Freshly isolated astrocyte (FIA) preparations: A useful single cell system for studying astrocyte properties

Kimelberg, Harold K.; Schools, Gary P.; Cai, Zhaohui; Zhou, Min

doi:10.1002/1097-4547(20000915)61:6<577::aid-jnr1>3.0.co;2-t

Cited by 31 publications

(25 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A concern with studies of cultured astrocytes is whether such studies accurately reflect physiological processes in vivo; thus, to begin to corroborate our studies in culture we used astrocytes freshly isolated from the hippocampus (33). After enzymatic treatment and trituration it is possible to isolate individual astrocytes from the hippocampus such that they maintain their fine ramified in vivo structure.…”

Section: Resultsmentioning

confidence: 99%

Fusion-related Release of Glutamate from Astrocytes

Zhang

Pangršič

Kreft

et al. 2004

Journal of Biological Chemistry

226

235

View full text Add to dashboard Cite

Although cell culture studies have implicated the presence of vesicle proteins in mediating the release of glutamate from astrocytes, definitive proof requires the identification of the glutamate release mechanism and the localization of this mechanism in astrocytes at synaptic locales. In cultured murine astrocytes we show an array of vesicle proteins, including SNARE proteins, and vesicular glutamate transporters that are required to fill vesicles with glutamate. Using immunocytochemistry and single-cell multiplex reverse transcription-PCR we demonstrate the presence of these proteins and their transcripts within astrocytes freshly isolated from the hippocampus. Moreover, immunoelectron microscopy demonstrates the presence of VGLUT1 in processes of astrocytes of the hippocampus. To determine whether calcium-dependent glutamate release is mediated by exocytosis, we expressed the SNARE motif of synaptobrevin II to prevent the formation of SNARE complexes, which reduces glutamate release from astrocytes. To further determine whether vesicular exocytosis mediates calcium-dependent glutamate release from astrocytes, we performed whole cell capacitance measurements from individual astrocytes and demonstrate an increase in whole cell capacitance, coincident with glutamate release. Together, these data allow us to conclude that astrocytes in situ express vesicle proteins necessary for filling vesicles with the chemical transmitter glutamate and that astrocytes release glutamate through a vesicle-or fusion-related mechanism.During the past decade there has been increasing evidence for both integrative and dynamic roles for astrocytes in the central nervous system. Following activation of G protein-coupled receptors, astrocytes exhibit calcium oscillations, leading to the release of the chemical transmitters glutamate and ATP (1-3). Studies in vitro and in brain slices have led to the hypothesis of tripartite synaptic transmission (4); neuronal activity causes elevations of synaptically associated calcium in astrocytes, which in turn leads to the release of chemical transmitters from these glial cells to locally modulate synaptic transmission (2, 5-7).The mechanisms mediating the release of these transmitters from astrocytes are, however, ill-defined and are still the subject of intense debate. At least three distinct release pathways have been proposed as mediating the calcium-dependent release of glutamate from astrocytes: the reversal of plasma membrane glutamate transporters, anion transporter mediate release mechanisms, and calcium-dependent exocytosis (8 -10). Because the release of glutamate is stimulated by calcium elevations and is not affected by glutamate transport inhibitors and because changes in cell volume have not been detected coincident with release, it has been proposed that this transmitter is released through a vesicle-mediated exocytotic pathway.Several observations made using cultured astrocytes support such a vesicle-mediated exocytotic mechanism of glutamate release, including the calcium depend...

show abstract

Section: Resultsmentioning

confidence: 99%

Fusion-related Release of Glutamate from Astrocytes

Zhang

Pangršič

Kreft

et al. 2004

Journal of Biological Chemistry

226

235

View full text Add to dashboard Cite

show abstract

“…Because astrocytes could de-differentiate while cultured, possibly leading to gene expression of VGLUTs, we next used freshly (acutely) isolated astrocytes that provide information of properties without the changes in gene expression (Kimelberg et al, 2000a(Kimelberg et al, ,b, 2001). Astrocytes were freshly isolated from rat visual cortices of 1-, 2-, 8-, and 55-d-old rats.…”

