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 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.
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References
Booher J, Sensenbrenner M (1972) Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2:97–105
Schousboe A (1980) Primary cultures of astrocytes from mammalian brain as a tool in neurochemical research. Cell Mol Biol Incl Cyto Enzymol 26:505–513
Hertz L, Juurlink BHJ, Szuchet S (1985) Cell cultures. In: Lajtha A (ed) Handbook of neurochemistry, vol 8, 2nd edn. Plenum Publishing Corporation, New York, pp 603–653
Waagepetersen HS, Sonnewald U, Schousboe A (2009) Energy and amino acid neurotransmitter metabolism in astrocytes. In: Parpura V, Haydon PG (eds) Astrocytes in (patho)physiology of the nervous system. Springer, Boston, pp 177–199
Westergaard N, Fosmark H, Schousboe A (1991) Metabolism and release of glutamate in cerebellar granule cells cocultured with astrocytes from cerebellum or cerebral cortex. J Neurochem 56:59–66
Waagepetersen HS, Sonnewald U, Larsson OM, Schousboe A (2000) A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons. J Neurochem 75:471–479
Eng LF, Vanderhaeghen JJ, Bignami A, Gerstl B (1971) An acidic protein isolated from fibrous astrocytes. Brain Res 28:351–354
Martinez-Hernandez A, Bell KP, Norenberg MD (1977) Glutamine synthetase: glial localization in brain. Science 195:1356–1358
Gegelashvili G, Schousboe A (1997) High affinity glutamate transporters: regulation of expression and activity. Mol Pharmacol 52:6–15
Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65:1–105
Bak LK, Sickmann HM, Schousboe A, Waagepetersen HS (2005) Activity of the lactate-alanine shuttle is independent of glutamate-glutamine cycle activity in cerebellar neuronal-astrocytic cultures. J Neurosci Res 79:88–96
Bak LK, Schousboe A, Sonnewald U, Waagepetersen HS (2006) Glucose is necessary to maintain neurotransmitter homeostasis during synaptic activity in cultured glutamatergic neurons. J Cereb Blood Flow Metab 26:1285–1297
Drejer J, Larsson OM, Schousboe A (1983) Characterization of uptake and release processes for d- and l-aspartate in primary cultures of astrocytes and cerebellar granule cells. Neurochem Res 8:231–243
Hertz L, Juurlink BHJ, Hertz E, Fosmark H, Schousboe A (1989) Preparation of primary cultures of mouse (rat) astrocytes. In: Shahar A, de Vellis J, Vernadakis A, Haber B (eds) A dissection and tissue culture manual of the nervous system. Alan R Liss, Inc., New York, pp 105–108
Hertz L, Juurlink BHJ, Fosmark H, Schousboe A (1982) Astrocytes in primary cultures. In: Pfeiffer SE (ed) Neuroscience approached through cell cultures. CRC Press, Boca Raton, pp 175–186
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Wilkinson GN (1961) Statistical estimations in enzyme kinetics. Biochem J 80:324–332
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Mawhinney TP, Robinett RS, Atalay A, Madson MA (1986) Analysis of amino acids as their tert-butyldimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry. J Chromatogr 358:231–242
Hertz L, Zielke HR (2004) Astrocytic control of glutamatergic activity: astrocytes as stars of the show. Trends Neurosci 27:735–743
Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary cultures of astrocytes and neurons. J Neurochem 41:1484–1487
Norenberg MD, Martinez-Hernandez A (1979) Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161:303–310
Juurlink BH, Schousboe A, Jørgensen OS, Hertz L (1981) Induction by hydrocortisone of glutamine synthetase in mouse primary astrocyte cultures. J Neurochem 36:136–142
Gegelashvili G, Danbolt NC, Schousboe A (1997) Neuronal soluble factors differentially regulate the expression of the GLT1 and GLAST glutamate transporters in cultured astroglia. J Neurochem 69:2612–2615
Gegelashvili G, Dehnes Y, Danbolt NC, Schousboe A (2000) The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms. Neurochem Int 37:163–170
Swanson RA, Liu J, Miller JW, Rothstein JD, Farrell K, Stein BA, Longuemare MC (1997) Neuronal regulation of glutamate transporter subtype expression in astrocytes. J Neurosci 17:932–940
Schlag BD, Vondrasek JR, Munir M, Kalandadze A, Zelenaia OA, Rothstein JD, Robinson MB (1998) Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons. Mol Pharmacol 53:355–369
Robinson MB (2002) Regulated trafficking of neurotransmitter transporters: common notes but different melodies. J Neurochem 80:1–11
