Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: Non-radioactive in situ hybridization and comparative immunocytochemistry

doi:10.1016/0306-4522(95)00477-7

Neuroscience

Volume 71, Issue 4, April 1996, Pages 989-1004

https://doi.org/10.1016/0306-4522(95)00477-7 Get rights and content

Abstract

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, epithelial 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.

Reference (51)

BignamiA. et al.
Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence
Brain Res.
(1972)
BurnetteW.N.
Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radionated protein A
Analyt. Biochem.
(1981)
ChomczynskiP. et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction
Analyt. Biochem.
(1987)
DanboltN.C. et al.
An [Na⁺ + K⁺] coupled L-glutamate transporter purified from rat brain is located in glial cell processes
Neuroscience
(1992)
DrenckhahnD. et al.
Production of polyclonal antibodies against proteins and peptides
Meth. cell Biol.
(1993)
FaggG.E. et al.
Amino acid neurotransmitters and their pathways in the mammalian central nervous system
Neuroscience
(1983)
HeadleyP.M. et al.
Excitatory amino acids and synaptic transmission: the evidence for a physiological function
Trends pharmac. Sci.
(1990)
KanaiY. et al.
The elusive transporters with a high affinity for glutamate
Trends Neurosci.
(1993)
KuglerP.
Enzymes involved in glutamatergic and GABAergic neurotranmission
Int. Rev. Cytol.
(1993)
KuglerP. et al.
Glutamate dehydrogenase in astrocytes of the rat dentate gyrus following lesion of the entorhinal cortex
Neuroscience
(1995)

LevyL.M. et al.

A monoclonal antibody raised against an [Na⁺ + K⁺]coupledl-glutamate transporter purified from rat brain confirms glial cell localization

Fedn Ear. biochem. Socs Lett.

(1993)

MaycoxP.R. et al.

Amino acid neurotransmission: spotlight on synaptic vesicles

Trends Neurosci.

(1990)

NichollsD.G. et al.

The release and uptake of excitatory amino acids

Trends pharmac. Sci.

(1990)

OertelW.H. et al.

Immunocytochemical localization of glutamate decar☐ylase in rat cerebellum with a new antiserum

Neuroscience

(1981)

RamachandranB. et al.

Differential expression of transporters for norepinephrine and glutamate in wild type, variant, and WNT1-expressing PC12 cells

J. biol. Chem.

(1993)

RothsteinJ.D. et al.

Localization of neuronal and glial glutamate transporters

Neuron

(1994)

SandovalM.E. et al.

Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers

Neuroscience

(1978)

SchousboeA. et al.

Differences in glutamate uptake in astrocytes cultured from different brain regions

Brain Res.

(1979)

ShashidharanP. et al.

Cloning and characterization of a glutamate transporter cDNA from human cerebellum

Biochim. biophys. Acta

(1993)

TanakaK.

Expression cloning of a rat glutamate transporter

Neurosci. Res.

(1993)

WürdigS. et al.

Histochemistry of glutamate metabolizing enzymes in the rat cerebellar cortex

Neurosci. Lett.

(1991)

AngererL.M. et al.

In situ hybridization to cellular RNA with radiolabelled RNA probes

AokiC. et al.

Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission

J. Neurosci.

(1987)

AsanE. et al.

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

Histochem. Cell Biol.

(1995)

AthertonE. et al.

Peptide synthesis. Part 2. Procedures for solid-phase synthesis using Nα-fluorenylmethoxycarbonylamino-acids on polyamide supports. Synthesis of substance P and of acyl carrier protein 65–74 decapeptide

J. Chem. Soc. Perkin

(1981)

Cited by (135)

