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J H Laake

Publications and source records attributed to J H Laake.

25 records · Page 2Linked to original sources
PubMed1995

Glutamine from glial cells is essential for the maintenance of the nerve terminal pool of glutamate: immunogold evidence from hippocampal slice cultures.

The immunogold labeling for glutamate and glutamine was studied at the electron microscopic level in hippocampal slice cultures following inhibition of L-glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming); EC 6.3.1.2]. In control cultures, glutamate-like immunoreactivity was highest in terminals, intermediate in pyramidal cell bodies, and low in glial cells. Glutamine-like immunoreactivity was high in glial cells, intermediate in pyramidal cell bodies, and low in terminals. After inhibition of glutamine synthetase with L-methionine sulfoximine, glutamate-like immunoreactivity was reduced by 52% in terminals and increased nearly four-fold in glia. Glutamine-like immunoreactivity was reduced by 66% in glia following L-methionine sulfoximine, but changed little in other compartments. In cultures that were treated with both L-methionine sulfoximine and glutamine (1.0 mM), glutamate-like immunoreactivity was maintained at control levels in terminals, whereas in glia glutamate-like immunoreactivity was increased and glutamine-like immunoreactivity was decreased to a similar extent as in cultures treated with L-methionine sulfoximine alone. We conclude that (a) glutamate accumulates in glia when the flux through glutamine synthetase is blocked, emphasizing the importance of this pathway for the handling of glutamate; and (b) glutamine is necessary for the maintenance of a normal level of glutamate in terminals, and neither reuptake nor de novo synthesis through pathways other than the glutaminase reaction is sufficient.

Animals↗
PubMed1993

Redistribution of glutamate and glutamine in slices of human neocortex exposed to combined hypoxia and glucose deprivation in vitro.

This study was undertaken to elucidate the roles of neurons and glial cells in the handling of glutamate and glutamine, a glutamate precursor, during cerebral ischemia. Slices (400-600 microns) from human neocortex obtained during surgery for epilepsy or brain tumors were incubated in artificial cerebrospinal fluid and subjected to 30 min of combined hypoxia and glucose deprivation (an in vitro model of brain ischemia). These slices, and control slices that had not been subjected to "ischemic" conditions, were then fixed and embedded. Ultrathin sections were processed according to a postembedding immunocytochemical method with polyclonal antibodies raised against glutamate or glutamine, followed by colloidal gold-labeled secondary antibodies. The gold particle densities over various tissue profiles were calculated from electron micrographs using a specially designed computer program. Combined hypoxia and glucose deprivation caused a reduced glutamate immunolabeling in neuronal somata, while that of glial processes increased. Following 1 h of recovery, the glutamate labeling of neuronal somata declined further to very low values, compared to control slices. The glutamate labeling of glial cells returned to normal levels following recovery. In axon terminals, no consistent change in the level of glutamate immunolabeling was observed. Immunolabeling of glutamine was low in both nerve terminals and neuronal somata in normal slices and was reduced to nondetectable levels in nerve terminals upon hypoxia and glucose deprivation. This treatment was also associated with a reduced glutamine immunolabeling in glial cells. Reversed glutamate uptake due to perturbations of the transmembrane ion concentrations and membrane potential probably contributes to the loss of neuronal glutamate under "ischemic" conditions. The increased glutamate labeling of glial cells under the same conditions can best be explained by assuming that glial cells resist a reversal of glutamate uptake, and that their ability to convert glutamate into glutamine is compromised due to the energy failure. The persistence of a nerve terminal pool of glutamate is compatible with recent biochemical data indicating that the exocytotic glutamate release is contingent on an adequate energy supply and therefore impeded during ischemia.

Brain Ischemia↗
PubMed1992

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei.

A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K+, 20 min) in the presence of a physiological or low Ca(2+)-concentration. Depolarization at physiological [Ca2+] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca2+ (10 mM Mg2+ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca(2+)-dependent component of glutamate release. By means of a double-labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate-like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca(2+)-dependent depletion of GABA-like immunoreactivity, but failed to change the level of glutamate-like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA-releasing terminals also contain a non-releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.

Animals↗
PubMed1990

Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis).

