Techniques to Investigate Mitochondrial Function in Neurons

350 Pages·2017·13.973 MB·English

by  Stefan Strack, Yuriy M. Usachev (eds.)

Neuromethods 123
Stefan Strack
Yuriy M. Usachev Editors
Techniques
to Investigate
Mitochondrial
Function
in Neurons
N
euromethods
Series Editor
Wolfgang Walz
University of Saskatchewan
Saskatoon, SK, Canada
For further volumes:
http://www.springer.com/series/7657
Techniques to Investigate
Mitochondrial Function
in Neurons
Edited by
Stefan Strack
Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Yuriy M. Usachev
Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Editors
Stefan Strack Yuriy M. Usachev
Department of Pharmacology Department of Pharmacology
University of Iowa Carver College of Medicine University of Iowa Carver College of Medicine
Iowa City, IA, USA Iowa City, IA, USA
ISSN 0893-2336 ISSN 1940-6045 (electronic)
Neuromethods
ISBN 978-1-4939-6888-6 ISBN 978-1-4939-6890-9 (eBook)
DOI 10.1007/978-1-4939-6890-9
Library of Congress Control Number: 2017934469
© Springer Science+Business Media LLC 2017
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Dedication
“To our families”
Preface to the Series
Experimental life sciences have two basic foundations: concepts and tools. The Neuromethods
series focuses on the tools and techniques unique to the investigation of the nervous system
and excitable cells. It will not, however, shortchange the concept side of things as care has
been taken to integrate these tools within the context of the concepts and questions under
investigation. In this way, the series is unique in that it not only collects protocols but also
includes theoretical background information and critiques which led to the methods and
their development. Thus it gives the reader a better understanding of the origin of the
techniques and their potential future development. The Neuromethods publishing program
strikes a balance between recent and exciting developments like those concerning new ani-
mal models of disease, imaging, in vivo methods, and more established techniques, includ-
ing, for example, immunocytochemistry and electrophysiological technologies. New
trainees in neurosciences still need a sound footing in these older methods in order to apply
a critical approach to their results.
Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods
series has been a success since its first volume published through Humana Press in 1985. The
series continues to flourish through many changes over the years. It is now published under
the umbrella of Springer Protocols. While methods involving brain research have changed a
lot since the series started, the publishing environment and technology have changed even
more radically. Neuromethods has the distinct layout and style of the Springer Protocols
program, designed specifically for readability and ease of reference in a laboratory setting.
The careful application of methods is potentially the most important step in the process
of scientific inquiry. In the past, new methodologies led the way in developing new disci-
plines in the biological and medical sciences. For example, Physiology emerged out of
Anatomy in the nineteenth century by harnessing new methods based on the newly discov-
ered phenomenon of electricity. Nowadays, the relationships between disciplines and meth-
ods are more complex. Methods are now widely shared between disciplines and research
areas. New developments in electronic publishing make it possible for scientists that
encounter new methods to quickly find sources of information electronically. The design of
individual volumes and chapters in this series takes this new access technology into account.
Springer Protocols makes it possible to download single protocols separately. In addition,
Springer makes its print-on-demand technology available globally. A print copy can there-
fore be acquired quickly and for a competitive price anywhere in the world.
Saskatoon, Canada Wolfgang Walz
vii
Preface
Mitochondria are double membrane-bound organelles present in nearly every eukaryotic
cell. Thought to originate from a primitive bacterial endosymbiont, mitochondria generate
most of the cellular supply of ATP and are therefore often referred to as the power plant of
the cell. In addition to their central role in metabolism, mitochondria generate and seques-
ter reactive oxygen species, serve as high capacity buffers of intracellular calcium, and medi-
ate programmed cell death (apoptosis).
Neurons are particularly dependent on proper mitochondrial function, because of the
high energy demands associated with the maintenance of ionic gradients during action
potential firing and synaptic transmission, the principal components of neuronal computa-
tion. As well, adult neurons are difficult to replace and have a complex cytoarchitecture
with dendrites and axons extending over considerable distances. For these reasons, mito-
chondrial quality control and transport are of unique importance in the nervous system.
Mounting evidence indicates that mitochondrial dysfunction is an essential contribut-
ing factor in several common neurodegenerative disorders, including Alzheimer disease,
amyotrophic lateral sclerosis, Huntington disease, and Parkinson disease. Moreover,
