High Affinity Uptake Of L-glutamate Research Articles

Terminal Glial Differentiation Involves Regulated Expression of the Excitatory Amino Acid Transporters in the Drosophila Embryonic CNS

The Drosophila excitatory amino acid transporters dEAAT1 and dEAAT2 are nervous-specific transmembrane proteins that mediate the high affinity uptake of l-glutamate or aspartate into cells. Here, we demonstrate by colocalization studies that both genes are expressed in discrete and partially overlapping subsets of differentiated glia and not in neurons in the embryonic central nervous system (CNS). We show that expression of these transporters is disrupted in mutant embryos deficient for the glial fate genes glial cells missing ( gcm) and reversed polarity ( repo). Conversely, ectopic expression of gcm in neuroblasts, which forces all nerve cells to adopt a glial fate, induces an ubiquitous expression of both EAAT genes in the nervous system. We also detected the dEAAT transcripts in the midline glia in late embryos and dEAAT2 in a few peripheral neurons in head sensory organs. Our results show that glia play a major role in excitatory amino acid transport in the Drosophila CNS and that regulated expression of the dEAAT genes contributes to generate the functional diversity of glial cells during embryonic development.

Selective loss of phorbol-12,13-dibutyrate-facilitated L-glutamate transport in forebrain neurons of aged rats.

Phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C, was used to evaluate potential age-related changes in phosphorylation-dependent facilitation of high-affinity L-glutamate uptake in the rat central nervous system (CNS). Forebrain homogenates from male Fischer-344/brown Norway F1 hybrid rats were separated into glia-enriched (glial plasmalemmal vesicles) and neuron-enriched fractions (synaptosomes) and assayed for sodium-dependent transport of L-[3H]glutamate. Glial fractions from rats aged 5, 25, 31, and 37 months exhibited similar rates of basal L-[3H]glutamate transport and demonstrated no significant age-related differences with respect to the maximal facilitatory effect of PDBu (1-100 microM). In contrast, neuronal fractions exhibited an age-related decline in both indices, with basal L-[3H]glutamate transport decreasing from 710+/-31 to 560+/-40 pmoL/mg protein/90 s for the 5- and 37-month groups, respectively (p < .03) and PDBu having a significantly attenuated effect in aged animals. Together, these results provide support for the hypothesis that aging is associated with a decrease in the number of neuronal L-glutamate transporters as well as a diminished capacity to up-regulate these transporters through a protein kinase C-dependent pathway.

Regional differences in the inhibition of l-glutamate and l-aspartate sodium-dependent high affinity uptake systems in rat CNS synaptosomes by l- trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy- d-aspartate and d-aspartate

The sodium-dependent high affinity transport of l-[ 3H]glutamate and l-[ 3H]aspartate into synaptosomal fractions prepared from three different regions was employed to investigate the inhibitors l- trans-pyrrolidine-2,4-dicarboxylate, threo-3-hydroxy- d-aspartate and d-aspartate. These substances showed regional heterogeneity as inhibitors of sodium-dependent high affinity uptake of l-glutamate and l-aspartate. l- trans-Pyrrolidine-2,4-dicarboxylate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate in the cortex (IC 50 8 μM vs l-glutamate and 13 μM vs l-aspartate) and cerebellum (IC 50 4 μM v l-glutamate and 8 μM vs l-aspartate). threo-3-Hydroxy- d-aspartate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate in the cortex (IC 50 9 μM vs l-glutamate and 13 μM vs l-aspartate) and hippocampus (IC 50 6 μM v l-glutamate and 11 μM v l-aspartate). d-Aspartate was a more potent inhibitor of the uptake of l-glutamate than of l-aspartate only in the cortex (IC 50 8 μM vs l-glutamate and 15 μM vs l-aspartate). These results thus support other evidence that there is regional heterogeneity in sodium-dependent high affinity acidic amino acid uptake sites in the brain.

Na+-dependent high affinity uptake of l-glutamate in primary cultures of human fibroblasts isolated from three different types of tissue

Cultured human fibroblasts isolated from embryonic muscle, skin and peripheral nerve tissues were found to accumulate [3H]L-glutamate by a Na+-dependent uptake process strongly inhibited by several glutamate/aspartate analogues including d- and l-aspartate, d- and l-thero-3-hydroxyaspartate and l-trans-pyrrolidine-2,4-dicarboxylate but not d-glutamate. It was also reduced by elevated concentrations of K+, Rb+ and Cs+. The values of Km's were 5–20 μM, well within the ‘high affinity’ region. Variations in the capacity (Vmax) of [3H]l-glutamate uptake did not correlate with the origin (muscle, skin or nerve tissue) of the fibroblasts. The uptake characteristics suggest that it is mediated by a transport system similar to that commonly observed only in brain tissue.

The Na(+)-dependent binding of [3H]L-aspartate in thaw-mounted sections of rat forebrain.

Binding of [3H]L-aspartate to thaw-mounted coronal sections of frozen rat forebrain was strong in grey regions of telencephalon (neocortex, hippocampus and neostriatum), but it was weaker and unevenly distributed in diencephalon. At low nanomolar concentrations of ligand used in the present studies, [3H]L-aspartate binding was strongly inhibited by L-threo-3-hydroxyaspartate and L-trans-pyrrolidine-2,4-dicarboxylate, compounds known to be substrate/inhibitors of the high affinity uptake of L-glutamate and L-aspartate. None of the typical ligands for the glutamate and aspartate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), N-methyl-D-aspartate and kainate, produced a strong enough inhibition (only CNQX at 100 microM weakly inhibited) of the Na(+)-dependent [3H]L-aspartate binding to suggest that [3H]L-aspartate was bound to the receptor binding sites. Furthermore, the binding was absolutely dependent on the presence of Na+ in the incubation medium. It is concluded that [3H]L-aspartate is a ligand suitable for autoradiographic studies of the distribution of Na(+)-dependent, high affinity uptake of acidic amino acids in the central nervous system (CNS). However, feasibility of using [3H]L-aspartate as a specific marker of glutamatergic and/or aspartergic synapses in the CNS requires further investigation.

Changes in neurotransmitter parameters in the brain induced by l-cysteine injections in the young rat

A single subcutaneous injection of l-cysteine (1.2 mg/g body wt.) to 4-day-old rats leads to atrophy of the brains examined 27–31 days later. The brains could be separated into two groups (type 1 and 2) on account of the degree of atrophy. Type-1 lesion, with a brain weight reduction of 20%, was dominated by a severe reduction in high-affinity uptake of l-glutamate in CNS regions receiving corticofugal fibers such as thalamus and striatum. Glutamate decarboxylase was only reduced in cortical structures. In type-2 lesion, with a severe brain atrophy of about 50%, high-affinity glutamate uptake was further reduced and there was a more pronounced reduction in glutamate decarboxylase activity in several brain regions. Cholinergic neurons were less affected by the lesion and the levels of choline acetyltransferase showed a relative increase in brain regions which partly compensated for their reduction in size.

Heterogeneity of high affinity uptake of L-glutamate and L-aspartate in the mammalian central nervous system

Characteristics of high affinity uptake of L-glutamate are examined in order to evaluate the possible use of the uptake of [ 3H]L-glutamate, [ 3H]L-aspartate or any other suitable [ 3H]-labelled substrate as a marker for glutamatergic and aspartergic synapses in autoradiographic studies in the mammalian brain. Review of data on substrate specificity indicates the presence of at least two high affinity uptake systems specific for acidic amino acids in the central nervous tissue; one which takes up L-glutamate and L-aspartate and the other which is selective for L-glutamate only. Studies on ionic requirements, too, point to the existence of at least two distinct uptake systems with high affinity for L-glutamate. The Na +-dependent uptake system(s) handle(s) both L-glutamate and L-aspartate whereas the Na +-independent uptake system(s) show(s) selectivity for L-glutamate only. Available data do not favour the Na +-dependent binding of [ 3H]D-aspartate to thaw-mounted sections of frozen brain tissue as a suitable marker for glutamatergic/aspartergic synaptic nerve endings. However, there are reasons — such as the results of lesion studies and the existence of uptake sites which have a higher affinity for L-aspartate than for D-aspartate — to suggest that Na +-dependent binding of [ 3H]L-aspartate, rather than that of [ 3H]D-aspartate, should be further investigated as a possible marker for the glutamatergic/aspartergic synapses in the autoradiographic studies using sections of frozen brain.

Effects of variations in ionic concentrations on high affinity uptake of l-glutamate in non-glutamatergic neurons and non-neuronal cells cultured from neonatal rat cortex

High affinity uptake of l-glutamate in the CNS is known to be strongly dependent on Na +. However, the stoichiometry and the mechanism of the relationship between Na + and l-glutamate at the putative transport site (i.e. whether one or two Na + are co-transported with one molecule of l-glutamate and in which order they bind to the site) have been the subject of debate because the results of quantitative studies on Na +-dependence of l-glutamate uptake have varied from one experimental model to another. This communication discusses ionic requirements of l-glutamate uptake in two culture systems derived from neonatal rat cortex—one containing no neurons and the other including, in addition to glial cells, GABAergic neurons. The present results are consistent with a model in which one molecule of l-glutamate is co-transported with either one or two Na + and no fixed order is required for the binding of Na + and l-glutamate to the transport site. Also, in contrast to other studies, l-glutamate uptake was found to be significantly reduced in the absence of Ca 2+ and was not affected by depolarizing concentrations of K +.

