Jon H. Laake

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

Abstract: 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 fourfold 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.

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

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.

Discrete cellular and subcellular localization of glutamine synthetase and the glutamate transporter GLAST in the rat vestibular end organ.

Glial cells play an important role in the removal and metabolism of synaptically released glutamate in the central nervous system (CNS). It is not clear how glutamate is handled at peripheral glutamate synapses, which are not associated with glia. Glutamate is a likely transmitter in the synapse between the hair cells and afferent dendrites of the vestibular end organ. Immunocytochemistry was performed to investigate the distribution at this site of the high affinity glutamate transporter GLAST and glutamate metabolizing enzyme glutamine synthetase. Confocal microscopy revealed that GLAST and glutamine synthetase were co-localized in supporting cells apposed to the immunonegative hair cells. Postembedding immunoelectron microscopy revealed that GLAST was heterogeneously distributed along the plasma membranes of the supporting cells, with higher concentrations basally (at the level of the afferent synapses) than apically. Both immunoreactivities were also present in non-neuronal cells in the vestibular ganglion. The present findings suggest that glutamate released at the afferent synapse of vestibular hair cells may be taken up by adjacent supporting cells and converted into glutamine. Thus, at this peripheral synapse, the supporting cells may carry out functions similar to those of glial cells in the CNS.

A quantitative electron microscopic immunocytochemical study of the distribution and synaptic handling of glutamate in rat hippocampus.

One of the major problems in glutamate immunocytochemistry has been the difficulty involved in separating immunocytochemical labelling due to metabolic glutamate from the labelling caused by transmitter glutamate. Another problem appears to be the accessibility of antigenic sites in conventional light microscopic preparations. In the present report, we have applied the primary glutamate antiserum onto ultrathin tissue sections, followed by the use of a colloidal gold detection system. The use of this postembedding immunogold procedure allows equal access of antibodies to all cellular compartments exposed at the section surface, allows quantitative assessment of the immunoreactivity, and affords a high resolution compatible with studies at the organelle level. When applied to slice preparations the immunogold procedure can be used to identify releasable pools of glutamate. These methodological advances have greatly increased the usefulness of glutamate immunocytochemistry as a tool to study putative glutamatergic terminals in the CNS.

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 μm) 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.

Distribution of glutamine-like immunoreactivity in the cerebellum of rat and baboon (Papio anubis) with reference to the issue of metabolic compartmentation

SummaryThe cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehydefixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabelled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.

Chapter 6 Molecular organization of cerebellar glutamate synapses

Publisher Summary This chapter discusses recent data on the synaptic localization of glutamate receptors and transporters, of glutamate itself, and of glutamine—the major precursor and metabolite of glutamate. Glutamate is the most prevalent excitatory transmitter in the cerebellum and mediates fast transmission in parallel and mossy fiber synapses and probably also in the third major excitatory fiber system formed by the climbing fibers. The operation of a glutamate–glutamine cycle requires that the glial cells be equipped with uptake mechanisms for glutamate. Several glutamate transporters have been cloned in recent years. The organization of key molecules at glutamatergic synapses in the rat cerebellar cortex is analyzed by high resolution immunocytochemical techniques using gold particles as markers. The distinct compartmentation of glutamate and glutamine is consistent with biochemical data indicating an active role of glia in the removal of released glutamate and in the supply of glutamine for de novo synthesis of transmitter glutamate.

Phosphate activated glutaminase is concentrated in mitochondria of sensory hair cells in rat inner ear: a high resolution immunogold study

Glutamate has been implicated in signal transmission between sensory hair cells and afferent fibers in the inner ear. However, the mechanisms responsible for glutamate replenishment in these cells are not known. Here we provide evidence that phosphate activated glutaminase, which is thought to be the predominant glutamate-synthesizing enzyme in the brain, is concentrated in all types of hair cell in the organ of Corti and vestibular epithelium. By use of two different antibodies (directed to the N and C terminus, respectively) it was shown that glutaminase is largely restricted to mitochondria and that part of the enzyme pool is associated with the inner membrane of this organelle. Quantitative analysis of immunogold labelled Lowicryl sections revealed that the level of glutaminase immunoreactivity in mitochondria of supporting cells is less than 15% of that in hair cell mitochondria. Using triple labelling for glutaminase, glutamate, and glutamine, evidence was provided of a positive correlation between the glutamate/glutamine ratio and the level of glutaminase immunoreactivity, suggesting that the glutaminase antibodies identify a functional enzyme pool. Our results strengthen the idea that glutamate is a hair cell transmitter and indicate that the sensory epithelia in the inner ear show a metabolic compartmentation analogous to that in the brain.

Light and Electron Microscopic Immunocytochemistry of Putative Neurotransmitter Amino Acids in the Cerebellum with Some Observations on the Distribution of Glutamine

Ramon y Cajal (1888, 1889) was the first to describe accurately the different cell types in the cerebellum and their interconnections. With the development of the electron microscope, the ultra-structural features of the different types of cells and synapses were soon characterized in great detail, so that today the synaptology of the cerebellar cortex must be regarded as well established (Mugnaini, 1972; Palay and Chan-Palay, 1974). In contrast, our understanding of the chemical nature of the cerebellar circuitries is still incomplete. Early biochemical studies and investigations based on immunocytochemistry of the gamma-aminobutyric acid (GABA) synthesizing enzyme, glutamic acid decarboxylase (GAD), strongly suggested that amino acids played major roles as transmitters in the cerebellum, as in other parts of the central nervous system (CNS) (for reviews see Mugnaini and Oertel, 1985; Ottersen and Storm-Mathisen, 1984a). However, it was not until recently that the neuroactive amino acids themselves could be visualized by immunocytochemistry (Storm-Mathisen et al, 1983), thus becoming amenable to precise anatomical analysis. In this chapter we show how amino acid immunocytochemistry has provided new insight in the organization of the amino acid transmitter systems in the cerebellum.