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P Kugler

Publications and source records attributed to P Kugler.

At least 19 recordsLinked to original sources
PubMed2001

Glutamate transporter expression in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

The glutamate transporters GLT-1 and GLAST localized in astrocytes are essential in limiting transmitter signalling and restricting harmful receptor overstimulation. To show changes in the expression of both transporters following lesion of the entorhinal cortex (and degeneration of the glutamatergic tractus perforans), quantitative microscopic in situ hybridization (ISH) using alkaline-phosphatase-labelled oligonucleotide probes was applied to the outer molecular layer of the hippocampal dentate gyrus of rats (termination field of the tractus perforans). Four groups of rats were studied: sham-operated controls, and animals 3, 14 and 60 days following unilateral electrolytic lesion of the entorhinal cortex. The postlesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of quantitative ISH data. Statistical analysis revealed that ipsilateral to the lesion side there was a significant decrease of the GLT-1 mRNA at every postlesional time-point and of the GLAST mRNA at 14 and 60 days postlesion. The maximal decrease was approximately 45% for GLT-1 and approximately 35% for GLAST. In the terminal field of the perforant path contralateral to the lesion side, no significant changes of ISH labelling were measured. The results were complemented by immunocytochemical data achieved using antibodies against synthetic GLT-1 and GLAST peptides. In accordance with ISH results, there was an obvious decrease of GLT-1 and GLAST immunostaining in the terminal field of the perforant path ipsilateral to the lesion side. From these data we conclude that, following a lesioning of the entorhinal cortex, the loss of glutamatergic synapses in the terminal field of the perforant path resulted in a strong downregulation of glutamate transporters in astrocytes. The decrease of synaptically released glutamate or of other neuronal factors could be involved in this downregulation.

ATP-Binding Cassette Transporters↗
PubMed1999

Glutamate transporter EAAC1 is expressed in neurons and glial cells in the rat nervous system.

Oligonucleotide and cRNA probes were used for non-radioactive in situ hybridization, carried out to identify the cell types in the nervous system of rat expressing the glutamate transporter EAAC1 mRNA. The results were compared with immunocytochemical data obtained using an antibody against a synthetic EAAC1 peptide. The present data confirm that EAAC1 is expressed in neurons of the CNS. Additionally, our findings indicate the localization of EAAC1 mRNA and protein in peripheral neurons (spinal ganglia) and in glial cells, i.e., oligodendrocytes in various white matter regions of the CNS, ependymal cells, and epithelial cells of the plexus choroideus of the four ventricles, as well as in satellite cells of spinal ganglia. Immunolabeling revealed a preferentially cytoplasmic staining of neurons and glial cells. The cytoplasmic staining was frequently granular, suggesting a localization of EAAC1 protein in vesicle membranes. A membrane localization of EAAC1 was also indicated by Western blotting, which showed immunoreactivity only in the 100,000 x g pellet of brain homogenate. We conclude that the glutamate transporter EAAC1 is not restricted to neurons but may also play an important role in glial cells, particularly in oligodendrocytes.

Amino Acid Sequence↗
PubMed1999

Cellular and regional expression of glutamate dehydrogenase in the rat nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry.

In the central nervous system glutamate dehydrogenase appears to be strongly involved in the metabolism of transmitter glutamate and plays a role in the pathogenesis of neurodegenerative disorders. In order to identify unequivocally the neural cell types expressing this enzyme, non-radioactive in situ hybridization, using a complementary RNA probe and oligonucleotide probes, was applied to sections of the rat central nervous system and, for comparison with peripheral neural cells, to cervical spinal ganglia. The results were complemented by immunocytochemical studies using a polyclonal antibody against purified glutamate dehydrodenase. Glutamate dehydrogenase messenger RNA was detectable at varying amounts in neurons and glial cells (i.e. astrocytes, oligodendrocytes, Bergmann glia, ependymal cells, epithelial cells of the plexus choroideus) throughout the central nervous system and in neurons and satellite cells of spinal ganglia. In some neuronal populations (e.g., pyramidal cells of the hippocampus, motoneurons of the spinal cord and spinal ganglia neurons) messenger RNA-labelling was higher than in other central nervous system neurons. This is remarkable because the immunostaining of neurons in the central nervous system regions studied was at best weak, whereas a predominantly high level of immunoreactivity was detected in astrocytes (and Bergmann glia). Thus, in neurons of the central nervous system, the detected levels of glutamate dehydrogenase messenger RNA and protein seem to be at variance whereas in peripheral neurons of spinal ganglia both in situ hybridization labelling and immunostaining are intense.