High affinity uptake of L-glutamate and γ-aminobutyric acid inDrosophila melanogaster

Preparations having properties resembling those of synaptosomes have been isolated from whole fly homogenates ofDrosophila melanogaster using ficoll gradient floatation technique. These have been characterized by marker enzymes and electron microscopy and binding of muscarinic antagenist3H Quinuclidinyl benzilate. An uptake system for neurotransmitter, a-Aminobutyric acid has been demonstrated in these preparations.

Autoradiographic demonstration of glutamate structures after stereotaxic injection of kainic acid in rat hippocampus

Autoradiography of high affinity uptake of l-glutamate in rat hippocampus after kainic acid lesion of CA3 and CA4 pyramidal cells has been used to identify the neurotransmitter of their ipsilateral connections. Glutamate or aspartate has tentatively been identified as the neurotransmitter of the projection from CA3 pyramidal cells to strata radiatum and oriens of CA1 and the projection from CA4 pyramidal cells to the inner parts of the molecular layer of area dentata.

Development of neurotransmitter parameters in lateral geniculate body, superior colliculus and visual cortex of the albino rat

The postnatal development of some neurotransmitter parameters was measured in lateral geniculate body, superior colliculus and visual cortex of the rat. The following parameters were studied: (i) high-affinity uptake of l-glutamate or d-aspartate as markers for glutamergic neurons; (ii) high-affinity uptake of GABA, which reflects both glial and neuronal uptake of GABA; (iii) HA β-alanine uptake as a marker for accumulation of GABA in glial structures; (iv) activity of glutamic acid decarboxylase which reflects GABAergic neurons; and (v) activity of choline acetyltransferase as a cholinergic marker. K m and V max were determined for high-affinity uptake of glutamate and GABA in newborn and adult animals. The possible glial influence on the uptake during development is discussed. In lateral geniculate body and visual cortex the HA glutamate uptake showed increasing activity from birth to adulthood, whereas in superior colliculus, the uptake was higher at birth, reaching a small significant peak after 12 days of age, and was then reduced to adult level. K m showed no such change between neonatal and adult animals. At birth, high-affinity GABA-uptake was similar to the adult level in superior colliculus and lateral geniculate body. In visual cortex, the uptake of GABA was 50% of adults. However, on day 15, the GABA uptake showed 2 to 3-fold higher activity in all regions when compared to adult level. K m for GABA uptake in neonatals and adults differed only in lateral geniculate body. High affinity uptake of β-alanine was 50–80% lower in adults than in newborn rats. Glutamate decarboxylase activity, however, increased continuously in all 3 regions examined. This was true also for choline acetyltransferase.

Kainic acid neurotoxicity; effect of systemic injection on neurotransmitter markers in different brain regions

Systemic injection of kainic acid (12 mg/kg) induces necrosis and neuronal degeneration in several brain regions. The most pronounced effects were observed in the piriform cortex, amygdaloid complex, hippocampus and septum. A good correlation between morphological changes and changes in some neurotransmitter markers was observed in these 4 areas. High affinity uptake of L-glutamate, as well as glutamate decarboxylase and choline acetyltransferase activities were reduced in the piriform cortex and amygdaloid complex whereas in the hippocampus and septum only the first two markers were reduced. No morphological changes or decrease in any of these neurotransmitter markers were observed in striatum or globus pallidus. A pronounced neuronal degeneration could be demonstrated in lateral thalamus and geniculate body, but this degeneration was not accompanied by any decrease in the transmitter markers tested.

Biochemical evidence for overlapping neocortical and allocortical glutamate projections to the nucleus accumbens and rostral caudatoputamen in the rat brain

The high affinity uptake of l-glutamate has been used to investigate the origin and distribution of putative glutamate fibers in restricted parts of the rostral caudatoputamen and the nucleus accumbens of the rat brain. Ablation of the frontal cortex reduced the glutamate uptake heavily (−77%) in the dorsal part of the ipsilateral caudatoputamen, but also led to significant decreases in the ventral parts of the ipsilateral caudatoputamen (−62% and −53%), in the ipsilateral nucleus accumbens (−25% and −18%) and in the contralateral dorsal part of the caudatoputamen (−21%). Lesion of the caudal neocortex reduced the glutamate uptake in the dorsal part of the ipsilateral caudatoputamen only (−23%). Lesions of the fimbria/fornix reduced the glutamate uptake in both parts of the ipsilateral nucleus accumbens (−46% and −34%) and by approximately 20% in the whole dorsoventral extent of the anterior caudatoputamen. The results indicate that the frontal neocortex distributes fibers which may use glutamate as neurotransmitter both to the whole ipsilateral caudatoputamen and to the nucleus accumbens, and also to the dorsal parts of the contralateral caudatoputamen. The caudal neocortex probably sends such fibers to the dorsal ipsilateral caudatoputamen, and the caudal allocortex sends such fibers through the fimbria/ fornix to the nucleus accumbens and the ventral part of the ipsilateral caudatoputamen. The results thus corroborate previous suggestions of close similarities between the nucleus accumbens and the ventral caudatoputamen.

Biochemical evidence for glutamate as a transmitter in hippocampal efferents to the basal forebrain and hypothalamus in the rat brain

The effects of bilateral transection of the fornix bundle on the high affinity uptake of glutamate and on the amino acid content in several nuclei of rat forebrain and hypothalamus were studied in order to investigate the possible role of glutamate as a transmitter of these fibres. This lesion decreased the high affinity uptake of l-glutamate by 60–70% in the mammillary body and lateral septum, and by 40–50% in the anterior diagonal band nucleus, the bed nucleus of the stria terminalis, the mediobasal hypothalamus and the nucleus accumbens. The content of endogenous glutamate in samples dissected from freeze-dried tissue also decreased significantly in these regions. Endogenous aspartate was slightly decreased in the anterior diagonal band nucleus and the mammillary body, but unchanged in the other regions. No significant changes were seen in the levels of serine, γ-aminobutyric acid, glutamine and taurine, except for an increase in glutamine and taurine in the bed nucleus of the stria terminalis. The high affinity uptake of γ-aminobutyric acid, tested in the bed nucleus of the stria terminalis, the mediobasal hypothalamus and the mammillary body, was unchanged after the lesion. The results indicate that allocortical efferents innervating subcortical nuclei through the fornix might use glutamate as a transmitter. The study further supports the concept that glutamate plays an important role as transmitter of several different corticofugal fibre systems in mammalian brain.

Effect of cyclic nucleotides on high affinity uptake of l-glutamate and taurine in glial and neuroblastoma cells

Cultured glial cells (clones NN and I 6) and neuroblastoma cells (clones M1 and M1NN) were used to study the effect of cyclic nucleotides on high affinity uptake of l-glutamate and taurine. Treatment of NN cells with a mixture of dBcAMP and BrcGMP for one or two days resulted in a more than 3-fold increase in the V max of l-glutamate high affinity uptake. Similar but smaller change was observed in a related glial clone (I 6). Effects of cyclic nucleotide derivatives on high affinity uptake of l-glutamate by neuroblastoma cells (clones M1 and M1NN) or of taurine by both glial or neuroblastoma cells were either smaller or absent. Some differences were found in kinetic parameters of uptake of l-glutamate and taurine by original and re-isolated cells, respectively. It is possible that these differences resulted from co-cultivation of glial and neuronal cells. However, since some values reported in this paper differ to some extent from those published previously, some other factors, such as subcloning, may have been responsible.

Evidence for glutamate as a neurotransmitter in the corticofugal fibres to the dorsal lateral geniculate body and the superior colliculus in rats

The high-affinity uptake of l-glutamate, d-aspartate and GABA were examined in homogenates from the dorsal lateral geniculate body and the superior colliculus after removal of the right visual cortex of adult rats. The high-affinity uptake of d-aspartate and l-glutamate were reduced by 53 and 75% respectively in the dorsal lateral geniculate body and by 46 and 53% respectively in the superior colliculus ipsilateral to the lesion. The uptake on the contralateral side was unaffected. The reductions were detected 3 days after the lesion and were maximally developed after 7 days. Subcellular fractionation showed that the main part of the uptake was confined to the synaptosomal fraction of both regions and that the reductions were most prominent in this fraction. The lesion was not accompanied by significant changes in high-affinity uptake of GABA nor in changes of choline acetyltransferase and glutamate decar☐ylase activities. The high-affinity l-glutamate uptake on the contralateral visual cortex was unchanged from control values. After ablation of the visual cortex the level of l-glutamate was reduced by 32 and 17% in the ipsilateral dorsal lateral geniculate body and superior colliculus, respectively. The levels of the other amino acids examined, including l-aspartate, were unchanged. Enucleation had no effect on the uptake of l-glutamate and of GABA in the dorsal lateral geniculate body or in the superior colliculus.

Effect of chronic pentobarbital treatment on high affinity uptake of L-glutamate by cultured glial cells

Effect of chronic pentobarbital treatment on high affinity uptake of L-glutamate by cultured glial cells