impaired mitochondrial dynamics and energetics have been implicated in peripheral dia-
betic neuropathy, chemotherapy-induced peripheral neuropathy, Charcot-Marie-Tooth
disease, and several other types of acquired and inherited peripheral neuropathies. A better
understanding of mitochondrial structure and function is therefore paramount to the devel-
opment of effective therapies for many major CNS and PNS diseases.
This book is intended as a laboratory manual for a wide range of researchers who study
mitochondria in the nervous system. The 16 chapters of this volume combine contributions
of leading investigators in the field and describe a broad spectrum of experimental approaches
for investigating structure, function, and transport of neuronal mitochondria in health and
disease. Many of these approaches were only recently developed and, to the best of our
knowledge, have never been assembled in book form. The state-of-the-art techniques com-
piled in this volume range from electron tomography-based 3D reconstruction of mito-
chondrial cristae to patch clamp recording from mitochondria in intact neurons. Several
chapters describe optical approaches based on the use of genetically engineered fluorescent
sensors for monitoring synaptic ATP and axonal ROS generation, mitochondrial Ca2+
cycling and pH changes, and mitochondrial dynamics and axonal trafficking in live neurons
in real time. With recent advancements in mass spectrometry, this book also includes a
chapter that details the use of mass spectrometry for mitochondrial proteomics analysis in
neurons. Additional chapters in this volume describe respirometry, NADH imaging, and
methods for studying pyruvate transport and mitophagy.
Each chapter focuses on a specific method for studying neuronal mitochondria and is
prefaced with an introduction to its underlying scientific principles and areas of implemen-
tation. The main sections of each chapter describe specific materials, reagents, tools, and
equipment required for a given technique, which is followed by a step-by-step protocol and
practical details for employing the method. Each chapter also contains a section that
ix
x Preface
discusses potential problems and their solutions associated with a specific method as well as
provides examples of results and outcomes for the described method.
We hope that this book will be a valuable practical resource for a broad range of inves-
tigators interested in the function of neuronal mitochondria in health and disease states and
would like to thank all the authors for their contribution to this book.
Iowa City, IA, USA Stefan Strack
Yuriy M. Usachev
Contents
Preface to the Series................................................. vii
Preface.......................................................... ix
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
1 Three-Dimensional Reconstruction of Neuronal Mitochondria
by Electron Tomography ........................................ 1
Guy Perkins
2 Measuring Mitochondrial Shape with ImageJ ......................... 31
Ronald A. Merrill, Kyle H. Flippo, and Stefan Strack
3 Live Imaging Mitochondrial Transport in Neurons..................... 49
Meredith M. Course, Chung-Han Hsieh, Pei-I Tsai,
Jennifer A. Codding- Bui, Atossa Shaltouki, and Xinnan Wang
4 Techniques to Investigate Bioenergetics of Mitochondria ................ 67
William I. Sivitz
5 Respirometry in Neurons ........................................ 95
Liang Zhang and Eugenia Trushina
6 Measuring ATP in Axons with FRET ............................... 115
Lauren Y. Shields, Bryce A. Mendelsohn, and Ken Nakamura
7 Characterizing Metabolic States Using Fluorescence Lifetime
Imaging Microscopy (FLIM) of NAD(P)H........................... 133
Thomas S. Blacker and Michael R. Duchen
8 Techniques for Simultaneous Mitochondrial and Cytosolic Ca2+ Imaging
in Neurons ................................................... 151
Jacob E. Rysted, Zhihong Lin, and Yuriy M. Usachev
9 Live Imaging of Mitochondrial ROS Production and Dynamic Redox
Balance in Neurons............................................. 179
Karolina Can, Sebastian Kügler, and Michael Müller
10 Monitoring of Permeability Transition Pore Openings
in Isolated Individual Brain Mitochondria............................ 199
Nickolay Brustovetsky and Tatiana Brustovetsky
11 Examination of Mitochondrial Ion Conductance by Patch Clamp
in Intact Neurons and Mitochondrial Membrane Preparations............. 211
Elizabeth A. Jonas and Nelli Mnatsakanyan
12 Monitoring of Permeability Transition Pore Openings in Mitochondria
of Cultured Neurons............................................ 239
Tatiana Brustovetsky and Nickolay Brustovetsky
13 Protocols for Assessing Mitophagy in Neuronal Cell Lines
and Primary Neurons ........................................... 249
Ruben K. Dagda and Monica Rice
xi
xii Contents
14 Examining Mitochondrial Function at Synapses In Situ.................. 279
Gregory T. Macleod and Maxim V. Ivannikov
15 Proteomic Analysis of Neuronal Mitochondria ........................ 299
Kelly L. Stauch and Howard S. Fox
16 Measuring Mitochondrial Pyruvate Oxidation......................... 321
Lawrence R. Gray, Alix A.J. Rouault, Lalita Oonthonpan,
Adam J. Rauckhorst, Julien A. Sebag, and Eric B. Taylor
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339