Animals↗
PubMed1997

Cellular and regional distribution of the glutamate transporter GLAST in the CNS of rats: nonradioactive in situ hybridization and comparative immunocytochemistry.

Oligonucleotide and cRNA probes were used for nonradioactive in situ hybridizations carried out to identify the neural cell types expressing the glutamate transporter GLAST mRNA in the rat CNS. Additionally, the regional distribution of GLAST mRNA-expressing cells was studied, and the results were complemented by immunocytochemical investigations using an antibody against a synthetic GLAST peptide. The findings documented that GLAST is expressed by Bergmann glia and by astrocytes throughout the CNS. The glial localization of GLAST mRNA was verified unequivocally by double-labeling with an astrocytic marker protein. Additionally, GLAST mRNA reactivity and GLAST immunoreactivity were found in ependymal cells. In other neural cell types of the CNS, GLAST expression was not detectable. A high level of astrocytic immunolabeling was observed in the entire gray matter of the brain, with variations in intensity in different regions. Those brain areas that are known to possess high glutamatergic activity and astrocytic glutamate metabolism stained intensely for both GLAST mRNA and GLAST protein. The latter observation suggests that the GLAST glutamate transporter participates in the regulation of extracellular glutamate concentrations, especially in brain areas receiving an intense glutamatergic innervation.

Amino Acid Transport System X-AG↗
PubMed1996

Astrocytes and Bergmann glia as an important site of nitric oxide synthase I.

In the central nervous system nitric oxide appears to be critically involved in a number of physiological and pathological processes. Although there is convincing evidence for expression of nitric oxide synthase in cultured glial cells, demonstration of this enzyme in glial cells in situ remained largely unsatisfactory. In the present study we applied immunostaining to freeze-dried sections of snap-frozen hippocampi and cerebella of rats and to sections of freeze-dried brain tissue in order to minimize diffusion artefacts and thus to obtain more precise information about the true in situ localization of nitric oxide synthase. Here we show that astrocytes and Bergmann glia react strongly with antibodies raised against cerebellar nitric oxide synthase and against a type I nitric oxide synthase-specific C-terminal peptide, respectively. This finding was further substantiated by histochemical localization of NADPH-diaphorase activity in astrocytes and Bergmann glia as well as by immunoreactivity of both types of glia cells with antibodies to the NADPH-delivering enzyme glucose-6-phosphate dehydrogenase. We conclude, that astrocytes are important sites of nitric oxide synthase I in brain, suggesting that these cells might use nitric oxide as gaseous messenger molecule for various aspects of glia-neuron signalling.

Animals↗
PubMed1996

Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: non-radioactive in situ hybridization and comparative immunocytochemistry.

Non-radioactive in situ hybridization using complementary RNA and oligonucleotide probes was applied in order to clearly identify the cell types expressing GLT1 and to show their regional distribution in the central nervous system of the rat. The results were compared with immunocytochemical data achieved using an antibody against a synthetic GLT1 peptide. The study showed that GLT1 was expressed in astrocytes and Bergmann glia which were identified by the detection of an astrocytic marker protein. Additionally, subsets of neurons in different brain regions (e.g., CA3/4 pyramidal cells of the hippocampus, endopiriform nucleus) were labelled by in situ hybridization. In other cell types of the central nervous system (oligodendrocytes, ependymal cells, epithelal cells of the choroid plexus, tanycytes), GLT1 expression was not detectable. The generally dense astrocytic immunolabelling of the gray matter of the brain showed an even higher intensity in regions reported to show high glutamatergic activity and astrocytic glutamate metabolism (e.g., the termination field of the glutamatergic perforant path in the hippocampus). On the basis of the cellular regional distribution of the GLT1 messenger RNA and protein demonstrated in the present study, it is reasonable to assume that this high affinity transporter is of importance for the maintenance of adequate extraneuronal glutamate levels.

