Skip to main content

Primary Cultures of Astrocytes and Neurons as Model Systems to Study the Metabolism and Metabolite Export from Brain Cells

  • Protocol
  • First Online:
Brain Energy Metabolism

Part of the book series: Neuromethods ((NM,volume 90))

Abstract

Primary cultures of astrocytes and neurons are frequently used to investigate metabolic properties of these two important brain cell types. Here we describe methods to generate primary cultures that are highly enriched in astrocytes or cerebellar granule neurons. These cultures are good model systems to investigate the basal metabolism of astrocytes and neurons as well as metabolite export from these cells. As examples for such studies, we describe here in detail the robust practical procedures that we use to investigate cellular glucose consumption and lactate production as well as the cellular contents and the export of glutathione from cultured brain cells. In this context we also describe some viability assays that are useful to confirm that a given experimental treatment is not toxic for cultured neural cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 139.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hirrlinger J, Dringen R (2010) The cytosolic redox state of astrocytes: maintenance, regulation and functional implications for metabolite trafficking. Brain Res Rev 63:177–188

    Article  PubMed  CAS  Google Scholar 

  2. Schmidt MM, Dringen R (2012) GSH synthesis and metabolism. In: Gruetter R, Choi IY (eds) Advances in neurobiology, vol 4: neural metabolism in vivo. Springer, New York, NY, pp 1029–1050

    Google Scholar 

  3. Parpura V, Heneka MT, Montana V, Oliet SH, Schousboe A, Haydon PG, Stout RF Jr, Spray DC, Reichenbach A, Pannicke T, Pekny M, Pekna M, Zorec R, Verkhratsky A (2012) Glial cells in (patho)physiology. J Neurochem 121: 4–27

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Lange SC, Bak LK, Waagepetersen HS, Schousboe A, Norenberg MD (2012) Primary cultures of astrocytes: their value in understanding astrocytes in health and disease. Neurochem Res 37:2569–2588

    Article  PubMed  CAS  Google Scholar 

  5. Hamprecht B, Löffler F (1985) Primary glial cultures as a model for studying hormone action. Methods Enzymol 109:341–345

    Article  PubMed  CAS  Google Scholar 

  6. Anggono V, Cousin MA, Robinson PJ (2008) Styryl dye-based synaptic vesicle recycling assay in cultured cerebellar granule neurons. Methods Mol Biol 457:333–345

    Article  PubMed  CAS  Google Scholar 

  7. Dringen R, Kussmaul L, Hamprecht B (1998) Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay. Brain Res Brain Res Protoc 2:223–228

    Article  PubMed  CAS  Google Scholar 

  8. Scheiber IF, Schmidt MM, Dringen R (2010) Zinc prevents the copper-induced damage of cultured astrocytes. Neurochem Int 57: 314–322

    Article  PubMed  CAS  Google Scholar 

  9. Gutterer JM, Dringen R, Hirrlinger J, Hamprecht B (1999) Purification of glutathione reductase from bovine brain, generation of an antiserum, and immunocytochemical localization of the enzyme in neural cells. J Neurochem 73:1422–1430

    Article  PubMed  CAS  Google Scholar 

  10. Dang TN, Bishop GM, Dringen R, Robinson SR (2010) The putative heme transporter HCP1 is expressed in cultured astrocytes and contributes to the uptake of hemin. Glia 58: 55–65

    Article  PubMed  Google Scholar 

  11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  12. Dobryszycka W, Owczarek H (1981) Effects of lead, copper, and zinc on the rat’s lactate dehydrogenase in vivo and in vitro. Arch Toxicol 48:21–27

    Article  PubMed  CAS  Google Scholar 

  13. Pamp K, Bramey T, Kirsch M, De Groot H, Petrat F (2005) NAD(H) enhances the Cu(II)-mediated inactivation of lactate dehydrogenase by increasing the accessibility of sulfhydryl groups. Free Radic Res 39:31–40

    Article  PubMed  CAS  Google Scholar 

  14. Hudson B, Upholt WB, Devinny J, Vinograd J (1969) The use of an ethidium analogue in the dye-buoyant density procedure for the isolation of closed circular DNA: the variation of the superhelix density of mitochondrial DNA. Proc Natl Acad Sci U S A 62:813–820

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Dringen R, Hamprecht B (1992) Glucose, insulin, and insulin-like growth factor-I regulate the glycogen-content of astroglia-rich primary cultures. J Neurochem 58:511–517

    Article  PubMed  CAS  Google Scholar 

  16. Liddell JR, Zwingmann C, Schmidt MM, Thiessen A, Leibfritz D, Robinson SR, Dringen R (2009) Sustained hydrogen peroxide stress decreases lactate production by cultured astrocytes. J Neurosci Res 87:2696–2708

