Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex
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Astrocyte cultures were prepared from cerebral cortex of new-born and 7-day-old mice and additionally, the cultures from new-born animals were passaged as secondary cultures. The cultures were characterized by immunostaining for the astrocyte markers glutamine synthetase (GS), glial fibrillary acidic protein, and the glutamate transporters EAAT1 and EAAT2. The cultures prepared from 7-day-old animals were additionally characterized metabolically using 13C-labeled glucose and glutamate as well as 15N-labeled glutamate as substrates. All types of cultures exhibited pronounced immunostaining of the astrocyte marker proteins. The metabolic pattern of the cultures from 7-day-old animals of the labeled substrates was comparable to that seen previously in astrocyte cultures prepared from new-born mouse brain showing pronounced glycolytic and oxidative metabolism of glucose. Glutamate was metabolized both via the GS pathway and oxidatively via the tricarboxylic acid cycle as expected. Additionally, glutamate underwent pronounced transamination to aspartate and alanine and the intracellular pools of alanine and pyruvate exhibited compartmentation. Altogether the results show that cultures prepared from cerebral cortex of 7-day-old mice have metabolic and functional properties indistinguishable from those of classical astrocyte cultures prepared from neocortex of new-born animals. This provides flexibility with regard to preparation and use of these cultures for a variety of purposes.
KeywordsGlutamate transporters Glial fibrillary acidic protein (GFAP) Glutamine synthetase (GS) Glucose and glutamate metabolism
The expert secretarial and technical assistance from Ms. Hanne Danø, Lene Vigh and Heidi Nielsen is cordially acknowledged. Prof. N. C. Danbolt, University of Oslo is acknowledged for providing us with antibodies recognizing the two glutamate transporters EAAT1 and EAAT2. The work has been financially supported by the Carlsberg Foundation (2009_01_0501) and the Danish Medical Research Council (09-063399). One of the authors (AS) would like to take this opportunity to add a personal thank you to Dr. Lajtha for his continuous support and help during several decades. This support has been of significant importance for my scientific career.
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