Skip to main content
SpringerLink
Log in

Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons

  • Protocol
  • First Online:
Techniques to Investigate Mitochondrial Function in Neurons

Part of the book series: Neuromethods ((NM,volume 123))

Abstract

Mitochondria are the most prominent cellular source of reactive oxygen species. As a by-product of cellular respiration, superoxide constantly escapes from the electron transport chain and is converted into other reactive oxygen and nitrogen species, which then may mediate downstream redox changes also in neighboring compartments and organelles. Such mitochondria-derived redox signals crucially contribute to the modulation of normal cell function but may as well cause random oxidative damage to various cellular constituents and evoke aberrant signaling. The resulting redox stress is considered to contribute to the onset and progression of various neuropathologies. Hence, there is a tremendous interest in mapping subcellular ROS levels as well as redox changes and to understand their spatiotemporal dynamics.

It is only since the development of genetically encoded fluorescent redox sensors that such analysis has become possible in a reliable manner. These advanced optical sensors overcome the severe disadvantages of oxidation -sensitive synthetic fluorescent dyes. For the first time they allow to monitor both reducing as well as oxidizing changes on the subcellular level, to decipher their detailed dynamics, and to quantify their very extent. In this chapter we summarize the properties of protein-based optical redox indicators and explain their superiority to synthetic dyes. Furthermore, we address the challenges of a proper and efficient delivery of the sensor-coding DNA, with a special emphasis on viral transduction and vector design. Finally, we give a detailed description of redox live-imaging applications in different neuronal preparations and point out to potential pitfalls.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Senior AE (1988) ATP synthesis by oxidative phosphorylation. Physiol Rev 68:177–231

    CAS  PubMed  Google Scholar 

  2. Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134:707–716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dröge W (2002) Free radicals in the physiological control of cell function. Physiol Rev 82:47–95

    Article  PubMed  Google Scholar 

  4. Brand MD (2010) The sites and topology of mitochondrial superoxide production. Exp Gerontol 45:466–472

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cadenas E, Davies KJ (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29:222–230

    Article  CAS  PubMed  Google Scholar 

  6. Lipton SA, Nicotera P (1998) Calcium, free radicals and excitotoxins in neuronal apoptosis. Cell Calcium 23:165–171

    Article  CAS  PubMed  Google Scholar 

  7. Kamsler A, Segal M (2003) Hydrogen peroxide modulation of synaptic plasticity. J Neurosci 23:269–276

    CAS  PubMed  Google Scholar 

  8. Atkins CM, Sweatt JD (1999) Reactive oxygen species mediate activity-dependent neuron-glia signaling in output fibers of the hippocampus. J Neurosci 19:7241–7248

    CAS  PubMed  Google Scholar 

  9. Finkel T (2011) Signal transduction by reactive oxygen species. J Cell Biol 194:7–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schulz JB, Beal MF (1994) Mitochondrial dysfunction in movement disorders. Curr Opin Neurol 7:333–339

    Article  CAS  PubMed  Google Scholar 

  11. Cooper JM, Schapira AH (1997) Mitochondrial dysfunction in neurodegeneration. J Bioenerg Biomembr 29:175–183

    Article  CAS  PubMed  Google Scholar 

  12. Dahl HH (1998) Getting to the nucleus of mitochondrial disorders: identification of respiratory chain-enzyme genes causing Leigh syndrome. Am J Hum Genet 63:1594–1597

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Behl C, Moosmann B (2002) Oxidative nerve cell death in Alzheimer’s disease and stroke: antioxidants as neuroprotective compounds. Biol Chem 383:521–536

    Article  CAS  PubMed  Google Scholar 

  14. Kong J, Xu Z (1998) Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J Neurosci 18:3241–3250

    CAS  PubMed  Google Scholar 

  15. Kovács R, Schuchmann S, Gabriel S, Kann O, Kardos J, Heinemann U (2002) Free radical-mediated cell damage after experimental status epilepticus in hippocampal slice culture. J Neurophsiol 88:2909–2918

    Article  Google Scholar 

  16. Großer E, Hirt U, Janc OA, Menzfeld C, Fischer M, Kempkes B, Vogelgesang S, Manzke TU, Opitz L, Salinas-Riester G, Müller M (2012) Oxidative burden and mitochondrial dysfunction in a mouse model of Rett syndrome. Neurobiol Dis 48:102–114

