Expression of high-affinity neuronal and glial glutamate transporters in the rat optic nerve

Recent studies have revealed that a dynamic axon-glial signaling occurs in the rat optic nerve, which is devoid of synapses. This interaction is postulated to be mediated by non-vesicular release of glutamate via a reversal of high-affinity glutamate transporters. Here we examined the expression of glial glutamate transporters (GLAST and GLT-1) and a neuronal transporter (EAAC1) in the rat optic nerve. RT-PCR analysis revealed the presence of mRNAs for GLT-1 and GLAST, but not EAAC1. RNase protection assays showed that of the two glial transporters, mRNA for GLAST was expressed at much higher level than was GLT-1. A similar expression pattern was found in primary astrocyte culture cells. GLAST mRNA level in the optic nerve was comparable to that in the cerebellum. Developmentally, GLAST mRNA level was highest at P2 and dropped slightly by adulthood. Nerve transection resulted in little or no change in mRNA levels for GLAST and GLT-1 assayed at 4 to 14 days post-transection, but GLAST mRNA level was decreased at 64 days. Western blot analysis revealed that the rat optic nerve showed immunoreactivity to antibodies against GLT-1, GLAST, and EAAC1. In conclusion, we suggest that glial and neuronal transporters are present in the rat optic nerve, where dynamic axon-glial interaction has been known to occur. In particular, the unusually high level of expression of GLAST in the optic nerve suggests a possible role for this glial transporter in protecting optic nerves from neurotoxicity during postnatal development.     

AMPA receptor protein in developing rat brain: glutamate receptor-1 expression and localization change at regional, cellular, and subcellular levels with maturation

We tested the hypothesis that the regional, cellular, and synaptic localizations of the glutamate receptor 1 (GluR 1) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor are regulated developmentally in rat brain. By immunoblotting, GluR1 was first detected in whole brain at embryonic day E15.5, and levels increased progressively during late embryonic (E20) and early postnatal (P2-P11) days. Regionally, GluR1 increased in cerebral cortex but decreased in striatum with postnatal maturation. These changes occurred in the presence of increased presynaptic maturation, as determined by synaptophysin detection. By immunocytochemistry, distinct cellular populations showed different temporal profiles of GluR1 expression during postnatal maturation. The neocortex and hippocampus showed a progressive maturation-related enrichment of GluR1, whereas the striatum showed a gradual reduction in GluR1 during maturation. In cerebellum, GluR1 protein was expressed transiently at restricted times postnatally by granule cells (P0-P11) and Purkinje cells (P13-P19), but by P21 and thereafter these neurons had sparse GluR1 immunoreactivity. By immunoelectron microscopy. GluR1 was found in neurites, specifically in both dendritic and axon terminal components of developing synapses. GluR1 was clustered at the plasma membrane of apparent growth cone appositions, neuronal cell bodies, and dendrites of developing neurons. The presence of GluR1 at presynaptic sites dissipated with synaptic maturation, as GluR1 became confined to the somatodendritic compartment as maturation progressed. We conclude that the regional expression as well as the cellular and synaptic localizations of the GluR1 are developmentally regulated and are different in immature and mature brain. Differences in glutamate receptor expression and synaptic localization in immature and mature brain may be relevant to the phenomenon that the perinatal and adult brain differ in their regional vulnerability to hypoxia-ischemia and excitotoxicity.     

Neuronal high-affinity glutamate transport in the rat central nervous system

EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.     

Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration?

Increasing evidence indicates that glutamate transporters are vulnerable to the action of biological oxidants, resulting in reduced uptake function. This effect could contribute to the build-up of neurotoxic extracellular glutamate levels, with major pathological consequences. Specific 'redox-sensing' elements, consisting of cysteine residues, have been identified in the structures of at least three transporter subtypes (GLT1, GLAST and EAAC1) and shown to regulate transport rate via thiol-disulphide redox interconversion. In this article, Davide Trotti, Niels Danbolt and Andrea Volterra discuss these findings in relation to the emerging view that in brain diseases oxidative and excitotoxic mechanisms might often operate in tight conjunction to induce neuronal damage. In particular, they review evidence suggesting a possible involvement of oxidative alterations of glutamate transporters in specific pathologies, including amyotrophic lateral sclerosis, Alzheimer's disease, brain trauma and ischaemia.     

Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression

The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, 35S-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.     

Distinct ontogeny of glucocorticoid and mineralocorticoid receptor and 11beta-hydroxysteroid dehydrogenase types I and II mRNAs in the fetal rat brain suggest a complex control of glucocorticoid actions

Glucocorticoids (GCs) act via intracellular mineralocorticoid (MR) and glucocorticoid receptors (GR). However, it has recently been recognized that GC access to receptors is determined by the presence of tissue-specific 11beta-hydroxysteroid dehydrogenases (11beta-HSDs) that catalyze the interconversion of active corticosterone and inert 11-dehydrocorticosterone. 11beta-HSD type 1 (11beta-HSD1) is a bidirectional enzyme in vitro that acts predominantly as a reductase (regenerating corticosterone) in intact neurons. In contrast, 11beta-HSD type 2 (11beta-HSD2) is a higher affinity exclusive dehydrogenase that excludes GCs from MR in the kidney, producing aldosterone-selectivity in vivo. We have examined the ontogeny of 11beta-HSD mRNAs and enzyme activity during prenatal brain development and correlated this with GR and MR mRNA development. These data reveal that (1) 11beta-HSD2 mRNA is highly expressed in all CNS regions during midgestation, but expression is dramatically reduced during the third trimester except in the thalamus and cerebellum; (2) 11beta-HSD2-like activity parallels closely the pattern of mRNA expression; (3) 11beta-HSD1 mRNA is absent from the CNS until the the third trimester, and activity is low or undectectable; and (4) GR mRNA is highly expressed throughout the brain from midgestation, but MR gene expression is absent until the last few days of gestation. High 11beta-HSD2 at midgestation may protect the developing brain from activation of GR by GCs. Late in gestation, repression of 11beta-HSD2 gene expression may allow increasing GC activation of GR and MR, permitting key GC-dependent neuronal and glial maturational events.     

Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N-terminal and central parts), GLAST (C-terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)-triphosphate (IP3)-receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na+-dependent "binding" of D-[3H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N-terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT.     

Localization of neuronal and glial glutamate transporters

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.     

Prostaglandin-H synthase-1 (PGHS-1) gene is expressed in specific neurons of the brain of the late gestation ovine fetus

Prostaglandin (PG) E2 acts on the brain stem to modulate breathing activity in the ovine fetus. The source of this PGE2 is unknown and we hypothesized that it is produced locally in the developing brain and functions in a paracrine and/or autocrine manner. The purpose of the present study was to establish whether prostaglandin-H synthase-1 (PGHS-1), a crucial enzyme in de novo prostaglandin synthesis, is present and its gene expressed in the ovine fetal brain. Immunohistochemical and molecular hybridization techniques were used to identify sites of PGHS-1 immunoreactivity and PGHS-1 mRNA expression respectively in the brain of the ovine fetus in late gestation (approximately 126 days gestation, term 145 days). PGHS-1 immunoreactivity was localized to specific regions of the fetal brain, including the cortex, hypothalamus, hippocampal formation, superior colliculus of the midbrain, parabrachial nucleus of the pons, and the reticular formation, raphe, nucleus of the solitary tract, and gracile and cuneate nuclei of the medulla. The relative abundance of PGHS-1 mRNA in selected brain regions, as determined by Northern blot analysis, correlated qualitatively with the number of PGHS-1 immunoreactive neurons identified in each region. In situ hybridization demonstrated PGHS-1 mRNA to be localized in the same neurons or nuclei as PGHS-1 immunoreactivity. These results indicate that PGHS-1 synthesized de novo in many brain regions including two that are important in respiratory control: the pneumotaxic center (parabrachial nucleus) and the dorsal respiratory group (nucleus tractus solitarius) suggesting that prostaglandins that modulate fetal respiratory activity are synthesized endogenously.     

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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.     

