Dopamine denervation of the prefrontal cortex increases expression of the astrocytic glutamate transporter GLT‐1

Both dopamine and glutamate are critically involved in cognitive processes such as working memory. Astrocytes, which express dopamine receptors, are essential elements in the termination of glutamatergic signaling: the astrocytic glutamate transporter GLT‐1 is responsible for > 90% of cortical glutamate uptake. The effect of dopamine depletion on glutamate transporters in the prefrontal cortex (PFC) remains unknown. In an effort to determine if astrocytes are a locus of cortical dopamine–glutamate interactions, we examined the effects of chronic dopamine denervation on PFC protein and mRNA levels of glutamate transporters. PFC dopamine denervation elicited a marked increase in GLT‐1 protein levels, but had no effect on levels of other glutamate transporters; high‐affinity glutamate transport was positively correlated with the extent of dopamine depletion. GLT‐1 gene expression was not altered. Our data suggest that dopamine depletion may lead to post‐translational modifications that result in increased expression and activity of GLT‐1 in PFC astrocytes.

     

Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex

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Monocular enucleation profoundly reduces secretogranin II expression in adult mouse visual cortex

Secretogranin II (ScgII), a member of the chromogranin family, is almost exclusively found in large dense core vesicles of a wide variety of endocrine and neuronal tissues, being stored together with many different neurotransmitters, peptide hormones and neuropeptides. In the brain ScgII is almost completely processed to secretoneurin, a peptide involved in neurite outgrowth, neuroprotection and neuronal plasticity. Furthermore, correlations with neurotransmitter release and a variety of neurological diseases were reported. In this study we examined possible changes in ScgII mRNA expression in the visual system of adult mice after removal of one eye. Mice were monocularly deprived of vision and sacrificed 1 day or 1, 3, 5 and 7 weeks after enucleation. Starting 1 day after the deprivation, a marked decrease of ScgII was visible in the contralateral visual cortex. Recovery initiated in the lateral supragranular visual cortex after 5 weeks of enucleation, but was far from complete in the 7 week animals, especially in the monocularly driven medial cortex. By comparison with the immediate early gene zif268, it was proven that ScgII cannot be categorized as an activity marker, but more likely plays a role in visual system plasticity by modulating a range of neurotransmitters and neuropeptides.     

Diversity of glutamate transporter expression and function in the mammalian retina

Summary. Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses. Between photoreceptors and second order neurons, increases in light intensity are signaled by decreases in the concentration of glutamate within the synaptic cleft. In such a system the precise control of glutamate in the synaptic cleft is thus essential and glutamate transporters are thought to contribute to this process. As demonstrated here, all neuronal and macroglial cells of the retina appear to express high-affinity glutamate transporters. GLAST1, GLT1, EAAC1 and EAAT5 are expressed in the retina and exhibit unique localisation and functional properties. In the present study we summarize retinal glutamate transporter expression, identify the major glutamate uptake site in the mammalian retina and discuss the possible functional roles of different glutamate transporter subtypes in glutamatergic neurotranmission in the retina. Received August 31, 1999 Accepted September 20, 1999     

Complementary neuronal and glial expression of two high-affinity glutamate transporter GLT1/EAAT2 forms in rat cerebral cortex

The glutamate transporter GLT1 is essential in limiting transmitter signaling and restricting harmful receptor overstimulation. It has been shown recently that GLT1 exists in two forms, the generic GLT1 and a 3'-end-spliced variant of GLT1 (GLT1v), both with similar transport characteristics. To differentiate clearly the cellular distribution of both GLT1 forms in the cortex, specific cRNA probes for non-radioactive in situ hybridization were generated and applied to adult rat brain sections. The results were complemented by western and northern blot analyses and by immunocytochemical investigations using specific peptide antibodies against both GLT1 forms. The study confirmed that generic GLT1 mRNA was expressed predominantly in astrocytes and, to a small extent, in neurons, whereas GLT1 protein was detected only in cell membranes of astrocytes. On the other hand, GLT1v mRNA and protein were demonstrated predominantly in neurons and in non-astrocytic glial cells irrespective of the cortical areas studied. A cytoplasmic granular staining of neurons and astrocytes predominated in the demonstration of GLT1v protein. It is concluded that the cellular expression of the two GLT1 forms is complementary. The cytoplasmic vesicular distribution of GLT1v may represent an endogenous protective mechanism to limit glutamate-induced excitotoxicity.     