    Article  PubMed  Google Scholar 

  17. Müller M, Can K (2014) Aberrant redox homoeostasis and mitochondrial dysfunction in Rett syndrome. Biochem Soc Trans 42:959–964

    Article  PubMed  Google Scholar 

  18. Chan PH (1996) Role of oxidants in ischemic brain damage. Stroke 27:1124–1129

    Article  CAS  PubMed  Google Scholar 

  19. Martin BL, Wu D, Jakes S, Graves DJ (1990) Chemical influences on the specificity of tyrosine phosphorylation. J Biol Chem 265:7108–7111

    CAS  PubMed  Google Scholar 

  20. Lipton SA, Choi YB, Takahashi H, Zhang D, Li W, Godzik A, Bankston LA (2002) Cysteine regulation of protein function--as exemplified by NMDA-receptor modulation. Trends Neurosci 25:474–480

    Article  CAS  PubMed  Google Scholar 

  21. Ostergaard H, Henriksen A, Hansen FG, Winther JR (2001) Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 20:5853–5862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Meyer AJ, Dick TP (2010) Fluorescent protein-based redox probes. Antioxid Redox Signal 13:621–650

    Article  CAS  PubMed  Google Scholar 

  23. Björnberg O, Ostergaard H, Winther JR (2006) Measuring intracellular redox conditions using GFP-based sensors. Antioxid Redox Signal 8:354–361

    Article  PubMed  Google Scholar 

  24. LeBel CP, Ischiropoulos H Bondy SC (1992) Evaluation of the probe 2′,7′-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem Res Toxicol 5:227–231

    Google Scholar 

  25. Gallop PM, Paz MA, Henson E, Latt SA (1984) Dynamic approaches to the delivery of reporter reagents into living cells. BioTechniques 2:32–36

    CAS  Google Scholar 

  26. Mohanty JG, Jaffe JS, Schulman ES, Raible DG (1997) A highly sensitive fluorescent micro-assay of H2O2 release from activated human leukocytes using a dihydroxyphenoxazine derivative. J Immunol Methods 202:133–141

    Article  CAS  PubMed  Google Scholar 

  27. Dugan LL, Sensi SL, Canzoniero LM, Handran SD, Rothman SM, Lin TS, Goldberg MP, Choi DW (1995) Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci 15:6377–6388

    CAS  PubMed  Google Scholar 

  28. Esposti MD, Hatzinisiriou I, McLennan H, Ralph S (1999) Bcl-2 and mitochondrial oxygen radicals. New approaches with reactive oxygen species-sensitive probes. J Biol Chem 274:29831–29837

    Article  CAS  PubMed  Google Scholar 

  29. Robinson KM, Janes MS, Pehar M, Monette JS, Ross MF, Hagen TM, Murphy MP, Beckman JS (2006) Selective fluorescent imaging of superoxide in vivo using ethidium-based probes. Proc Natl Acad Sci U S A 103:15038–15043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen CS, Gee KR (2000) Redox-dependent trafficking of 2,3,4,5, 6-pentafluorodihydrotetramethylrosamine, a novel fluorogenic indicator of cellular oxidative activity. Free Radic Biol Med 28:1266–1278

    Article  CAS  PubMed  Google Scholar 

  31. Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053

    Article  CAS  PubMed  Google Scholar 

  32. Belousov VV, Fradkov AF, Lukyanov KA, Staroverov DB, Shakhbazov KS, Terskikh AV, Lukyanov S (2006) Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat Methods 3:281–286

    Article  CAS  PubMed  Google Scholar 

  33. Guzy RD, Hoyos B, Robin E, Chen H, Liu L, Mansfield KD, Simon MC, Hammerling U, Schumacker PT (2005) Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing. Cell Metab 1:401–408

    Article  CAS  PubMed  Google Scholar 

  34. Kolossov VL, Spring BQ, Sokolowski A, Conour JE, Clegg RM, Kenis PJ, Gaskins HR (2008) Engineering redox-sensitive linkers for genetically encoded FRET-based biosensors. Exp Biol Med 233:238–248

