Immunocytochemical localization of the amino acid neurotransmitters in the chicken retina

Abstract

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.

Molecular Phenotyping of Retinal Ganglion Cells

“…Each chip was flat embedded in epoxy resin on a glass slide; a small rectangular sample was scribed from the slide (Stell and Lightfoot, 1975) and sectioned serially at 250 nm onto 12-spot Teflon-coated slides (C el-Line, Fisher Scientific). The immunocytochemical and IgG production procedures were as described previously (Marc et al, 1990(Marc et al, , 1995 using the silver-intensification protocol of Kalloniatis and Fletcher (1993). The samples were serially probed with IgGs targeting aspartate, glutamate, glutamine, glycine, GABA, taurine, and AGB obtained from Signature Immunologics Inc. (Salt Lake C ity, UT).…”

Excitotoxicity in the Enteric Nervous System

“…Glutamate-immunoreactive structures were identified by immunocytochemistry with rabbit antibodies to glutamate (Marc et al, 1990;Kalloniatis and Fletcher, 1993). Rabbit antisera raised against glutamate conjugated by glutaraldehyde to thyroglobulin were obtained from Chemicon.…”

Glutamate uptake in retinal glial cells during diabetes

“…D-Aspartate immunocytochemistry The post-embedding immunocytochemical procedure used was that described by Kalloniatis and Fletcher [32]. The tissue sections were etched in 1:5 solution of sodium ethoxide/ethanol rinsed in a graded series of methanol washes and placed in phosphate buffer (0.1 mol/l PB).…”

“…The slides were then rinsed several times in phosphate buffered saline (PBS) and fixed in 1% glutaraldehyde in 0.1 mol/l phosphate buffer for 10 min. The immunogold was visualised by silver intensification [32,33].…”