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Brain Energy Metabolism pp 1–24Cite as

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

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

Abstract

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.

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References

  1. Waagepetersen HS et al (2002) Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons. Neurochem Res 27(11):1431–1437

    Article  PubMed  CAS  Google Scholar 

  2. Waagepetersen HS et al (1999) Synthesis of vesicular GABA from glutamine involves TCA cycle metabolism in neocortical neurons. J Neurosci Res 57(3):342–349

    Article  PubMed  CAS  Google Scholar 

  3. Yu AC et al (1984) Metabolic fate of [14C]- glutamine in mouse cerebral neurons in primary cultures. J Neurosci Res 11(4):351–357

    Article  PubMed  CAS  Google Scholar 

  4. Olstad E et al (2007) Pyruvate recycling in cultured neurons from cerebellum. J Neurosci Res 85(15):3318–3325

    Article  PubMed  CAS  Google Scholar 

  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): 811–820

    PubMed  CAS  Google Scholar 

  6. Sonnewald U et al (2004) First direct demonstration of extensive GABA synthesis in mouse cerebellar neuronal cultures. J Neurochem 91(4):796–803

    Article  PubMed  CAS  Google Scholar 

  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): 1306–1312

    Article  PubMed  CAS  Google Scholar 

  8. Lai JC et al (1977) Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization. J Neurochem 28(3):625–631

    Article  PubMed  CAS  Google Scholar 

  9. Lai JC, Clark JB (1976) Preparation and properties of mitochondria derived from synaptosomes. Biochem J 154(2):423–432

    PubMed  CAS  PubMed Central  Google Scholar 

  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, NJ

    Google Scholar 

  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):291–300

    Article  PubMed  CAS  Google Scholar 

  12. Lai JC, Clark JB (1979) Preparation of synaptic and nonsynaptic mitochondria from mammalian brain. Methods Enzymol 55:51–60

    Article  PubMed  CAS  Google Scholar 

  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):300–309

    Article  PubMed  CAS  Google Scholar 

  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):320–329

    Article  PubMed  CAS  Google Scholar 

  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):451–459

    Article  PubMed  CAS  Google Scholar 

  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):43–50

    Article  PubMed  CAS  Google Scholar 

  17. Sims NR, Anderson MF (2008) Isolation of mitochondria from rat brain using Percoll density gradient centrifugation. Nat Protoc 3(7): 1228–1239

    Article  PubMed  CAS  Google Scholar 

  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):95–101

    Article  PubMed  CAS  Google Scholar 

  19. Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55(2):698–707

    Article  PubMed  CAS  Google Scholar 

  20. Wang X et al (2011) Isolation of brain mitochondria from neonatal mice. J Neurochem 119(6):1253–1261

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  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):47–52

    PubMed  CAS  Google Scholar 

  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):226–232

    Article  PubMed  CAS  Google Scholar 

  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):1490–1502

    Article  PubMed  CAS  Google Scholar 

  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):127–135

    Article  PubMed  CAS  Google Scholar 

  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–C176

    PubMed  CAS  Google Scholar 

  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):747–754

    Article  PubMed  CAS  Google Scholar 

  27. Lai JC (1992) Oxidative metabolism in neuronal and non-neuronal mitochondria. Can J Physiol Pharmacol 70(Suppl):S130–S137

    Article  PubMed  CAS  Google Scholar 

  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): 136–143

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Cheeseman AJ, Clark JB (1988) Influence of the malate-aspartate shuttle on oxidative metabolism in synaptosomes. J Neurochem 50(5):1559–1565

    Article  PubMed  CAS  Google Scholar 

  30. Tildon JT, Roeder LM, Stevenson JH (1985) Substrate oxidation by isolated rat brain mitochondria and synaptosomes. J Neurosci Res 14(2):207–215

    Article  PubMed  CAS  Google Scholar 

  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):380–390

    Article  PubMed  CAS  Google Scholar 

  32. Smith PK et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1): 76–85

    Article  PubMed  CAS  Google Scholar 

  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):2613–2626

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  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):229–241

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pediatrics and Program in Neuroscience, University of Maryland School of Medicine, 655 W. Baltimore Street, BRB Room 13-019, Baltimore, MD, 21201, USA

    Mary C. McKenna Ph.D. & Irene B. Hopkins

Authors
  1. Mary C. McKenna Ph.D.
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  2. Irene B. Hopkins
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Corresponding author

Correspondence to Mary C. McKenna Ph.D. .

Editor information

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

Appendices

Appendix 1: Sample Reaction Mixture sheet

figure afigure a

Appendix 2: Sample Protocol Sheet

Experimental protocol sheet—tube set up

14C Substrate

Litter

 

Vol

Vol of tissue

Total

 

Number

Tubes

Rx mix

Synaptosomes

Vol in tube

1-GLC

A

1–6

0.6

0.1

0.7

6-GLC

A

7–12

0.6

0.1

0.7

U-GLU

A

13–18

0.6

0.1

0.7

U-LAC

A

19–24

0.6

0.1

0.7

1-GLC

B

25–30

0.6

0.1

0.7

6-GLC

B

31–36

0.6

0.1

0.7

U-GLU

B

37–42

0.6

0.1

0.7

U-LAC

B

43–48

0.6

0.1

0.7

1-GLC

C

49–54

0.6

0.1

0.7

6-GLC

C

55–60

0.6

0.1

0.7

U-GLU

C

61–66

0.6

0.1

0.7

U-LAC

C

67–72

0.6

0.1

0.7

No-tissue blanksa

   

Vol of water

 

1-GLC

–

73–77

0.6

0.1

0.7

6-GLC

–

78–82

0.6

0.1

0.7

U-GLU

–

83–87

0.6

0.1

0.7

U-LAC

–

88–92

0.6

0.1

0.7

  1. aNote that the center wells from these no-tissue blanks are counted (10 min per tube) before the experimental tubes when samples are placed in the scintillation counter (see Appendix 3). This is done for ease in setting up the excel spreadsheet for calculating the data (see Appendix 3 below)

Appendix 3: Sample Calculations

figure bfigure b

*Data from Synap E could not be used because the protein content was much higher than the range of 0.2-0.4 mg per 0.2 ml

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McKenna, M.C., Hopkins, I.B. (2014). Determination of CO2 Production in Subcellular Preparations Like Synaptosomes and Isolated Mitochondria Using 14C-Labeled Substrates and Radioactive CO2 Measurements. 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_1

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  • DOI: https://doi.org/10.1007/978-1-4939-1059-5_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1058-8

  • Online ISBN: 978-1-4939-1059-5

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