Section: Resultsmentioning

confidence: 99%

Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes

Montana¹,

Ni²,

Sunjara³

et al. 2004

J. Neurosci.

340

353

View full text Add to dashboard Cite

Astrocytes exhibit excitability based on variations of their intracellular Ca 2ϩ concentrations, which leads to glutamate release, that in turn can signal to adjacent neurons. This glutamate-mediated astrocyte-neuron signaling occurs at physiological intracellular Ca 2ϩ levels in astrocytes and includes modulation of synaptic transmission. The mechanism underlying Ca 2ϩ -dependent glutamate release from astrocytes is most likely exocytosis, because astrocytes express the protein components of the soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptors complex, including synaptobrevin 2, syntaxin, and synaptosome-associated protein of 23 kDa. Although these proteins mediate Ca 2ϩ -dependent glutamate release from astrocytes, it is not well understood whether astrocytes express functional vesicular glutamate transporters (VGLUTs) that are critical for vesicle refilling. Here, we find in cultured and freshly isolated astrocytes the presence of brain-specific Na ϩ -dependent inorganic phosphate cotransporter and differentiation-associated Na ϩ -dependent inorganic phosphate cotransporter that have recently been identified as VGLUTs 1 and 2. Indirect immunocytochemistry showed a punctate pattern of VGLUT immunoreactivity throughout the entire cell body and processes, whereas pharmacological inhibition of VGLUTs abolished mechanically and agonist-evoked Ca 2ϩ -dependent glutamate release from astrocytes. Taken together, these data indicate that VGLUTs play a functional role in exocytotic glutamate release from astrocytes.

show abstract

“…To ask whether this outcome represented neuronal or glial localization, we used individual identified cells freshly isolated from living hip- pocampi. Within 45 min after killing animals, cells were isolated, at which point they maintained their in vivo properties (33). By using pGFAP-GFP-transgenic mice in which the human GFAP promoter drives the expression of GFP selectively in astrocytes (ref.…”

Section: Resultsmentioning

confidence: 99%

Synaptotagmin IV regulates glial glutamate release

Zhang

Fukuda

Bockstaele

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

195

168

View full text Add to dashboard Cite

Calcium-binding synaptotagmins (Syts) are membrane proteins that are conserved from nematode to human. Fifteen Syts (Syts I-XV) have been identified in mammalian species. Syt I has been well studied and is a candidate for the Ca 2؉ -sensor that triggers evoked exocytosis underlying fast synaptic transmission. Whereas the functions of the other Syts are unclear, Syt IV is of particular interest because it is rapidly up-regulated after chronic depolarization or seizures, and because null mutations exhibit deficits in fine motor coordination and hippocampus-dependent memory. Screening Syts I-XIII, which are enriched in brain, we find that Syt IV is located in processes of astroglia in situ. Reduction of Syt IV in astrocytes by RNA interference decreases Ca 2؉ -dependent glutamate release, a gliotransmission pathway that regulates synaptic transmission. Mutants of the C2B domain, the only putative Ca 2؉ -binding domain in Syt IV, act in a dominant-negative fashion over Ca 2؉ -regulated glial glutamate release, but not gliotransmission induced by changes in osmolarity. Because we find that Syt IV is expressed predominantly by astrocytes and is not in the presynaptic terminals of the hippocampus, and because Syt IV knockout mice exhibit hippocampal-based memory deficits, our data raise the intriguing possibility that Syt IV-mediated gliotransmission contributes to hippocampal-based memory.

show abstract

Freshly isolated astrocyte (FIA) preparations: A useful single cell system for studying astrocyte properties

Cited by 31 publications

References 73 publications

Fusion-related Release of Glutamate from Astrocytes

Fusion-related Release of Glutamate from Astrocytes

Vesicular Glutamate Transporter-Dependent Glutamate Release from Astrocytes

Synaptotagmin IV regulates glial glutamate release

Contact Info

Product

Resources

About