Long-term exposure to high glucose induces changes in the expression of AMPA receptor subunits and glutamate transmission in primary cultured cortical neurons
2022, Biochemical and Biophysical Research Communications
Hyperglycemia, which occurs under the diabetic conditions, induces serious diabetic complications. Diabetic encephalopathy has been defined as one of the major complications of diabetes, and is characterized by neurochemical and neurodegenerative changes. However, little is known about the effect of long-term exposure to high glucose on neuronal cells. In the present study, we showed that exposure to glutamate (100 mM) for 7 days induced toxicity in primary cortical neurons using the MTT assay. Additionally, high glucose increased the sensitivity of AMPA- or NMDA-induced neurotoxicity, and decreased extracellular glutamate levels in primary cortical neurons. In Western blot analyses, the protein levels of the GluA1 and GluA2 subunits of the AMPA receptor as well as synaptophysin in neurons treated with high glucose were significantly increased compared with the control (25 mM glucose). Therefore, long-term exposure to high glucose induced neuronal death through the disruption of glutamate homeostasis.
Novel aspects of glutamine synthetase in ammonia homeostasis: Glutamine synthetase and ammonia
2020, Neurochemistry International
Elevated blood ammonia (hyperammonemia) is believed to be a major contributor to the neurological sequelae following severe liver disease. Ammonia is cleared via two main mechanisms, the urea cycle pathway and the glutamine synthetase reaction. Recent studies of genetically modified animals confirm the importance of the urea cycle, but also suggest that the glutamine synthetase reaction is more important than previously recognized. While the liver clears about two-thirds of the body's ammonia via the combined action of the urea cycle and glutamine synthetase, extrahepatic tissues do not express all the components required for performing a complete urea cycle and therefore depend on the glutamine synthetase reaction for ammonia clearance. The brain is particularly vulnerable to the effects of hyperammonemia, which include impaired extracellular potassium buffering and brain edema. Moreover, the glutamine synthetase reaction is intimately linked to the metabolism of the excitatory and inhibitory neurotransmitters glutamate and gamma aminobutyric acid (GABA), implicating a key role for this enzyme in neurotransmission. This review discusses the emerging roles of glutamine synthetase in brain pathophysiology, particularly aspects related to ammonia homeostasis and hepatic encephalopathy.
Axon-terminals expressing EAAT2 (GLT-1; Slc1a2) are common in the forebrain and not limited to the hippocampus
2019, Neurochemistry International
The excitatory amino acid transporter type 2 (EAAT2) represents the major mechanism for removal of extracellular glutamate. In the hippocampus, there is some EAAT2 in axon-terminals, whereas most of the protein is found in astroglia. The functional importance of the neuronal EAAT2 is unknown, and it is debated whether EAAT2-expressing nerve terminals are present in other parts of the brain. Here we selectively deleted the EAAT2 gene in neurons (by crossing EAAT2-flox mice with synapsin 1-Cre mice in the C57B6 background). To reduce interference from astroglial EAAT2, we measured glutamate accumulation in crude tissue homogenates. EAAT2 proteins levels were measured by immunoblotting. Although synapsin 1-Cre mediated gene deletion only reduced the forebrain tissue content of EAAT2 protein to 95.5 ± 3.4% of wild-type (littermate) controls, the glutamate accumulation in homogenates of neocortex, hippocampus, striatum and thalamus were nevertheless diminished to, respectively, 54 ± 4, 46 ± 3, 46 ± 2 and 65 ± 7% of controls (average ± SEM, n = 3 pairs of littermates). GABA uptake was unaffected. After injection of U-¹³C-glucose, lack of neuronal EAAT2 resulted in higher ¹³C-labeling of glutamine and GABA in the hippocampus suggesting that neuronal EAAT2 is partly short-circuiting the glutamate-glutamine cycle in wild-type mice. Crossing synapsin 1-Cre mice with Ai9 reporter mice revealed that Cre-mediated excision occurred efficiently in hippocampus CA3, but less efficiently in other regions and hardly at all in the cerebellum. Conclusions: (1) EAAT2 is expressed in nerve terminals in multiple brain regions. (2) The uptake catalyzed by neuronal EAAT2 plays a role in glutamate metabolism, at least in the hippocampus. (3) Synapsin 1-Cre does not delete floxed genes in all neurons, and the contribution of neuronal EAAT2 is therefore likely to be larger than revealed in the present study.
Neuronal vs glial glutamate uptake: Resolving the conundrum
2016, Neurochemistry International
Citation Excerpt :
The astrocytes were intensely labeled, while the neurons appeared unlabeled. Labeling was not even detected in glutamatergic axon-terminals in the stratum radiatum of the CA1 hippocampus (Danbolt et al., 1992; Lehre et al., 1995) despite high levels of EAAT2 mRNA in CA3 pyramidal cells from which they originate (Torp et al., 1994, 1997; Schmitt et al., 1996). Subsequent immunocytochemical studies confirmed this conclusion (e.g. Rothstein et al., 1994; Schmitt et al., 1996; Milton et al., 1997; Kugler and Schmitt, 2003; Berger et al., 2005; Holmseth et al., 2009), while in-situ hybridization studies added that EAAT2 mRNA was also present in several neuronal populations (Berger and Hediger, 1998, 2000, 2001).
Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox.
GLT-1: The elusive presynaptic glutamate transporter
2016, Neurochemistry International
Historically, glutamate uptake in the CNS was mainly attributed to glial cells for three reasons: 1) none of the glutamate transporters were found to be located in presynaptic terminals of excitatory synapses; 2) the putative glial transporters, GLT-1 and GLAST are expressed at high levels in astrocytes; 3) studies of the constitutive GLT-1 knockout as well as pharmacological studies demonstrated that >90% of glutamate uptake into forebrain synaptosomes is mediated by the operation of GLT-1. Here we summarize the history leading up to the recognition of GLT-1a as a presynaptic glutamate transporter. A major issue now is understanding the physiological and pathophysiological significance of the expression of GLT-1 in presynaptic terminals. To elucidate the cell-type specific functions of GLT-1, a conditional knockout was generated with which to inactivate the GLT-1 gene in different cell types using Cre/lox technology. Astrocytic knockout led to an 80% reduction of GLT-1 expression, resulting in intractable seizures and early mortality as seen also in the constitutive knockout. Neuronal knockout was associated with no obvious phenotype. Surprisingly, synaptosomal uptake capacity (V_max) was found to be significantly reduced, by 40%, in the neuronal knockout, indicating that the contribution of neuronal GLT-1 to synaptosomal uptake is disproportionate to its protein expression (5–10%). Conversely, the contribution of astrocytic GLT-1 to synaptosomal uptake was much lower than expected. In contrast, the loss of uptake into liposomes prepared from brain protein from astrocyte and neuronal knockouts was proportionate with the loss of GLT-1 protein, suggesting that a large portion of GLT-1 in astrocytic membranes in synaptosomal preparations is not functional, possibly because of a failure to reseal. These results suggest the need to reinterpret many previous studies using synaptosomal uptake to investigate glutamate transport itself as well as changes in glutamate homeostasis associated with normal functions, neurodegeneration, and response to drugs.
Role of Glutamate Transport in Alcohol Withdrawal
2016, Neuropathology of Drug Addictions and Substance Misuse
Glutamate is the major excitatory neurotransmitter in the brain. However, excessive glutamate can lead to neurotoxicity. Evidence suggests that a hyperglutamatergic brain state is the core pathology behind alcohol withdrawal syndrome (AWS). This hyperglutamatergic state results from upregulation of glutamate receptors, dysregulation of glutamate metabolism, and reduction of synaptic glutamate clearance. Numerous genetic factors along this pathway could modulate the vulnerability or severity of AWS. Benzodiazepines, which facilitate inhibitory neurotransmission, are standard therapeutic agents for acute AWS. While effective in reducing withdrawal severity, benzodiazepines carry risks of sedation, respiratory suppression, and dependence, and they do not correct the persistent hyperglutamatergic state associated with relapse. Data show that ceftriaxone, a β-lactam antibiotic, upregulates excitatory amino acid transporter 2 (EAAT2) and attenuates withdrawal manifestations in preclinical models. These findings underscore the importance of glutamatergic mechanisms in AWS and suggest that glutamate reuptake may represent a novel target for acute withdrawal and relapse prevention.