    Article  PubMed  CAS  Google Scholar 

  17. Dringen R, Gebhardt R, Hamprecht B (1993) Glycogen in astrocytes: possible function as lactate supply for neighboring cells. Brain Res 623:208–214

    Article  PubMed  CAS  Google Scholar 

  18. Schmidt MM, Dringen R (2009) Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes. Front Neuroenergetics 1:1–10

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502–522

    Article  PubMed  CAS  Google Scholar 

  20. Dringen R, Hamprecht B (1996) Glutathione content as an indicator for the presence of metabolic pathways of amino acids in astroglial cultures. J Neurochem 67:1375–1382

    Article  PubMed  CAS  Google Scholar 

  21. Dringen R, Kranich O, Hamprecht B (1997) The γ-glutamyl transpeptidase inhibitor acivicin preserves glutathione released by astroglial cells in culture. Neurochem Res 22:727–733

    Article  PubMed  CAS  Google Scholar 

  22. Hirrlinger J, Dringen R (2005) Multidrug resistance protein 1-mediated export of glutathione and glutathione disulfide from brain astrocytes. Methods Enzymol 400:395–409

    Article  PubMed  CAS  Google Scholar 

  23. Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione-reductase and 2-vinylpyridine. Anal Biochem 106:207–212

    Article  PubMed  CAS  Google Scholar 

  24. Rahman B, Kussmaul L, Hamprecht B, Dringen R (2000) Glycogen is mobilized during the disposal of peroxides by cultured astroglial cells from rat brain. Neurosci Lett 290:169–172

    Article  PubMed  CAS  Google Scholar 

  25. Allaman I, Pellerin L, Magistretti PJ (2004) Glucocorticoids modulate neurotransmitter-induced glycogen metabolism in cultured cortical astrocytes. J Neurochem 88:900–908

    Article  PubMed  CAS  Google Scholar 

  26. Waagepetersen HS, Sonnewald U, Schousboe A (2003) Compartmentation of glutamine, glutamate, and GABA metabolism in neurons and astrocytes: functional implications. Neuroscientist 9:398–403

    Article  PubMed  CAS  Google Scholar 

  27. Wilhelm F, Hirrlinger J (2011) The NAD+/NADH redox state in astrocytes: independent control of the NAD+ and NADH content. J Neurosci Res 89:1956–1964

    Article  PubMed  CAS  Google Scholar 

  28. Wilhelm F, Hirrlinger J (2012) Multifunctional roles of NAD+ and NADH in astrocytes. Neurochem Res 37:2317–2325

    Article  PubMed  CAS  Google Scholar 

  29. Schmidlin A, Wiesinger H (1995) Stimulation of arginine transport and nitric oxide production by lipopolysaccharide is mediated by different signaling pathways in astrocytes. J Neurochem 65:590–594

    Article  PubMed  CAS  Google Scholar 

  30. Minich T, Yokota S, Dringen R (2003) Cytosolic and mitochondrial isoforms of NADP+-dependent isocitrate dehydrogenases are expressed in cultured rat neurons, astrocytes, oligodendrocytes and microglial cells. J Neurochem 86:605–614

    Article  PubMed  CAS  Google Scholar 

  31. Hirrlinger J, Resch A, Gutterer JM, Dringen R (2002) Oligodendroglial cells in culture effectively dispose of exogenous hydrogen peroxide: comparison with cultured neurones, astroglial and microglial cells. J Neurochem 82: 635–644

    Article  PubMed  CAS  Google Scholar 

  32. Dauth S, Schmidt MM, Rehders M, Dietz F, Kelm S, Dringen R, Brix K (2012) Characterization and metabolism of astroglia-rich primary cultures from cathepsin K-deficient mice. Biol Chem 393:959–970

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We very much like to thank Dr. Bernd Hamprecht (Tuebingen, Germany) and Dr. Mike Cousin (Edinburgh, Scotland) for the opportunities to learn the methods to prepare APCs and CGNCs, respectively, in their laboratories.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ralf Dringen .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Tulpule, K., Hohnholt, M.C., Hirrlinger, J., Dringen, R. (2014). Primary Cultures of Astrocytes and Neurons as Model Systems to Study the Metabolism and Metabolite Export from Brain Cells. In: Hirrlinger, J., Waagepetersen, H. (eds) Brain Energy Metabolism. Neuromethods, vol 90. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1059-5_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1059-5_3

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1058-8

  • Online ISBN: 978-1-4939-1059-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics