, transferrin is located primarily in neuritic layers. Later in development, when levels again increase, newly forming photoreceptor outer segments are strongly transferrin positive. These findings are discussed in light of developmental events occurring during retinal maturation.

Lucifer Yellow labeling of embryonic chick retinal amacrine cells in vitro.

Lucifer Yellow (LY) selectively labels embryonic chick amacrine cells from days 11 until 14 in vivo. Its usefulness as an in vitro marker was investigated. In vivo labeling and subsequent culturing was not possible due to dye leakage. Neurons, however, could be labeled at various times in vitro. The number of neurons labeled with LY in vitro was consistent with the number of neurons expected to be labeled and increased when selected areas of the retina known to be rich in LY-labeled neurons were used in culturing. Neurons plated at times when labeling was not found in vivo (Embryonic day 8, E8) began to label only at times that were equivalent to times when labeling was found in vivo (E10-E11). This suggests that the selectivity of labeling is preserved in vitro and that LY can be used as an in vitro marker for retinal amacrine cells.

High affinity binding of transferrin in cultures of embryonic neurons from the chick retina

Immunohistochemical and autoradiographic analysis of neuronal cultures from embryonic day 8 (E8) and day 11 (E11) chick retina indicate that transferrin receptors and binding sites are present on soma and neurites. Cultures maintained in the presence of transferrin expressed elevated transferrin binding due to an increase in the number of transferrin receptors. Cultures from E11 neural retina exhibited a decrease in transferrin binding when compared to E8 cultures. This appears to be due to a decrease in the number of binding sites. Neurons maintained in a transferrin-free medium supplemented with 0.4 microM of iron sulfate generally expressed slight increases in transferrin binding.