Characterization of glucose transporter isoforms in the adult and developing human eye

The expression of glucose transporter isoforms (Glut 1, Glut 3, Glut 4, and Glut 5) in the human eye was investigated at various ages ranging between 8 weeks gestation (first trimester) and adult using Western blot and immunohistochemical analyses. Glut 1 and Glut 3 expression and cellular localization patterns were similar to those of human brain. Glut 1 (50-kilodalton protein) was expressed by epithelial cells (retinal pigmented epithelium, choroidal, iridial, and pars planus), which form the blood-eye barrier, retinal Mueller cells, the lens fiber cells, iridial microvascular endothelial cells, and to a lesser extent by the outer segments of the photoreceptor cells in the adult eye. This pattern was conserved throughout development and was evident as early as 8 weeks gestation. In addition, the endothelial cells of vitreous hyaloid vessels expressed Glut 1 at 8 weeks gestation. Glut 3 (50 to 55-kilodalton protein) immunoreactivity was observed only in the adult inner synaptic layer of the retina. Neither Glut 4 nor Glut 5 was expressed in any occular tissue at any age examined. These results suggest that Glut 1 is the main glucose transporter of the human eye and that it is ontogenically conserved. In contrast, Glut 3 is associated with selective neuronal processes, and its expression is developmentally altered.     

The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain

The role of transporters in shaping the glutamate concentration in the extracellular space after synaptic release is controversial because of their slow cycling and because diffusion alone gives a rapid removal. The transporter densities have been measured electrophysiologically, but these data are from immature brains and do not give precise information on the concentrations of the individual transporter subtypes. Here we show by quantitative immunoblotting that the numbers of the astroglial glutamate transporters GLAST (EAAT1) and GLT (EAAT2) are 3200 and 12,000 per micrometer3 tissue in the stratum radiatum of adult rat hippocampus (CA1) and 18,000 and 2800 in the cerebellar molecular layer, respectively. The total astroglial cell surface is 1.4 and 3.8 m2/cm3 in the two regions, respectively, implying average densities of GLAST and GLT molecules in the membranes around 2300 and 8500 micrometer-2 in the former and 4700 and 740 micrometer-2 in the latter region. The total concentration of glial glutamate transporters in both regions corresponds to three to five times the estimated number of glutamate molecules in one synaptic vesicle from each of all glutamatergic synapses. However, the role of glial glutamate transporters in limiting synaptic spillover is likely to vary between the two regions because of differences in the distribution of astroglia. Synapses are completely ensheathed and separated from each other by astroglia in the cerebellar molecular layer. In contrast, synapses in hippocampus (stratum radiatum) are only contacted by astroglia and are often found side by side without intervening glial processes.     

Enhancement of extracellular glutamate scavenge system in injured motoneurons

An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate-uptake kinetics. GS-transfected human embryonic kidney cells showed an up-regulation in glutamate-uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate-uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.     

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The excitatory effect of presynaptically released glutamate is tightly regulated and terminated by high affinity sodium-dependent glutamate transporters. The regulation of the glial glutamate transporter GLT-1 is potentially important in synaptic modulation. Using astroglial cultures prepared from the rat cerebral cortex, we found that the delta-opioid receptor agonist [D-pen2,D-pen5]-enkephalin decreases and glutamate increases the expression of the GLT-1 transporter mRNA. Corresponding changes in the uptake kinetics were found after incubation for 48 h with the respective agonists when glial glutamate uptake was measured in primary astroglial cultures. The data suggest that long-term receptor activation induces alterations in glial glutamate uptake properties.     

Ionic conductivity, transference numbers, composition and mobility of ions in cross-linked lysozyme crystals

Basic fibroblast growth factor (bFGF) gene expression as well as its immunoreactivity were studied after partial unilateral hemitransection of the rat brain during a time course of 24 h, 72 h, 7 and 14 days. The mechanical injury resulted in a global increase of bFGF gene expression at the 24-h time interval. This global increase was seen at the ipsilateral site at the level of the lesion as well as rostral to the lesion in the ipsilateral hemisphere. The upregulation in bFGF gene expression was in most of the areas investigated due to an upregulation in glial cells as seen by means of nonradioactive in situ hybridization compared with immunocytochemistry for glial fibrillary acidic protein (GFAP). Basic FGF immunoreactivity (IR) was increased around the lesion in glial cell nuclei 7 days after the injury. This increase was also detected in GFAP positive glial cells surrounding small vessels in the lesioned area. Moreover, in the present paper we demonstrate increased tenascin immunoreactivity in the lesioned area 7 days after injury. The tenascin IR was increased at the edges of the lesion as well as in vessel like structures. The tenascin IR was partially codistributed with GFAP IR in the lesioned area. The lesion was also characterized by an increase in vimentin IR as well as in laminin IR. It is suggested that the observed changes in the expression of bFGF, matrix proteins (laminin, tenascin) and intermediate filaments (vimentin) are involved in (a) tissue repair, (b) protection of neuronal cells from excitotoxic influences and (c) formation of new vessels in the lesioned area.     

Serotonin transporter messenger RNA in the developing rat brain: early expression in serotonergic neurons and transient expression in non-serotonergic neurons

Serotonin has been shown to affect the development of the mammalian nervous system. The serotonin transporter is a major factor in regulating extracellular serotonin levels. Using in situ hybridization histochemistry the rat serotonin transporter messenger RNA was localized during embryogenesis, the first four weeks postnatally and adulthood. Three general classes of serotonin transporter messenger RNA expression patterns were observed: (i) early detection with continued expression through adult age, (ii) transient expression colocalized with vesicular monoamine transporter 2 messenger RNA but with no detectable tryptophan hydroxylase immunoreactivity, and (iii) transient expression in the apparent absence of both vesicular monoamine transporter 2 messenger RNA and tryptophan hydroxylase immunoreactivity. For example, hybridization for serotonin transporter messenger RNA was strong in serotonin cell body-containing areas beginning early in gestation, and remained intense through adulthood. Immunoreactivity for tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, was completely overlapping with the presence of serotonin transporter messenger RNA in raphe nuclei postnatally. Sensory relay systems including the ventrobasal nucleus (somatosensory), lateral and medial geniculate nuclei (visual and auditory, respectively) as well as trigeminal, cochlear and solitary nuclei were representative of the second class of observations. In general, the limbic system expressed serotonin transporter messenger RNA in the third pattern with various limbic structures differing in the timing of expression. Septum, olfactory areas and the developing hippocampus contained serotonin transporter messenger RNA early in the developing brain. Other regions such as cingulate and frontopolar cortex exhibited hybridization peri- and postnatally, respectively. Several hypothalamic nuclei and pituitary transiently expressed serotonin transporter messenger RNA either postnatally or perinatally, respectively. If the observed patterns correlate with functional protein expression, distinct classes of serotonin transporter messenger RNA expression may reflect different functional roles for the serotonin transporter and serotonin, itself. Since the serotonin transporter is a target for a number of addictive substances including cocaine and amphetamine derivatives as well as antidepressants, transient expression of the serotonin transporter might suggest a window of vulnerability of associated cells to fetal drug exposure. Re-uptake, storage and re-release from non-serotonergic neurons might serve as a feedback mechanism from target neurons to serotonergic neurons. Alternatively, the transient expression of serotonin transporter messenger RNA may reflect critical periods important for tight regulation of extracellular serotonin in several brain regions, and may indicate previously unappreciated roles for serotonin as a developmental cue.     

Reactive astrocytes express nitric oxide synthase in the spinal cord of transgenic mice expressing a human Cu/Zn SOD mutation

The distribution of the neuronal isoform of nitric oxide synthase (nNOS) in the spinal cord of transgenic mice expressing a mutated human copper/zinc superoxide dismutase gene was enhanced when investigated by immunocytochemistry. Immunocytochemistry showed intensely stained NOS-immunoreactive (IR) glial cells with the appearance of astrocytes in the spinal cord and brain stem of transgenic mice, but none were observed at these sites in control mice. Using antisera directed against GFAP, the specific marker for astrocyte, the glial cells were confirmed by immunocytochemistry to be astrocytes. This immunocytochemical evidence suggests that nitric oxide may mediate glutamate neurotoxicity, and this study provides the first in vivo evidence that nitric oxide may be implicated in the pathologic process of human familial amyotrophic lateral sclerosis.     

Phosphorylation and modulation of brain glutamate transporters by protein kinase C

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.