Differential expression of the glutamate transporter GLT-1 in pancreas

The glutamate uptake transporter GLT-1 is best understood for its critical role in preventing brain seizures. Increasing evidence argues that GLT-1 also modulates, and is modulated by, metabolic processes that influence glucose homeostasis. To investigate further the potential role of GLT-1 in these regards, the authors examined GLT-1 expression in pancreas and found that mature multimeric GLT-1 protein is stably expressed in the pancreas of wild-type, but not GLT-1 knockout, mice. There are three primary functional carboxyl-terminus GLT-1 splice variants, called GLT-1a, b, and c. Brain and liver express all three variants; however, the pancreas expresses GLT-1a and GLT-1b but not GLT-1c. Quantitative real time-PCR further revealed that while GLT-1a is the predominant GLT-1 splice variant in brain and liver, GLT-1b is the most abundant splice variant expressed in pancreas. Confocal microscopy and immunohistochemistry showed that GLT-1a and GLT-1b are expressed in both islet β- and α-cells. GLT-1b was also expressed in exocrine ductal domains. Finally, glutamine synthetase was coexpressed with GLT-1 in islets, which suggests that, as with liver and brain, one possible role of GLT-1 in the pancreas is to support glutamine synthesis.     

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.     

GLUT 1-glucose transporter protein in adult and fetal mouse lung

We observed approximately 45-50 kD GLUT 1 protein in mouse lung homogenates and demonstrated a greater abundance in fetus compared to adult. In situ immunohistochemical analysis demonstrated GLUT 1 expression only in the perineural sheath of nerves. While the trapped fetal red blood cells expressed GLUT 1 abundantly, adult red blood cells were devoid of GLUT 1. No GLUT 1 was evident in fetal and adult lung alveolar and bronchiolar epithelial cells, vascular endothelial cells and the lung mesenchymal elements. Thus, GLUT 1 is not the major lung glucose transporter.     

Spreading depression-induced preconditioning in the mouse cortex: differential changes in the protein expression of ionotropic nicotinic acetylcholine and glutamate receptors

Preconditioning of the cerebral cortex was induced in mice by repeated cortical spreading depression (CSD), and the major ionotropic glutamate (GluRs) and nicotinic acetylcholine receptor (nAChRs) subunits were compared by quantitative immunoblotting between sham- and preconditioned cortex, 24 h after treatment. A 30% reduction in alpha-amino-3-hydroxy-5-methyl-4-iso- xazolepropionate (AMPA) GluR1 and 2 subunit immunoreactivities was observed in the preconditioned cortex (p < 0.03), but there was no significant change in the NMDA receptor subunits, NR1, NR2A and NR2B. A 12-15-fold increase in alpha7 nAChR subunit expression following in vivo CSD (p < 0.001) was by far the most remarkable change associated with preconditioning. In contrast, the alpha4 nAChR subunit was not altered. These data point to the alpha7 nAChR as a potential new target for neuroprotection because preconditioning increases consistently the tolerance of the brain to acute insults such as ischaemia. These data complement recent studies implicating alpha7 nAChR overexpression in the amelioration of chronic neuropathologies, notably Alzheimer's disease (AD).     

Glycoprotein expression in foetal and adult mouse cerebellum

1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.     

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system. This study was supported by the Johannes und Frieda Marohn-Stiftung, Erlangen.     

Glucose transporter gene expression in early mouse embryos.

The glucose transporter (GLUT) isoforms responsible for glucose uptake in early mouse embryos have been identified. GLUT 1, the isoform present in nearly every tissue examined including adult brain and erythrocytes, is expressed throughout preimplantation development. GLUT 2, which is normally present in adult liver, kidney, intestine and pancreatic beta cells is expressed from the 8-cell stage onward. GLUT 4, an insulin-recruitable isoform, which is expressed in adult fat and muscle, is not expressed at any stage of preimplantation development or in early postimplantation stage embryos. Genetic mapping studies of glucose transporters in the mouse show that Glut-1 is located on chromosome 4, Glut-2 on chromosome 3, Glut-3 on chromosome 6, and Glut-4 on chromosome 11.     

Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.     

Estimating the glutamate transporter surface density in distinct sub-cellular compartments of mouse hippocampal astrocytes

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.     

Plasticity and regulatory mechanisms of Hox gene expression in mouse neural crest cells

In amniotes, the developmental potentials of neural crest cells differ between the cranium and the trunk. These differences may be attributable to the different expression patterns of Hox genes between cranial and trunk neural crest cells. However, little is known about the factors that control Hox genes expression in neural crest cells. The present data demonstrate that retinoic acid (RA) treatment and the activation of Wnt signaling induce Hoxa2 and Hoxd9 expression, respectively, in mouse mesencephalic neural crest cells, which never express Hox genes in vivo. Furthermore, Wnt signaling suppresses the induction of Hoxa2. We also demonstrate that these factors participate in the maintenance of Hoxa2 and Hoxd9 expression in mouse trunk neural crest cells. Our results suggest that RA and Wnt signaling function as environmental factors that regulate the expression of Hoxa2 and Hoxd9 in mouse neural crest cells.