    Article  CAS  Google Scholar 

  35. Yano T, Oku M, Akeyama N, Itoyama A, Yurimoto H, Kuge S, Fujiki Y, Sakai Y (2010) A novel fluorescent sensor protein for visualization of redox states in the cytoplasm and in peroxisomes. Mol Cell Biol 30:3758–3766

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Funke F, Gerich FJ, Müller M (2011) Dynamic, semi-quantitative imaging of intracellular ROS levels and redox status in rat hippocampal neurons. Neuroimage 54:2590–2602

    Article  CAS  PubMed  Google Scholar 

  37. Foster KA, Galeffi F, Gerich FJ, Turner DA, Müller M (2006) Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration. Prog Neurobiol 79:136–171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279:22284–22293

    Article  CAS  PubMed  Google Scholar 

  39. Enyedi B, Varnai P, Geiszt M (2010) Redox state of the endoplasmic reticulum is controlled by Ero1L-alpha and intraluminal calcium. Antioxid Redox Signal 13:721–729

    Article  CAS  PubMed  Google Scholar 

  40. Gutscher M, Pauleau AL, Marty L, Brach T, Wabnitz GH, Samstag Y, Meyer AJ, Dick TP (2008) Real-time imaging of the intracellular glutathione redox potential. Nat Methods 5:553–559

    Article  CAS  PubMed  Google Scholar 

  41. Lentz TB, Gray SJ, Samulski RJ (2012) Viral vectors for gene delivery to the central nervous system. Neurobiol Dis 48:179–188

    Article  CAS  PubMed  Google Scholar 

  42. Machida CA (ed) (2003) Viral vectors for gene therapy, Methods in molecular medicine, vol 76. Humana Press, Totowa, NJ

    Google Scholar 

  43. Zhang F, Gradinaru V, Adamantidis AR, Durand R, Airan RD, de Lecea L, Deisseroth K (2010) Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat Protoc 5:439–456

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Delzor A, Escartin C, Deglon N (2013) Lentiviral vectors: a powerful tool to target astrocytes in vivo. Curr Drug Targets 14:1336–1346

    Article  CAS  PubMed  Google Scholar 

  45. Delzor A, Dufour N, Petit F, Guillermier M, Houitte D, Auregan G, Brouillet E, Hantraye P, Deglon N (2012) Restricted transgene expression in the brain with cell-type specific neuronal promoters. Hum Gene Ther Methods 23:242–254

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Wu Z, Asokan A, Samulski RJ (2006) Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 14:316–327

    Article  CAS  PubMed  Google Scholar 

  47. Shevtsova Z, Malik JM, Michel U, Bähr M, Kügler S (2005) Promoters and serotypes: targeting of adeno-associated virus vectors for gene transfer in the rat central nervous system in vitro and in vivo. Exp Physiol 90:53–59

    Article  CAS  PubMed  Google Scholar 

  48. Drinkut A, Tereshchenko Y, Schulz JB, Bähr M, Kügler S (2012) Efficient gene therapy for Parkinson’s disease using astrocytes as hosts for localized neurotrophic factor delivery. Mol Ther 20:534–543

    Article  CAS  PubMed  Google Scholar 

  49. Ayuso E, Mingozzi F, Bosch F (2010) Production, purification and characterization of adeno-associated vectors. Curr Gene Ther 10:423–436

    Article  CAS  PubMed  Google Scholar 

  50. Ehrengruber MU (2002) Alphaviral vectors for gene transfer into neurons. Mol Neurobiol 26:183–201

    Article  CAS  PubMed  Google Scholar 

  51. Shevtsova Z, Malik JM, Michel U, Schöll U, Bähr M, Kügler S (2006) Evaluation of epitope tags for protein detection after in vivo CNS gene transfer. Eur J Neurosci 23:1961–1969

    Article  CAS  PubMed  Google Scholar 

  52. Lingor P, Schöll U, Bähr M, Kügler S (2005) Functional applications of novel Semliki Forest virus vectors are limited by vector toxicity in cultures of primary neurons in vitro and in the substantia nigra in vivo. Exp Brain Res 161:335–342

