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Metabolic Mapping of Astrocytes and Neurons in Culture Using Stable Isotopes and Gas Chromatography-Mass Spectrometry (GC-MS)

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Brain Energy Metabolism

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

Abstract

The experimental procedure involved in metabolic mapping of astrocytes and neurons in culture using stable isotopes and gas chromatography-mass spectrometry (GC-MS) comprises several steps each of which has to be thoroughly considered in order to obtain the desired information. In this chapter we describe the different aspects to be considered when designing an incubation experiment to provide information about cellular metabolism in vitro, i.e., incubation time, incubation medium, and which isotope to employ. The labeling patterns obtained in several metabolites following metabolism of a variety of 13C or 15N labeled precursors through different metabolic pathways are depicted in order to provide sufficient insight to enable the reader to select the best suited precursor. The cell extraction and sample preparation procedures required before GC-MS analysis are described as are the subsequent integration of chromatograms and the calculations needed to correct for natural abundance and to obtain percentage labeling of M, M + 1, M + 2, etc. in a compound. Also, the cell culturing procedure for preparing primary cultures of neurons and astrocytes and also co-cultures of these cell types isolated from either mouse cerebral cortex or cerebellum is described in detail.

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Abbreviations

CSF:

Cerebrospinal fluid

dBcAMP:

dibutyryl cyclic AMP

DMF:

N,N-Dimethylformamide

f.c.:

Final concentration

GABA-T:

GABA transaminase

GAD:

Glutamate decarboxylase

GC-MS:

Gas chromatography-mass spectrometry

GDH:

Glutamate dehydrogenase

GS:

Glutamine synthetase

LAF hood:

Laminar flow hood

MCL:

Molecular carbon labeling

MS:

Mass spectrometry

MSTFA:

N-methyl-N-(trimethylsilyl)trifluoroacetamide

MTBSTFA:

N-methyl-N-(tert.-butyldimethylsilyl)trifluoroactamide

NMR:

Nuclear magnetic resonance

PC:

Pyruvate carboxylase

PDH:

Pyruvate dehydrogenase

PEPCK:

Phosphoenolpyruvate carboxykinase

PPP:

Pentose phosphate pathway

TCA:

Tricarboxylic acid

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Author information

Authors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Jagtvej 160, 2100, Copenhagen, Denmark

    Anne B. Walls, Lasse K. Bak & Arne Schousboe

  2. Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology, Trondheim, Norway

    Ursula Sonnewald

  3. Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Helle S. Waagepetersen

Authors
  1. Anne B. Walls
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  2. Lasse K. Bak
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  3. Ursula Sonnewald
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  4. Arne Schousboe
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  5. Helle S. Waagepetersen
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Corresponding author

Correspondence to Anne B. Walls .

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Editors and Affiliations

  1. Carl-Ludwig-Institute for Physiology, University of Leipzig, Leipzig, Germany

    Johannes Hirrlinger

  2. Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

    Helle S. Waagepetersen

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Walls, A.B., Bak, L.K., Sonnewald, U., Schousboe, A., Waagepetersen, H.S. (2014). Metabolic Mapping of Astrocytes and Neurons in Culture Using Stable Isotopes and Gas Chromatography-Mass Spectrometry (GC-MS). 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_4

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  • DOI: https://doi.org/10.1007/978-1-4939-1059-5_4

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