ATP-Binding Cassette Transporters↗
PubMed1995

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue.

In situ hybridization (ISH) of somatostatin (SOM) mRNA was carried out on sections of rat brain using an alkaline phosphatase (AP) coupled oligonucleotide probe. Different hybridization and AP development conditions were tested for qualitative and quantitative detection of target mRNA on sections of unfixed tissue. Hybridization signal intensities after 24 h of hybridization were high. Comparison with adjacent formaldehyde-fixed tissue sections and hybridization for various lengths of time (2-42 h) indicated that in unfixed tissue retention of SOM mRNA was at least as high as after fixation, and that the mRNA was not degraded during hybridization. The use of tetranitroblue instead of nitroblue tetrazolium chloride in the AP detection medium provided a superior signal-to-noise ratio, and medium stability was improved for quantitative studies on unfixed sections by adding 10% polyvinyl alcohol at pH 8.5. Microphotometric measurements of mean optical densities (MOD) of the formazan reaction product in a defined area within individual neurons of the lateral central amygdaloid nucleus showed a linear increase over the first 23 h of AP reaction time. The mean MOD values per neuron were comparably high in various equally thick sections of the nucleus and increased with section thickness in a linear manner. The findings indicate that the ISH and detection reagents penetrate the entire section and that there is a linear relationship between the amount of AP reaction product measured and the amount of mRNA present in the measured area. Thus, ISH using an AP-coupled oligonucleotide on sections of unfixed tissue appears suitable for quantitative mRNA detection.

Alkaline Phosphatase↗
PubMed1995

Glutamate dehydrogenase in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex.

Applying quantitative microscopic histochemistry, the activity of the mitochondrial glutamate dehydrogenase which is localized in astrocytes was determined in the molecular layer of the dentate gyrus of the rat hippocampus. This hippocampal region contains the important terminations of the glutamatergic perforant path. For comparison, determinations of the mitochondrial succinate dehydrogenase were performed, which is localized preferentially in terminals and dendrites. Two age groups of animals were examined: young adults (three months old) and aged subjects (24 months old). Both age groups were divided into controls, and animals killed three, 21 and 90 days following unilateral electrolytic lesion of the entorhinal cortex. The post-lesional shrinkage of the terminal field of the perforant path, ipsilateral to the lesion side, was determined and considered in the evaluation of enzymatic data. Statistic analysis revealed that ipsilateral to the lesion side there was a significant decrease of glutamate and succinate dehydrogenase activities in the terminal field of the perforant path three, 21 and 90 days following lesion. It is reasonable to assume that the decrease of succinate dehydrogenase activity (50-60%) was caused by the loss of mitochondria localized in degenerating terminals, whereas the decrease of glutamate dehydrogenase activity (20-30%) was related to the decrease of glutamatergic transmission following lesion. In the terminal field of the perforant path contralateral to the lesion side both significant increases and decreases of enzyme activities were measured following lesion. From these results it is concluded that the hippocampus contralateral to the lesion side cannot be considered as an appropriate intraindividual control. The comparison between young and aged animals showed no differences in the demonstration of glutamate dehydrogenase and only restricted differences in the activity level of succinate dehydrogenase post-lesion. Therefore, it is reasonable to assume that the post-lesional reactivity of the enzymes studied was very similar in both age groups.

Age Factors↗
PubMed1994

Glucose-6-phosphate dehydrogenase is enriched in oligodendrocytes of the rat spinal cord. Enzyme histochemical and immunocytochemical studies.

