Co-localization of Glutamic Acid Decarboxylase and Phosphate-activated Glutaminase in Neurons of Lateral Reticular Nucleus in Feline Thalamus
ABSTRACT Immunohistochemical methods were used to label singly and/or in combination glutamic acid decarboxylase (GAD, the sole synthesizing enzyme for the inhibitory neurotransmitter gamma-aminobutyric acid) and phosphate-activated glutaminase (GLN, a synthesizing enzyme for glutamate) in neurons of lateral reticular nucleus (LRN) of thalamus of adult cats. (1) GAD- and GLN-immunoreactivity (IR) exhibited matching regional patterns of organization within LRN. (2) GAD- and GLN-IR co-localized within most if not all LRN neuronal cell bodies as shown by light microscopy. (3) GAD- and GLN-IR had distinct subcellular localizations in LRN neurons as shown by correlative light/electron microscopy. LRN neurons are important conceptual models where strongly inhibitory cells receive predominant excitatory glutamatergic afferents (from neocortex). Consistent with known actions of intermediary astrocytes, LRN neurons demonstrate GLN enrichment synergistically coupled with glutamatergic innervation to supplement the glutamate pool for GABA synthesis (via GAD) and for metabolic utilization (via the GABA shunt/tricarboxylic acid cycle) but not, apparently, for excitatory neurotransmission.
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- "Furthermore, in this work novel data support expression of both isoforms in a scarcity of Purkinje cells and in many GABAergic neurons at the molecular layer of the cerebellum, which point toward GA as an important biosynthetic source of Glu at inhibitory synapses indeed, even though early studies did not detect KGA immunoreactivity in GABAergic neurons of neocortex from rodents and monkey (Kaneko et al., 1992; Kaneko and Mitzuno, 1994; Altschuler et al., 1985, 1984; Donoghue et al., 1985). However , more recent studies presented immunocytochemical evidence of the localization of KGA in GABAergic neurons in the cat visual cortex (Van der Gucht et al., 2003) and thalamus (Fisher, 2007), whereby suggesting that Gln can be a metabolic precursor for GABA synthesis. Interestingly, coexpression of KGA and glutamic acid decarboxylase (GAD; EC 22.214.171.124) "
ABSTRACT: The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes. GLIA 2014Glia 03/2015; 63(3):365-382. DOI:10.1002/glia.22758 · 6.03 Impact Factor
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ABSTRACT: A defining histopathologic feature of Taylor-type cortical dysplasia (CD) is the presence of cytomegalic neurons and balloon cells. Most cytomegalic neurons appear to be pyramidal-shaped and glutamatergic. The present study demonstrates the presence of cytomegalic GABAergic interneurons in a subset of pediatric patients with severe CD. Cortical tissue samples from children with mild, severe, and non-CD pathologies were examined using morphologic and electrophysiologic techniques. By using in vitro slices, cytomegalic cells with characteristics consistent with interneurons were found in 6 of 10 patients with severe CD. Biocytin labeling demonstrated that cytomegalic interneurons had more dendrites than normal-appearing interneurons. Whole-cell patch clamp recordings showed that cytomegalic interneurons had increased membrane capacitance and time constant compared with normal-appearing interneurons. They also displayed signs of cellular hyperexcitability, evidenced by increased firing rates, decreased action potential inactivation, and the occurrence of spontaneous membrane depolarizations. Single-cell reverse transcription-polymerase chain reaction and immunohistochemistry for GABAergic markers provided further evidence that these cells were probably cytomegalic interneurons. The pathophysiologic significance of GABAergic cytomegalic interneurons in severe CD tissue is unknown, but they could inhibit glutamatergic cytomegalic pyramidal neurons, or contribute to the synchronization of neuronal networks and the propagation of ictal activity in a subset of pediatric patients with severe CD.Journal of Neuropathology and Experimental Neurology 07/2007; 66(6):491-504. DOI:10.1097/01.jnen.0000240473.50661.d8 · 4.37 Impact Factor
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ABSTRACT: A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.Neurochemical Research 08/2008; 33(7):1341-5. DOI:10.1007/s11064-008-9589-9 · 2.55 Impact Factor