The Number of Glutamate Transporter Subtype Molecules at Glutamatergic Synapses: Chemical and Stereological Quantification in Young Adult Rat Brain

Lehre, Knut P.; Danbolt, N

doi:10.1523/jneurosci.18-21-08751.1998

Cited by 617 publications

(598 citation statements)

References 67 publications

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“…Maintenance of resting levels far below the Km-values of the transporters (Danbolt, 2001;Conti et al, 2004) requires a vast excess of transporters (Bergles and Jahr, 1997; Otis and Kavanaugh, 2000;Herman and Jahr, 2007) in agreement with biochemical measurements of transporter concentrations (Lehre and Danbolt, 1998;Holmseth et al, 2012b).…”

Section: The Expression Levels Required For Physiologically Significamentioning

confidence: 71%

“…It can be due to the same antibodies as those recognizing the antigen under study (Danbolt et al, 1998;Holmseth et al, 2005;Wilson et al, 1996). Consequently, even monoclonal antibodies can display cross-reactivity.…”

Section: [Figure 1 About Here]mentioning

confidence: 99%

“…Consequently, even monoclonal antibodies can display cross-reactivity. In fact, when we made monoclonal antibodies to EAAT2 (Levy et al, 1993) we also isolated clones producing polyreactive antibodies (Danbolt et al, 1998). The cross-reactivity is often unexpected.…”

Section: [Figure 1 About Here]mentioning

confidence: 99%

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Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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Immunocytochemistry and Western blotting are still major methods for protein localization, but they rely on the specificity of the antibodies. Validation of antibody specificity remains challenging mostly because ideal negative controls are often unavailable. Further, immunochemical labeling patterns are also influenced by a number of other factors such as postmortem changes, fixation procedures and blocking agents as well as the general assay conditions (e.g., buffers, temperature, etc.). Western blotting similarly depends on tissue collection and sample preparation as well as the electrophoretic separation, transfer to blotting membranes and the immunochemical probing of immobilized molecules. Publication of inaccurate information on protein distribution has downstream consequences for other researchers because the interpretation of physiological and pharmacological observations depends on information on where ion channels, receptors, enzymes or transporters are located. Despite numerous reports, some of which are strongly worded, erroneous localization data are being published. Here we describe the extent of the problem and illustrate the nature of the pitfalls with examples from studies of neurotransmitter transporters. We explain the importance of supplementing immunochemical observations with other measurements (e.g., mRNA levels and distribution, protein activity, mass spectrometry, electrophysiological recordings, etc.) and why quantitative considerations are integral parts of the quality control. Further, we propose a practical strategy for researchers who plan to embark on a localization study. We also share our thoughts about guidelines for quality control. GLIA 2016;64:2045–2064

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Section: The Expression Levels Required For Physiologically Significamentioning

confidence: 71%

Section: [Figure 1 About Here]mentioning

confidence: 99%

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Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Danbolt

Zhou

Furness

et al. 2016

Glia

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“…Excitatory amino acids transporter 1 (EAAT1, also known as GLAST) is the major glutamate transporter in the cerebellum (Lehre and Danbolt, 1998). It is expressed strongly by astrocytes in the molecular layer of the cerebellum and is at highest density on Bergmann glia.…”

Section: Introductionmentioning

confidence: 99%

“…The role of neuron-glia interactions is also being investigated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (Sheldon and Robinson, 2007). Moreover, glial dysfunction is likely to contribute to the pathogenesis of cerebellar ataxia in a mouse model of spinocerebellar ataxia type 7 (SCA7) (Custer et al, 2006).Excitatory amino acids transporter 1 (EAAT1, also known as GLAST) is the major glutamate transporter in the cerebellum (Lehre and Danbolt, 1998). It is expressed strongly by astrocytes in the molecular layer of the cerebellum and is at highest density on Bergmann glia.…”

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confidence: 99%

Reactive astrocytosis and glial glutamate transporter clustering are early changes in a spinocerebellar ataxia type 1 transgenic mouse model

Giovannoni

Maggio²,

Bianco³

et al. 2007

Neuron Glia Biol.