Glucose-6-phosphate dehydrogenase (G6PD) was localized in rat spinal cord by catalytic enzyme histochemistry and immunocytochemistry. G6PD detected by either method was shown to be strongly enriched in cell bodies and processes of oligodendrocytes, whereas in the compact myelin G6PD was not detected. The enzyme histochemical procedure for the demonstration of G6PD was also adapted for microphotometric measurements of G6PD activity in the spinal cord white matter. There was a linear relationship between G6PD activity and section thickness up to 14 microns and between G6PD activity and reaction time up to 5-6 min as demonstrated by kinetic and end-point measurements. Significantly lower activities were measured in end-point measurements than in kinetic measurements because of formazan loss during rinsing. Methoxyphenazine methosulphate as an exogenous electron carrier and sodium azide as a blocker of the respiratory chain significantly increased the demonstrable G6PD activity. The Km was 0.62 mM and the Vmax 3 mumol glucose-6-phosphate/cm3 wet tissue and per min at 25 degrees C. It is concluded that G6PD in oligodendrocytes may be important for the generation of NADPH required for lipid biosynthesis related to myelogenesis, and reduction of glutathione required for protection of membrane sulphydryl groups.

Animals↗
PubMed1994

Nitric oxide synthase and NADP-linked glucose-6-phosphate dehydrogenase are co-localized in brush cells of rat stomach and pancreas.

The epithelia of the respiratory and gastrointestinal tract and their appendages contain a distinct population of disseminated epithelial cells called brush cells or caveolated cells. On the basis of their structure, it was suggested that brush cells might serve as chemo- or volume receptors that play a role in certain aspects of gastrointestinal and bronchopulmonary secretion or motility. In the present study we provide first clues to a possible function of this widespread epithelial cell type. Brush cells of the rat gastric cardia and major pancreatic duct display strong immunoreactivity for nitric oxide synthase (NOS) and also exhibit high activity of NADPH-diaphorase. This NADPH-oxidizing activity was previously shown to be mediated by a specific domain of the sequence of the NOS. NADPH, in turn, appears to be delivered by glucose-6-phosphate dehydrogenase, which we found in brush cells at particularly high levels. We conclude that brush cells of the stomach and pancreas may represent a specialized population of paracrine cells that use nitric oxide as a messenger molecule to control certain gastrointestinal functions.

Amino Acid Oxidoreductases↗
PubMed1993

In situ measurements of enzyme activities in the brain.

The present review focuses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver alpha-ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of alpha-ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires alpha-ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.

4-Aminobutyrate Transaminase↗
PubMed1993

Acetylcholinesterase activity and post-lesional plasticity in the hippocampus of young and aged rats.

Applying quantitative microscopic histochemistry, the activity of acetylcholinesterase was determined in the various layers of the rat hippocampus at three different levels along the rostrocaudal extent. Two age groups of animals were examined: young adults (two to three months old) and aged subjects (26 months old). Young adults were divided into controls, and animals killed eight and 35 days following bilateral ibotenate lesioning of the medial septum-diagonal band complex. Aged rats were divided into controls and animals 35 days post-lesion. Analysis of variance revealed that the mean acetylcholinesterase activities of the entire hippocampus of individuals were not significantly different between young and aged rats when averaged across controls and 35 days post-lesion. There was a significant decrease of acetylcholinesterase activity (-52%) in young adults eight days post-lesion as compared to controls, but a significant increase (+63%) took place until 35 days post-lesion as compared to eight days post-lesion. Significantly lower activities existed, however, in young (-22%) and aged rats (-18%) 35 days post-lesion as compared to controls. This decrease in mean activity was not age dependent. As acetylcholinesterase is considered to be a good cholinergic indicator in the hippocampus, the results suggest a homotypic collateral sprouting from spared cholinergic afferents following ibotenate lesion of the medial septum-diagonal band complex in young and aged rats. Based on the data obtained, it is reasonable to assume that there was no difference in the post-lesional plasticity of neuronal acetylcholinesterase between young adult and aged rats.

