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Techniques for Simultaneous Mitochondrial and Cytosolic Ca2+ Imaging in Neurons

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Techniques to Investigate Mitochondrial Function in Neurons

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

Abstract

Mitochondria efficiently buffer Ca2+ influx during excitation, which limits the amplitude of cytosolic Ca2+ rise, and then slowly release Ca2+ back into the cytosol thereby extending the duration of cytosolic Ca2+ response. This mitochondrial Ca2+ cycling helps shape Ca2+ transients and regulates Ca2+-dependent functions in neurons such as excitability, synaptic plasticity, bioenergetics, and survival. Therefore identifying the molecular components of mitochondrial Ca2+ transport in neurons and defining their pharmacological and functional properties presents an important and challenging task for neuroscientists. Fulfilling this task requires a set of tools for simple and reliable measurement of Ca2+ concentration inside and outside mitochondria, i.e., within the mitochondrial matrix and cytosol, respectively. In this chapter we describe instrumentation and techniques for simultaneous measurements of mitoc hondrial and cytosolic Ca2+ concentration in central and peripheral neurons by using synthetic (e.g., Fura-2 ) and genetic (e.g., mito-R-GECO1 ) fluorescent Ca2+ indicators. We include detailed protocols for preparing primary cultures of hippocampal and dorsal root ganglion (DRG) sensory neurons, gene transfer into these cells and the use of fluorescent microscopy and patch clam p for comprehensive characterization of Ca2+ fluxes across the mitochondrial and plasma membranes.

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Acknowledgments

This work was supported by NIH/NINDS grants NS087068 and NS096246. J.E.R. was supported by a predoctoral fellowship through the American Heart Association, Midwest Affiliate Grant 15PRE25310013.

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Authors and Affiliations

  1. Department of Pharmacology, University of Iowa Carver College of Medicine, 2-450 BSB, 51 Newton Road, Iowa City, IA, 52242, USA

    Jacob E. Rysted, Zhihong Lin & Yuriy M. Usachev

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  1. Jacob E. Rysted
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  2. Zhihong Lin
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  3. Yuriy M. Usachev
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Correspondence to Yuriy M. Usachev .

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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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Rysted, J.E., Lin, Z., Usachev, Y.M. (2017). Techniques for Simultaneous Mitochondrial and Cytosolic Ca2+ Imaging 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_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_8

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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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