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Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeats within the coding sequence of the ataxin-1 protein. In the present study, we used a conditional transgenic mouse model of SCA1 to investigate very early molecular and morphological changes related to the behavioral phenotype. In mice with neural deficits detected by rotarod performance, and simultaneous spatial impairments in exploratory activity and uncoordinated gait, we observed both significant altered expression and patchy distribution of excitatory amino acids transporter 1. The molecular changes observed in astroglial compartments correlate with changes in synapse morphology; synapses have a dramatic reduction of the synaptic area external to the postsynaptic density. By contrast, Purkinje cells demonstrate preserved structure. In addition, severe reactive astrocytosis matches changes in the glial glutamate transporter and synapse morphology. We propose these morpho-molecular changes are the cause of altered synaptic transmission, which, in turn, determines the onset of the neurological symptoms by altering the synaptic transmission in the cerebellar cortex of transgenic animals. This model might be suitable for testing drugs that target activated glial cells in order to reduce CNS inflammation.Keywords: EAAT1, synaptic plasticity, SCA1, neurodegeneration INTRODUCTIONThe contribution of non-neuronal cells to the pathogenesis of neurodegenerative diseases has been described although the mechanism remains poorly understood. The role of neuron-glia interactions is also being investigated in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (Sheldon and Robinson, 2007). Moreover, glial dysfunction is likely to contribute to the pathogenesis of cerebellar ataxia in a mouse model of spinocerebellar ataxia type 7 (SCA7) (Custer et al., 2006).Excitatory amino acids transporter 1 (EAAT1, also known as GLAST) is the major glutamate transporter in the cerebellum (Lehre and Danbolt, 1998). It is expressed strongly by astrocytes in the molecular layer of the cerebellum and is at highest density on Bergmann glia. EAAT1 is not detected in neurons (Ginsberg et al., 1995). EAAT1 present on the plasma membrane of the astrocytic processes and cell bodies (Chaudhry et al., 1995). Targeting of EAAT1 is regulated carefully on astroglial membranes facing the neuropil, large dendrites, cell bodies and capillary endothelium (Danbolt, 2001). Therefore, its distribution follows the morphological changes of astrocytes during inflammatory processes. Recently, in a mouse model of reactive astrocytosis in the spinal cord, it has been reported that this glial process includes a marked proteolytic cascade having as substrates glial transporters. Subsequent changes in neurotransmitter uptake represent the basis of morphological and functional changes that sustain central plasticity . Moreover, glial activation involves changes in cell phenotype and gene expression that might trig...

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Distribution of the glutamate transporters GLAST and GLT-1 in rat circumventricular organs, meninges, and dorsal root ganglia

Berger¹,

Hediger²

2000

J. Comp. Neurol.

105

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The glial glutamate transporters GLAST and GLT‐1 are primarily responsible for the removal of glutamate from brain extracellular fluid. This study compares the distribution of GLAST and GLT‐1 expression in the circumventricular organs of the brain, in the meninges, and in the dorsal root ganglion. By using a highly sensitive nonisotopic in situ hybridization method and immunostaining, we demonstrate marked differences in the expression patterns for the two transporters. In the three sensory circumventricular organs that contain neuronal elements, i.e., the subfornical organ, the vascular organ of the lamina terminalis, and the area postrema, GLAST is strongly expressed, whereas GLT‐1 is faintly expressed or absent. Both transporters are absent from the choroid plexus, and only GLAST mRNA is found in the subcommisural organ. In the pineal gland, GLAST is expressed by astrocytic cells near the pineal stalk, whereas GLT‐1 is expressed by pinealocytes throughout the gland. In the pituitary gland, GLAST is likely expressed by folliculo‐stellate cells in the anterior lobe, by a group of astrocyte‐like cells and by marginal cells in the intermediate lobe, and by pituicytes in the posterior lobe, whereas GLT‐1 is expressed only by the astrocyte‐like cells in the intermediate lobe. Finally, GLAST, but not GLT‐1, is expressed by specific layers of the meninges, and by satellite cells in the dorsal root ganglion. These results show that GLAST is the primary glutamate transporter in the circumventricular organs. The data provide further evidence that these two glutamate transporters fulfill markedly different functions in the nervous system. J. Comp. Neurol. 421:385–399, 2000. © 2000 Wiley‐Liss, Inc.

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The Number of Glutamate Transporter Subtype Molecules at Glutamatergic Synapses: Chemical and Stereological Quantification in Young Adult Rat Brain

Cited by 617 publications

References 67 publications

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

Reactive astrocytosis and glial glutamate transporter clustering are early changes in a spinocerebellar ataxia type 1 transgenic mouse model

Distribution of the glutamate transporters GLAST and GLT-1 in rat circumventricular organs, meninges, and dorsal root ganglia

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