Determination of CO2 Production in Subcellular Preparations Like Synaptosomes and Isolated Mitochondria Using 14C-Labeled Substrates and Radioactive CO2 Measurements

  • Mary C. McKennaEmail author
  • Irene B. Hopkins
Part of the Neuromethods book series (NM, volume 90)


The rate of 14CO2 production from 14C-labeled substrates is an indication of how actively the substrate is used for energy production by that tissue. This chapter focuses on the methods for determining the rates of 14CO2 production from freshly isolated synaptosomes and mitochondria from brain. The techniques for the isolation of synaptosomes from different age rat and/or mouse brain, and for the isolation of mitochondria are described in detail. Information is provided about how to set up the experiments for determining the rate of 14CO2 production (oxidation) from 14C-labeled substrates. Detailed information is given on performing the experiments and calculating the data. The techniques for performing substrate competition studies are also given. This chapter provides straightforward information about the useful techniques of determining the rates of 14CO2 production from any labeled 14C-substrate used by the brain for energy production. This technique can provide valuable information about substrate use in synaptosomes and mitochondria isolated from normal and/or abnormal brain.

Key words

Oxidation 14CO2 release Substrate competition Substrate metabolism Subcellular fractionation Synaptosomes Mitochondria Radioisotopes 


  1. 1.
    Waagepetersen HS et al (2002) Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons. Neurochem Res 27(11):1431–1437PubMedCrossRefGoogle Scholar
  2. 2.
    Waagepetersen HS et al (1999) Synthesis of vesicular GABA from glutamine involves TCA cycle metabolism in neocortical neurons. J Neurosci Res 57(3):342–349PubMedCrossRefGoogle Scholar
  3. 3.
    Yu AC et al (1984) Metabolic fate of [14C]- glutamine in mouse cerebral neurons in primary cultures. J Neurosci Res 11(4):351–357PubMedCrossRefGoogle Scholar
  4. 4.
    Olstad E et al (2007) Pyruvate recycling in cultured neurons from cerebellum. J Neurosci Res 85(15):3318–3325PubMedCrossRefGoogle Scholar
  5. 5.
    Olstad E, Qu H, Sonnewald U (2007) Glutamate is preferred over glutamine for intermediary metabolism in cultured cerebellar neurons. J Cereb Blood Flow Metab 27(4): 811–820PubMedGoogle Scholar
  6. 6.
    Sonnewald U et al (2004) First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures. J Neurochem 91(4):796–803PubMedCrossRefGoogle Scholar
  7. 7.
    Leong SF et al (1984) The activities of some energy-metabolising enzymes in nonsynaptic (free) and synaptic mitochondria derived from selected brain regions. J Neurochem 42(5): 1306–1312PubMedCrossRefGoogle Scholar
  8. 8.
    Lai JC et al (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J Neurochem 28(3):625–631PubMedCrossRefGoogle Scholar
  9. 9.
    Lai JC, Clark JB (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem J 154(2):423–432PubMedPubMedCentralGoogle Scholar
  10. 10.
    Lai JCK, Clark JB (1989) Isolation and characterization of synaptic and nonsynaptic mitochondria from mammalian brain. In: Boulton AA, Baker GB, Butterworth R (eds) Neuromethods, vol 11, Carbohydrates and energy metabolism (pp. 43–98). Humana Press, Clifton, NJGoogle Scholar
  11. 11.
    McKenna MC et al (1994) Energy metabolism in cortical synaptic terminals from weanling and mature rat brain: evidence for multiple compartments of tricarboxylic acid cycle activity. Dev Neurosci 16(5–6):291–300PubMedCrossRefGoogle Scholar
  12. 12.
    Lai JC, Clark JB (1979) Preparation of synaptic and nonsynaptic mitochondria from mammalian brain. Methods Enzymol 55:51–60PubMedCrossRefGoogle Scholar
  13. 13.
    McKenna MC et al (1998) Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity. Dev Neurosci 20(4–5):300–309PubMedCrossRefGoogle Scholar
  14. 14.
    McKenna MC et al (1993) Regulation of energy metabolism in synaptic terminals and cultured rat brain astrocytes: differences revealed using aminooxyacetate. Dev Neurosci 15(3–5):320–329PubMedCrossRefGoogle Scholar