View all citing articles on Scopus

View full text

Expression of the glutamate transporter GLT1 in neural cells of the rat central nervous system: Non-radioactive in situ hybridization and comparative immunocytochemistry

Abstract

Brain Res.

Analyt. Biochem.

Analyt. Biochem.

Neuroscience

Meth. cell Biol.

Neuroscience

Trends pharmac. Sci.

Trends Neurosci.

Int. Rev. Cytol.

Neuroscience

Fedn Ear. biochem. Socs Lett.

Trends Neurosci.

Trends pharmac. Sci.

Neuroscience

J. biol. Chem.

Neuron

Neuroscience

Brain Res.

Biochim. biophys. Acta

Neurosci. Res.

Neurosci. Lett.

In situ hybridization to cellular RNA with radiolabelled RNA probes

Regional distribution of astrocytes with intense immunoreactivity for glutamate dehydrogenase in rat brain: implications for neuron-glia interactions in glutamate transmission

J. Neurosci.

Qualitative and quantitative detection of alkaline phosphatase coupled to an oligonucleotide probe for somatostatin mRNA after in situ hybridization using unfixed rat brain tissue

Histochem. Cell Biol.

Peptide synthesis. Part 2. Procedures for solid-phase synthesis using Nα-fluorenylmethoxycarbonylamino-acids on polyamide supports. Synthesis of substance P and of acyl carrier protein 65–74 decapeptide

J. Chem. Soc. Perkin