AB132 Sigma-AldrichAnti-Aspartate Antibody

Recent studies have proposed that glutamate corelease by mesostriatal dopamine (DA) neurons regulates behavioral activation by psychostimulants. How and when glutamate release by DA neurons might play this role remains unclear. Considering evidence for early expression of the type 2 vesicular glutamate transporter in mesencephalic DA neurons, we hypothesized that this cophenotype is particularly important during development. Using a conditional gene knock-out approach to selectively disrupt the Vglut2 gene in mouse DA neurons, we obtained in vitro and in vivo evidence for reduced growth and survival of mesencephalic DA neurons, associated with a decrease in the density of DA innervation in the nucleus accumbens, reduced activity-dependent DA release, and impaired motor behavior. These findings provide strong evidence for a functional role of the glutamatergic cophenotype in the development of mesencephalic DA neurons, opening new perspectives into the pathophysiology of neurodegenerative disorders involving the mesostriatal DA system.

PURPOSE: Retinal detachment induces neurochemical changes in the neural retina over a span of days to weeks. However, little information is available on the acute response in the retina to detachment. METHODS: Distribution of the neurotransmitters glutamate, glycine, and gamma-aminobutyric acid (GABA) and the metabolic amino acids aspartate and glutamine was examined immunocytochemically from 5 to 30 minutes and at 3 hours after retinal detachment in a salamander eyecup preparation. RESULTS: Glutamate showed a rapid depletion from neuronal cell bodies in detached retina, whereas Müller cells, which normally sequester and metabolize glutamate, showed increased immunolabeling for glutamine. Changes occurred exclusively in detached retinal regions of the eyecup. Aspartate, a precursor for glutamate synthesis, also showed decreased labeling in neuronal cell bodies in detached retinal regions, although these changes were not as striking as those observed for glutamate. In contrast, the distributions of the inhibitory amino acid neurotransmitters glycine and GABA were not affected appreciably by acute retinal detachment. CONCLUSIONS: These results indicate that retinal detachment induces rapid, localized alterations in the glutamatergic system of the neural retina that are consistent with a massive efflux of neuronal glutamate and concomitant alterations in glutamate metabolism. An acute efflux of neuronal glutamate in detached retina could contribute to excitotoxicity and to the initiation of structural alterations and changes in gene expression; it is also consistent with reported neurochemical changes associated with longer term retinal detachment.

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.

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.

An immunological approach to visualize aspartate in the rat brain was attempted by raising antibodies against this acidic amino acid. Using an adapted ELISA method, their specificity was tested by competition experiments between aspartate conjugated via glutaraldehyde to various protein-carriers and either non-conjugated aspartate or conjugated amino acids, preincubated with anti-aspartate antibodies. Their titer and specificity were found high enough to allow their use in immunocytochemistry which demonstrated the presence of a large number of aspartate-containing cell-bodies in many areas of the brain.