  15. 15.
    McKenna MC et al (2000) Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells. Neurochem Int 36(4–5):451–459PubMedCrossRefGoogle Scholar
  16. 16.
    Sonnewald U, McKenna M (2002) Metabolic compartmentation in cortical synaptosomes: influence of glucose and preferential incorporation of endogenous glutamate into GABA. Neurochem Res 27(1–2):43–50PubMedCrossRefGoogle Scholar
  17. 17.
    Sims NR, Anderson MF (2008) Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3(7): 1228–1239PubMedCrossRefGoogle Scholar
  18. 18.
    Anderson MF, Sims NR (2000) Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Brain Res Protoc 5(1):95–101PubMedCrossRefGoogle Scholar
  19. 19.
    Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55(2):698–707PubMedCrossRefGoogle Scholar
  20. 20.
    Wang X et al (2011) Isolation of brain mitochondria from neonatal mice. J Neurochem 119(6):1253–1261PubMedCrossRefPubMedCentralGoogle Scholar
  21. 21.
    Tildon JT et al (1996) Modulation of central nervous system metabolism by macromolecules: effects of albumin and histones on glucose oxidation by synaptosomes. J Assoc Acad Minor Phys 7(2):47–52PubMedGoogle Scholar
  22. 22.
    Tildon JT, McKenna MC, Stevenson JH (1993) Differential effects of serum protein(s) on substrate oxidation by isolated synaptosomes and cultured rat brain astrocytes. Dev Neurosci 15(3–5):226–232PubMedCrossRefGoogle Scholar
  23. 23.
    Dienel GA, Wang RY, Cruz NF (2002) Generalized sensory stimulation of conscious rats increases labeling of oxidative pathways of glucose metabolism when the brain glucose-oxygen uptake ratio rises. J Cereb Blood Flow Metab 22(12):1490–1502PubMedCrossRefGoogle Scholar
  24. 24.
    Tildon JT, Stevenson JH, Roeder LM (1987) Serum effects on substrate oxidation by dissociated brain cells: possible sites of action. Brain Res 403(1):127–135PubMedCrossRefGoogle Scholar
  25. 25.
    Hawkins RA et al (1985) Cerebral glucose use measured with [14C]glucose labeled in the 1, 2, or 6 position. Am J Physiol 248(1 Pt 1): C170–C176PubMedGoogle Scholar
  26. 26.
    McKenna MC, Hopkins IB, Carey A (2001) Alpha-cyano-4-hydroxycinnamate decreases both glucose and lactate metabolism in neurons and astrocytes: implications for lactate as an energy substrate for neurons. J Neurosci Res 66(5):747–754PubMedCrossRefGoogle Scholar
  27. 27.
    Lai JC (1992) Oxidative metabolism in neuronal and non-neuronal mitochondria. Can J Physiol Pharmacol 70(Suppl):S130–S137PubMedCrossRefGoogle Scholar
  28. 28.
    Kristian T et al (2006) Isolation of mitochondria with high respiratory control from primary cultures of neurons and astrocytes using nitrogen cavitation. J Neurosci Methods 152(1–2): 136–143PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Cheeseman AJ, Clark JB (1988) Influence of the malate-aspartate shuttle on oxidative metabolism in synaptosomes. J Neurochem 50(5):1559–1565PubMedCrossRefGoogle Scholar
  30. 30.
    Tildon JT, Roeder LM, Stevenson JH (1985) Substrate oxidation by isolated rat brain mitochondria and synaptosomes. J Neurosci Res 14(2):207–215PubMedCrossRefGoogle Scholar
  31. 31.
    McKenna MC et al (1996) New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes. Dev Neurosci 18(5–6):380–390PubMedCrossRefGoogle Scholar
  32. 32.
    Smith PK et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76–85PubMedCrossRefGoogle Scholar
  33. 33.
    McKenna MC (2012) Substrate competition studies demonstrate oxidative metabolism of glucose, glutamate, glutamine, lactate and 3-hydroxybutyrate in cortical astrocytes from rat brain. Neurochem Res 37(11):2613–2626PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    McKenna MC et al (2000) Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals. Neurochem Int 37(2–3):229–241PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Pediatrics and Program in NeuroscienceUniversity of Maryland School of MedicineBaltimoreUSA

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