O’Shea RD, Lau CL, Farso MC, Diwakarla S, Zagami CJ, Svendsen BB, Feeney SJ, Callaway JK, Jones NM, Pow DV, Danbolt NC, Jarrott B, Beart PM (2006) Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: evidence for delayed up-regulation and redistribution of GLT-1. Neurochem Int 48:604–610
Hertz L, Schousboe A, Boechler N, Mukerji S, Fedoroff S (1978) Kinetic characteristics of the glutamate uptake into normal astrocytes in cultures. Neurochem Res 3:1–14
Hertz L, Peng L, Dienel GA (2007) Energy metabolism in astrocytes: high rate of oxidative metabolism and spatiotemporal dependence on glycolysis/glycogenolysis. J Cereb Blood Flow Metab 27:219–249
Hassel B, Westergaard N, Schousboe A, Fonnum F (1995) Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate. Neurochem Res 20:413–420
Sonnewald U, Westergaard N, Schousboe A (1997) Glutamate transport and metabolism in astrocytes. Glia 21:56–63
Westergaard N, Drejer J, Schousboe A, Sonnewald U (1996) Evaluation of the importance of transamination versus deamination in astrocytic metabolism of [U-13C]glutamate. Glia 17:160–168
Sonnewald U, Westergaard N, Krane J, Unsgard G, Petersen SB, Schousboe A (1991) First direct demonstration of preferential release of citrate from astrocytes using [13C]NMR spectroscopy of cultured neurons and astrocytes. Neurosci Lett 128:235–239
Sonnewald U, Westergaard N, Hassel B, Muller TB, Unsgard G, Fonnum F, Hertz L, Schousboe A, Petersen SB (1993) NMR spectroscopic studies of 13C acetate and 13C glucose metabolism in neocortical astrocytes: evidence for mitochondrial heterogeneity. Dev Neurosci 15:351–358
Leo GC, Driscoll BF, Shank RP, Kaufman E (1993) Analysis of [1–13C]d-glucose metabolism in cultured astrocytes and neurons using nuclear magnetic resonance spectroscopy. Dev Neurosci 15:282–288
Waagepetersen HS, Sonnewald U, Larsson OM, Schousboe A (2001) Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes. Glia 35:246–252
Lajtha A, Berl S, Waelsch H (1959) Amino acid and protein metabolism of the brain-IV. The metabolism of glutamic acid. J Neurochem 3:322–332
Berl S, Lajtha A, Waelsch H (1961) Amino acid and protein metabolism-VI. Cerebral compartments of glutamic acid metabolism. J Neurochem 7:186–197
Walz W, Mukerji S (1988) Lactate release from cultured astrocytes and neurons: a comparison. Glia 1:366–370
Schousboe A, Westergaard N, Waagepetersen HS, Larsson OM, Bakken IJ, Sonnewald U (1997) Trafficking between glia and neurons of TCA cycle intermediates and related metabolites. Glia 21:99–105
Hassel B, Sonnewald U, Unsgård G, Fonnum F (1994) NMR spectroscopy of cultured astrocytes: effects of glutamine and the gliotoxin fluorocitrate. J Neurochem 62:2187–2194
Lovatt D, Sonnewald U, Waagepetersen HS, Schousboe A, He W, Lin JH, Han X, Takano T, Wang S, Sim FJ, Goldman SA, Nedergaard M (2007) The transcriptome and metabolic gene signature of protoplasmic astrocytes in the adult murine cortex. J Neurosci 27:12255–12266
Sonnewald U, Weatergaard N, Petersen SB, Unsgård G, Schousboe A (1993) Metabolism of [U-13C]glutamate in astrocytes studied by 13C NMR spectroscopy: Incorporation of more label into lactate than into glutamine demonstrates the importance of the TCA cycle. J Neurochem 61:1179–1182
McKenna MC, Sonnewald U, Huang X, Stevenson J, Zielke HR (1996) Exogenous glutamate concentration regulates the metabolic fate of glutamate in astrocytes. J Neurochem 66:386–393
Schousboe A, Svenneby G, Hertz L (1977) Uptake and metabolism of glutamate in astrocytes cultured from dissociated mouse brain hemispheres. J Neurochem 29:999–1005
Westergaard N, Varming T, Peng L, Sonnewald U, Hertz L, Schousboe A (1993) Uptake, release, and metabolism of alanine in neurons and astrocytes in primary cultures. J Neurosci Res 35:540–545
Acknowledgments
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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Special Issue: In Honor of Dr. Abel Lajtha.
Dorte M. Skytt and Karsten K. Madsen have equally contributed to this study.
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Skytt, D.M., Madsen, K.K., Pajęcka, K. et al. Characterization of Primary and Secondary Cultures of Astrocytes Prepared from Mouse Cerebral Cortex. Neurochem Res 35, 2043–2052 (2010). https://doi.org/10.1007/s11064-010-0329-6
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DOI: https://doi.org/10.1007/s11064-010-0329-6