A post-embedding immunogold procedure was used to analyse, in a semiquantitative manner, the distributions of aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system in rats and baboons. The neurons in the inferior olive were uniformly labelled for aspartate as well as glutamate, indicating a 100% co-localization of these two amino acids in the cell bodies. The level of glutamate-like immunoreactivity in the climbing fibre terminals was similar to that in the parent cell bodies, as judged by a computer-assisted calculation of gold particle densities. In contrast, the level of aspartate-like immunoreactivity in the climbing fibre terminals was only one-seventh of that of the olivary neurons. No differences were found between the hemispheres and vermis. Nerve terminals in the inferior olive were generally moderately labelled with the aspartate antiserum, as were cell bodies of astrocytes. With a few exceptions, the results obtained in baboons were similar to those in rats. Notably, no evidence was found of an enrichment of aspartate-like immunoreactivity in climbing fibres. The present results do not support previous data suggesting that aspartate is the transmitter of the climbing fibres but indicate that glutamate or another excitatory compound should be considered as candidate for this role. Our findings show that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.

Afferent Pathways↗
PubMed1990

Demonstration of a releasable pool of glutamate in cerebellar mossy and parallel fibre terminals by means of light and electron microscopic immunocytochemistry.

The chemical substance(s) responsible for the fast signalling in the mossy fibre to granule cell synapses in the cerebellum has not been identified, although recent studies suggest that glutamate is a strong candidate. In the present investigation, this issue was explored by means of a quantitative electron microscopic immunocytochemical procedure. Ultrathin sections of plastic-embedded rat cerebella were treated with an antiserum specific for glutaraldehyde-fixed glutamate, followed by a secondary antibody coupled to colloidal gold particles. The gold particle density over mossy fibre terminals was assessed in tissue that had been rapidly fixed by perfusion, as well as in tissue that had been incubated in artificial cerebrospinal fluid in vitro before immersion fixation. In both preparations the mossy fibres appeared as the most intensely glutamate-immunoreactive profile type in the cerebellar cortex, and the parallel fibre terminals were also strongly labelled. Corresponding results were obtained at the light microscopic level. Most of the immunoreactivity in the mossy and parallel fibre terminals could be depleted in a Ca(+)-dependent manner by depolarization with a high K+ concentration. These data suggest that the mossy and parallel fibre terminals contain a glutamate pool that behaves as a transmitter pool.

Animals↗
PubMed1986

Metabolism and transport of amino acids studied by immunocytochemistry.

The immunocytochemical method for demonstrating amino acids makes it possible to study accumulation and depletion of amino acids in individual tissue compartments resulting from experimental manipulations. We have incubated hippocampal slices in oxygenated Krebs solution, containing various additives, under basal conditions and during synaptic release of transmitters evoked by elevated K+ concentrations or by veratrine. Immunoreactivities for glutamate (Glu-LI), aspartate (Asp-LI), glutamine (Gln-LI), gamma-amino-butyrate (GABA-LI) and taurine (Tau-LI) have been demonstrated by specific antibodies after fixation of the slices in glutaraldehyde. Prolonged depolarisation depleted Glu-LI, Asp-LI and Gln-LI from nerve-ending-like structures. GABA-LI was less affected and Tau-LI not affected at all. The depletion of immunoreactivities could be prevented by metabolic precursors of transmitter amino acids, notably glutamine. This effect of glutamine was abolished by inhibiting glutaminase with diazooxonorleucine. Glu-LI, Asp-LI, GABA-LI and Gln-LI accumulated in astroglial cells during conditions of prolonged depolarization-induced release. The accumulation of GABA-LI in glia was strongly increased by inhibition of aminotransferases by aminooxyacetic acid. The described changes in Glu-LI were prevented by low Ca2+/high Mg2+, and promoted when the glial enzyme glutamine synthetase was inhibited by methionine sulfoximine. D-Aspartate, a metabolically inert competitive inhibitor/substrate for high affinity uptake of glutamate, inhibited the accumulation of Glu-LI in glia. The results confirm the biochemically derived theories on metabolic compartmentation in nervous tissue, and add knowledge on the dynamics of the cellular distribution of amino acids. They also indicate the possibilities offered by the present approach for studying metabolism and pharmacology at the cellular level.

Amino Acids↗