Acetylcholinesterase↗
PubMed1992

Mitochondrial enzymes related to glutamate and GABA metabolism in the hippocampus of young and aged rats: a quantitative histochemical study.

Quantitative histochemistry (scanning microphotometry) was used to determine the activities of the mitochondrial enzymes NAD-linked isocitrate dehydrogenase (EC 1.1.1.41), L-glutamate dehydrogenase (EC 1.4.1.3) and GABA transaminase (EC 2.6.1.19) in various layers of the hippocampus (middle one third) of young (3-4 months old) and memory-impaired aged rats (28-30 months old). For comparison, determinations of cytochrome c oxidase (EC 1.9.3.1) as a marker for mitochondria and energy metabolism were also performed. The study showed that there was a layered reaction pattern in the hippocampus and that the cellular distribution and the levels of enzyme activity were different. However, the activities of the different enzymes (excepting GABA transaminase and cytochrome c oxidase) were significantly correlated in the hippocampus in both age groups. Age-dependent changes were only observed for NAD-linked isocitrate dehydrogenase and GABA transaminase (significant increases of activities in some layers of the hippocampus, preferentially in the terminal field of the perforant path). From the present study it is concluded that, 1. the enzymatic complement of mitochondria in neurons and glia depends upon layer specific metabolic processes of the hippocampus (also with respect to glutamatergic and GABAergic terminal fields) indicating a layer specific interaction of the enzymes studied to produce or catabolize glutamate and GABA, and 2. the age dependent changes of the studied enzymes are very restricted.

4-Aminobutyrate Transaminase↗
PubMed1991

Histochemistry of glutamate metabolizing enzymes in the rat cerebellar cortex.

Applying catalytic enzyme histochemistry, glutamate dehydrogenase (GDH) and phosphate activated glutaminase (PAG) were demonstrated at the light microscopic level, and aspartate aminotransferase (AAT) was detected at the electron microscopic level. GDH staining appeared preferentially in glial cells (Bergmann glia and astrocytes), whereas AAT was localized only in neuronal structures. Cytoplasmic AAT was demonstrated in the perikarya and terminal plexus of basket cells, in the perikarya of stellate cells, in about 60% of the granule cells, in mossy fiber boutons, in numerous small boutons in the molecular layer, and in the axoplasm of numerous myelinated and unmyelinated fibers. PAG was observed in both neuronal structures (perikarya of granule and Purkinje cells) and in astrocytes and Bergmann glia.

Animals↗
PubMed1991

Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases.

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.

Animals↗
PubMed1991

Microphotometric determination of enzymes in brain sections. IV. Isocitrate dehydrogenases.

A polyvinyl alcohol-(PVA) containing incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of NAD- and NADP-linked isocitrate dehydrogenases (ICDHs) in cryostat sections of the rat hippocampus. The following incubation medium is recommended for the quantification of NAD- and NADP- (differences in brackets) ICDHs: 100 mM DL-isocitrate, 10 mM sodium azide, 5 mM (4 mM) nitroblue tetrazolium (NBT), 7 mM NAD (4 mM NADP), 10 mM magnesium chloride, 0.25 mM phenazine methosulfate (PMS), with or without 5 mM ADP (without ADP), 23% PVA in 0.05 M Hepes buffer; the final pH was 7.5. With these incubation media a linear response of the reactions lasted at least 20 min. In kinetic and end-point measurements the same level of activities was demonstrable. The use of NaN3 (as a blocker of the respiratory chain) and PMS (as artificial electron carrier) was indispensible for the transfer of all reduction equivalents in the dehydrogenase reactions to the tetrazolium salt NBT. Furthermore, the activation by magnesium ions and the need of PVA to avoid diffusion artefacts of the loosely bound ICDHs were clearly shown. It is concluded that the quantification of ICDHs in situ could be a valuable tool for neurochemical investigations because ICDHs play a role not only in the substrate flux through the tricarboxylic acid cycle but also in providing alpha-ketoglutarate for the formation of glutamate which is an important amino acid in the brain.

Animals↗