    Article  CAS  PubMed  Google Scholar 

  53. Wagener KC, Kolbrink B, Dietrich K, Kizina KM, Terwitte LS, Kempkes B, Bao G, Müller M (2016) Redox-indicator mice stably expressing genetically-encoded neuronal roGFP: versatile tools to decipher subcellular redox dynamics in neuropathophysiology. Antioxid Redox Signal 25:41–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Weller J, Kizina KM, Can K, Bao G, Müller M (2014) Response properties of the genetically encoded optical H2O2 sensor HyPer. Free Radic Biol Med 76C:227–241

    Article  Google Scholar 

  55. Stoppini L, Buchs PA, Muller D (1991) A simple method for organotypic cultures of nervous tissue. J Neurosci Methods 37:173–182

    Article  CAS  PubMed  Google Scholar 

  56. Malgaroli A, Tsien RW (1992) Glutamate-induced long-term potentiation of the frequency of miniature synaptic currents in cultured hippocampal neurons. Nature 357:134–139

    Article  CAS  PubMed  Google Scholar 

  57. Can K, Toloe J, Kügler S, Müller M (2014) Aberrant redox homeostasis in Rett syndrome affects cytosol and mitochondria. Soc Neurosci Abstr 515.17

    Google Scholar 

  58. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    CAS  PubMed  Google Scholar 

  59. Müller M, Mironov SL, Ivannikov MV, Schmidt J, Richter DW (2005) Mitochondrial organization and motility probed by two-photon microscopy in cultured mouse brainstem neurons. Exp Cell Res 303:114–127

    PubMed  Google Scholar 

  60. Guzman JN, Sanchez-Padilla J, Wokosin D, Kondapalli J, Ilijic E, Schumacker PT, Surmeier DJ (2010) Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1. Nature 468:696–700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Xu X, von Lohneysen K, Soldau K, Noack D, Vu A, Friedman JS (2011) A novel approach for in vivo measurement of mouse red cell redox status. Blood 118:3694–3697

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Breckwoldt MO, Pfister FM, Bradley PM, Marinkovic P, Williams PR, Brill MS, Plomer B, Schmalz A, St Clair DK, Naumann R, Griesbeck O, Schwarzländer M, Godinho L, Bareyre FM, Dick TP, Kerschensteiner M, Misgeld T (2014) Multiparametric optical analysis of mitochondrial redox signals during neuronal physiology and pathology in vivo. Nat Med 20:555–560

    Article  CAS  PubMed  Google Scholar 

  63. Wolf AM, Nishimaki K, Kamimura N, Ohta S (2014) Real-time monitoring of oxidative stress in live mouse skin. J Invest Dermatol 134:1701–1709

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Belinda Kempkes for excellent technical assistance, and we are grateful to Professor S. James Remington, Institute of Molecular Biology, University of Oregon, Eugene OR USA, for making available to us the plasmids expressing roGFP1 redox-sensitive proteins. Our research was funded by the Cluster of Excellence and Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB) and the International Rett Syndrome Foundation (IRSF, grant #2817) as well as by the University Medical Center Göttingen and the State of Lower Saxony (large scale equipment grant INST 1525/14-1 FUGG).

Author information

Authors and Affiliations

  1. Zentrum Physiologie und Pathophysiologie, Institut für Neuro- und Sinnesphysiologie, Universitätsmedizin Göttingen, Humboldtallee 23, Göttingen, D-37073, Germany

    Karolina Can & Michael Müller

  2. Cluster of Excellence and Research Center Nanomicroscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany

    Karolina Can, Sebastian Kügler & Michael Müller

  3. Klinik für Neurologie, Universitätsmedizin Göttingen, Göttingen, Germany

    Sebastian Kügler

Authors
  1. Karolina Can
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sebastian Kügler
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Müller .

Editor information

Editors and Affiliations

  1. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Stefan Strack

  2. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Yuriy M. Usachev

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Can, K., Kügler, S., Müller, M. (2017). Live Imaging of Mitochondrial ROS Production and Dynamic Redox Balance in Neurons. In: Strack, S., Usachev, Y. (eds) Techniques to Investigate Mitochondrial Function in Neurons. Neuromethods, vol 123. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6890-9_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6888-6

  • Online ISBN: 978-1-4939-6890-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation