Quantitative ultrastructural localization of glutamate dehydrogenase in the rat cerebellar cortex

Abstract

Glutamate dehydrogenase is one of the main enzymes involved in the formation and metabolism of the neurotransmitter glutamate. In the present study we investigated the enzyme ultrastructurally in the cerebellar cortex, a region rich in well defined glutamatergic neurons, by pre-embedding immunocytochemical staining (peroxidase-antiperoxidase), as well as by post-embedding immunogold labelling employing a new system for quantitation and for specificity testing under the conditions of the immunocytochemical procedure. A new antiserum against immunologically purified bovine liver glutamate dehydrogenase or antibodies isolated from this by affinity chromatography were used in rats fixed by perfusion with aldehydes. The pre-embedding method displayed peroxidase reaction preferentially in mitochondria of astroglial cells (including the Bergmann glia). Mitochondria of neuronal tissue elements were usually free of peroxidase-reaction product. Extra-mitochondrial staining was not observed. The post-embedding immunogold method was employed to overcome penetration problems and allow semiquantitative analysis of localization and specificity. The highest densities of gold particles were found over the mitochondria in astroglial cell elements (including the Bergmann glia). Mitochondria in cell bodies of Bergmann glia had a lower particle density than those in astrocytic processes. In the latter, analysis of frequency distribution revealed no evidence of a population of mitochondria lacking glutamate dehydrogenase, but suggested the presence of populations with different levels of immunoreactivity. Comparison with the labelling of embedded bovine liver glutamate dehydrogenase indicated that the enzyme constitutes a high proportion (10%) of the total matrix protein of these mitochondria. A weaker but significant labelling was found in oligodendrocytes of the white matter. The labelling of mitochondria in neuronal elements including glutamatergic mossy fibre terminals was of the order of 15% of that in astroglial mitochondria. No difference was detected between glutamatergic neurons (mossy and parallel fibres, granular cells) and non-glutamatergic neurons (Purkinje cells). The particle density over non-mitochondrial areas was very close to background over empty resin. The results, obtained with different methods of tissue and antibody preparation, agree to show that the present form of glutamate dehydrogenase is restricted to mitochondria and preferentially localized in astrocytes.

Substitution of Ser for Arg-443 in the Regulatory Domain of Human Housekeeping (GLUD1) Glutamate Dehydrogenase Virtually Abolishes Basal Activity and Markedly Alters the Activation of the Enzyme by ADP and l-Leucine

“…It is well known that the glutamate neurotransmitter action is terminated by uptake into glial cells (10). In the salamander Mü ller cells (a specialized type of glial cells found in the retina), glutamate uptake generated significant intracellular acidification (the pH dropped from approximately 7.2 to approximately 7.0) due to countertransport of OH Ϫ (33,34).…”

Steady-state in Vivo Glutamate Dehydrogenase Activity in Rat Brain Measured by 15N NMR

“…One group of rats (Group I) was given infusion of 15 NH 4 Cl (Cambridge Isotopes: Ͼ97% enriched in 15 N in 1 M aqueous solution at pH 7.4) at a rate of 2.3 Ϯ 0.04 mmol/h/kg body weight. The infusion was continued (a) for the time indicated up to 6 h, or (b) for a fixed period of 3.1 Ϯ 0.18 h, followed by 14 1 The abbreviations used are: GDH, glutamate dehydrogenase; GS, glutamine synthetase; GABA, ␥-aminobutyric acid; Glx, glutamate ϩ glutamine.…”

“…ϩ infusion or 14 NH 4 ϩ chase. With the anesthetized rat still breathing, the cranium was opened by scissor dissection and the brain was removed in toto and rapidly frozen in liquid nitrogen for preparation of a perchloric acid extract, as described previously (21).…”

“…15 N NMR was previously used for noninvasive monitoring of 15 N-metabolites to determine in vivo glutamine synthetase (GS) (19) and phosphate-activated glutaminase (20) [2][3][4][5][6][7][8][9][10][11][12][13][14][15] N]glutamine were quantified in vitro for better spectral resolution. The results are discussed in relation to the role of GDH in glutamate replenishment.…”

“…Biochemical and histochemical studies show higher GDH levels in neurons (10 -12), while immunocytochemical studies show highest GDH immunoreactivity in astrocytes (13) with only 15% reactivity in neurons (14). Moreover, a recent review of glutamate metabolism in mammalian brain (6) suggests that the rapid labeling of brain glutamate after intravenous injection of 14 C-labeled glucose (15,16) and the slow labeling of brain glutamate after short term 15 …”