Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex

Abstract

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 (13)C-labeled glucose and glutamate as well as (15)N-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.

The glutamate transporter, GLAST, participates in a macromolecular complex that supports glutamate metabolism

“…In this study we measured the stoichiomentry of CO 2 produced from transported glutamate. Previous studies using astrocytes (Farinelli and Nicklas, 1992; McKenna et al, 1996; Rao and Murthy, 1993; Skytt et al, 2010; Sonnewald et al, 1997; Waniewski and Martin, 1986; Yu et al, 1982), synaptosomes (Yudkoff et al, 1994), and brain slices (El Hage et al, 2011) have examined the catabolism of glutamate and its conversion to CO 2 with conflicting results. Some studies found a relatively small portion of glutamate flux through the tricarboxylic acid (TCA) cycle, whereas others found a large portion of glutamate entering the TCA cycle.…”

The chemokine, macrophage inflammatory protein-2γ, reduces the expression of glutamate transporter-1 on astrocytes and increases neuronal sensitivity to glutamate excitotoxicity

“…Primary astrocytes were prepared from neonatal (<24-hours old) SJL/J mouse brains using methods similar to those described previously [16,17]. Primary glial cell cultures were maintained in (D)MEM (Gibco, Carlsbad, CA, USA) supplemented with 10% FCS, 6 mg/ml glucose, and 5 μg/ml bovine pancreas insulin (Sigma-Aldrich, St. Louis, MO, USA), referred to as complete medium, in 10% CO 2 at 37°C.…”

Primary Cultures of Astrocytes: Their Value in Understanding Astrocytes in Health and Disease

“…Neuronal stem cells tend to be confined to the periventricular area, thus this is not a major issue for preparation of astrocytes from other brain regions. In view of the above, investigations of GFAP expression can be supplemented with other astrocyte-specific markers such as GS (20), pyruvate carboxylase (PC) (24, 25), excitatory amino acid transporter 2 (GLT-1) (26) or aldehyde dehydrogenase 1 family member L1 (Aldh1L1)(6). …”