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Examining Mitochondrial Function at Synapses In Situ

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Techniques to Investigate Mitochondrial Function in Neurons

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

Abstract

Synaptic mitochondria are exposed to an environment quite unlike any other subcellular environment. The cytosolic proton concentration ([H+]c) in the presynaptic compartment can increase several fold within seconds during a burst of action potentials (APs) while the cytosolic free Ca2+ concentration ([Ca2+]c) can increase several fold within milliseconds of a single AP. To understand how mitochondria function under such dynamic conditions they must be examined in the context of these small synaptic compartments. Here, we describe the application of fluorescent reporters to examine mitochondrial function in situ at the Drosophila melanogaster (fruit fly) larval neuromuscular junction (NMJ). Emphasis is placed on genetically encoded fluorescent (GEF) probes, rather than chemical fluorescent probes, due to the large range of GEF-probes now available and their specificity of targeting. We describe how best to prepare NMJs for ex vivo interrogation, apply stimuli to motor axons, and collect and analyze fluorescence data. We summarize the probes that have been used successfully at the Drosophila NMJ to monitor changes in mitochondrial [Ca2+], [H+], [O2·−], [ATP] and voltage across the inner mitochondrial membrane (IMM). Lastly, for those who wish to generate transgenic Drosophila to express other GEF-probes, we list useful genetic reagents and signal sequences known to be effective at targeting GEF-probes to mitochondria in Drosophila. Overall, the techniques described here should provide a starting point for a diverse array of researchers who may wish to use GEF-probes targeted to mitochondria in Drosophila either as screening tools or as reporters of mitochondrial function at synapses in situ.

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Acknowledgments

G.T.M. was supported by NIH NINDS awards NS061914 and NS083031.

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Authors and Affiliations

  1. Department of Biological Sciences & Wilkes Honors College, Florida Atlantic University, Jupiter, FL, USA

    Gregory T. Macleod

  2. Department of Neuroscience & Physiology, NYU School of Medicine, New York, NY, USA

    Maxim V. Ivannikov

Authors
  1. Gregory T. Macleod
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  2. Maxim V. Ivannikov
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Corresponding author

Correspondence to Gregory T. Macleod .

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

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Macleod, G.T., Ivannikov, M.V. (2017). Examining Mitochondrial Function at Synapses In Situ. 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_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6888-6

  • Online ISBN: 978-1-4939-6890-9

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