AB5018 Sigma-AldrichAnti-Glutamate (No Glutaraldehyde) Antibody

Lampreys are one of the two surviving groups of the agnathan (jawless) stages in vertebrate evolution and are thus ideal candidates for elucidating the evolution of visual systems. This study investigated the retinal amino acid neurochemistry of the southern hemisphere lamprey Geotria australis during the downstream migration of the young, recently-metamorphosed juveniles to the sea and during the upstream migration of the fully-grown and sexually-maturing adults to their spawning areas. Glutamate and taurine were distributed throughout the retina, whilst GABA and glycine were confined to neurons of the inner retina matching patterns seen in most other vertebrates. Glutamine and aspartate immunoreactivity was closely matched to Müller cell morphology. Between the migratory phases, few differences were observed in the distribution of major neurotransmitters i.e. glutamate, GABA and glycine, but changes in amino acids associated with retinal metabolism i.e. glutamine and aspartate, were evident. Taurine immunoreactivity was mostly conserved between migrant stages, consistent with its role in primary cell functions such as osmoregulation. Further investigation of glutamate signalling using the probe agmatine (AGB) to map cation channel permeability revealed entry of AGB into photoreceptors and horizontal cells followed by accumulation in inner retinal neurons. Similarities in AGB profiles between upstream and downstream migrant of G. australis confirmed the conservation of glutamate neurotransmission. Finally, calcium binding proteins, calbindin and calretinin were localized to the inner retina whilst recoverin was localized to photoreceptors. Overall, conservation of major amino acid neurotransmitters and calcium-associated proteins in the lamprey retina confirms these elements as essential features of the vertebrate visual system. On the other hand, metabolic elements of the retina such as neurotransmitter precursor amino acids and Müller cells are more sensitive to environmental changes associated with migration.

The viability and functions of the corneal epithelium are dependent in large measure on the active uptake of nutrients, growth factors, and amino acids from stroma and tear. The present study presents the cellular distribution(s) of glutamate, the Na(+)-dependent glutamate/aspartate transporters (excitatory amino acid transporters; EAAT1-5), Na(+)-independent glutamate/cystine exchanger (Xc(-) antiporter) subunits (xCT light chain and 4F2hc heavy chain), glutamine synthetase (GS), and gamma-glutamyltranspeptidase (GGT) in human corneal epithelium.

Changes in glutathione contents occur in plants during environmental stress situations, such as pathogen attack, as the formation of reactive oxygen species leads to the activation of the antioxidative defence system. As glutathione is synthesized out of its constituents cysteine, glycine, and glutamate the availability of these components will limit glutathione synthesis in plants especially during stress situations and therefore the ability of the plant to fight oxidative stress. To gain a deeper insight into possible limitations of glutathione synthesis during pathogen attack the present investigations were aimed to study how the subcellular distribution of glutathione precursors correlates with the subcellular distribution of glutathione during virus attack in plants. Selective antibodies against cysteine, glutamate, and glycine were used to study the impact of Zucchini yellow mosaic virus (ZYMV) infection on glutathione precursor contents within different cell compartments of cells from Cucurbita pepo (L.) plants with the transmission electron microscope (TEM). Generally, levels of cysteine and glutamate were found to be strongly decreased in most cell compartments of younger and older leaves including glutathione-producing cell compartments such as plastids and the cytosol. The strongest decrease of cysteine was found in plastids (- 54 %) and mitochondria (- 51 %) of younger leaves and in vacuoles (- 37 %) and plastids (- 29 %) of older leaves. The strongest decrease of glutamate in younger leaves occurred in peroxisomes (- 67 %) and nuclei (- 58 %) and in peroxisomes (- 64 %) and plastids (- 52 %) of the older ones. Glycine levels were found to be strongly decreased (- 63 % in mitochondria and - 53 % in plastids) in most cell compartments of older leaves and strongly increased (about 50 % in plastids and peroxisomes) in all cell compartments of the younger ones. These results indicate that low glycine contents in the older leaves were responsible for low levels of glutathione in these organs during ZYMV infection rather than limited amounts of cysteine or glutamate. Glutathione precursors were virtually absent in cell walls and intercellular spaces and play therefore no important role during ZYMV attack in the apoplast. While glutamate was absent in vacuoles, elevated levels of glycine (up to 30 %) and decreased cysteine contents (up to - 37 %) were observed in vacuoles during ZYMV infection. The impact of the present results on the current knowledge about glutathione synthesis and degradation on the cellular level during ZYMV infection are discussed.

OBJECTIVE: To determine whether taurine and glutamate contents are reduced in damaged photoreceptors in dogs with primary glaucoma (PG) in a manner consistent with an ischemia-like release of both of these amino acids from damaged cells. SAMPLE POPULATION: Retinas from 6 dogs with PG and 3 control dogs. PROCEDURE: Serial, semithin sections of each canine retina were stained with toluidine blue to identify damaged photoreceptors or via immunogold techniques to quantify taurine and glutamate content in retinal cells. RESULTS: Regions with a thin outer nuclear layer and pathologic nuclear changes in photoreceptors were evident in retinas of dogs with PG. The density of immunostaining for taurine in damaged photoreceptors was significantly reduced to (mean +/- SEM) 37.5 +/- 2.6% of the density in adjacent undamaged photoreceptors. Photoreceptors with decreased taurine immunostaining also had decreased glutamate immunostaining, consistent with ischemia-like release of both of these amino acids from damaged cells. Immunostaining for glutamate, but not taurine, was increased in presumptive radial glial cells (i.e., Miller cells) in damaged regions, consistent with an ischemia-induced redistribution of amino acids in dogs with PG. CONCLUSIONS AND CLINICAL RELEVANCE: Retinal damage in dogs with PG includes ischemia-like losses of taurine and glutamate from photoreceptors and accumulation of glutamate, but not taurine, in nearby Müller cells. These changes are consistent with glutamate release and depletion of intracellular taurine in damaged regions, perhaps contributing to progressive damage in these areas.

In prior studies that used transneuronal transport of isogenic recombinants of pseudorabies virus, we established that medial medullary reticular formation (MRF) neurons sent collateralized projections to both diaphragm and abdominal muscle motoneurons. Furthermore, inactivation of MRF neurons in cats and ferrets increased the excitability of diaphragm and abdominal motoneurons, suggesting that MRF neurons controlling respiratory activity are inhibitory. To test this hypothesis, the present study determined the neurochemical phenotypes of MRF premotor respiratory neurons in the ferret by using immunohistochemical procedures. Dual-labeling immunohistochemistry combining pseudorabies virus injections into respiratory muscles with the detection of glutamic acid decarboxylase-like immunoreactive and glutamate-like immunoreactive cells showed that both GABAergic and glutamatergic MRF neurons project to respiratory motoneurons, although the latter are more common. These data suggest that the role of the MRF in respiratory regulation is multifaceted, as this region provides both inhibitory and excitatory influences on motoneuron activity.

5-HT transporters (SERTs) are transiently expressed in thalamocortical neurons during development, permitting these glutamatergic neurons to co-release 5-HT as a "borrowed" transmitter. The high level of SERT expression in these neurons is likely important in the serotonergic modulation of neocortical circuits and provides a system for examining endogenous SERT regulation. We tested the hypothesis that developmental expression of SERT in thalamocortical neurons is regulated by psychostimulants that are agonists and antagonists of SERT. Cultured thalamocortical neurons from embryonic day 18 rats were examined for SERT expression until P15. In untreated cultures, SERT protein levels peaked at postnatal day 3 (P3) and were absent by P10. Chronic treatment with SERT substrates (5-HT, 3,4-methylenedioxymethamphetamine) increased both peak SERT protein levels (fourfold) and the time course of SERT expression. SERT substrates also shifted the relative functional expression of SERT by redistributing intracellular SERT protein to the plasma membrane. The subcellular redistribution was prevented by PKC activators. SERT antagonists (e.g., fluoxetine, cocaine) reduced total SERT expression levels and the time course of SERT expression. These data (1) show that endogenous SERT is differentially regulated by 5-HT and psychostimulants, (2) indicate that SERT modulation occurs via changes in both total SERT protein levels and subcellular redistribution of the transporter, and (3) suggest that some of the actions of drugs of abuse in neocortical development may be attributable to alterations in SERT expression and concomitant changes in 5-HT signaling.