Guide to Research Techniques in Neuroscience - Matt Carter, Jennifer C. Shieh

Guide to Research Techniques in Neuroscience (eBook)

Matt Carter, Jennifer C. Shieh (Autoren)

eBook Download: PDF | EPUB

2015 | 2. Auflage
418 Seiten
Elsevier Science (Verlag)
978-0-12-800597-2 (ISBN)

This volume addresses that gap, gathering multidisciplinary knowledge and providing tools for understanding the neuroscience techniques that are essential to the field, and allowing the reader to design experiments in a variety of neuroscience disciplines.

Written to provide a 'hands-on' approach for graduate students, postdocs, or anyone new to the neurosciences
Techniques within one field are compared, allowing readers to select the best techniques for their own work
Includes key articles, books, and protocols for additional detailed study
Data analysis boxes in each chapter help with data interpretation and offer guidelines on how best to represent results
Walk-through boxes guide readers step-by-step through experiments

Matt Carter, PhD, is currently Assistant Professor of Biology at Williams College. His previous position was as a post-doctoral fellow in Richard Palmiter's lab at the University of Washington using optogenetic techniques to study neural circuitry. He has authored the first edition of this book (Elsevier, 2009) as well as Designing Science Presentations: A Visual Guide to Figures, Papers, Slides, Posters, and More (Elsevier, 2012). He was the awardee of Stanford University's Walter J. Gores Award for Excellence in Teaching, and two-time recipient of the Stanford School of Medicine's Excellence in Teaching Award. He currently teaches courses at Williams in both Topics in Neuroscience as well as Neural Systems and Circuits.
Former: carterme@u.washington.edu

Neuroscience is, by definition, a multidisciplinary field: some scientists study genes and proteins at the molecular level while others study neural circuitry using electrophysiology and high-resolution optics. A single topic can be studied using techniques from genetics, imaging, biochemistry, or electrophysiology. Therefore, it can be daunting for young scientists or anyone new to neuroscience to learn how to read the primary literature and develop their own experiments. This volume addresses that gap, gathering multidisciplinary knowledge and providing tools for understanding the neuroscience techniques that are essential to the field, and allowing the reader to design experiments in a variety of neuroscience disciplines. Written to provide a "e;hands-on"e; approach for graduate students, postdocs, or anyone new to the neurosciences Techniques within one field are compared, allowing readers to select the best techniques for their own work Includes key articles, books, and protocols for additional detailed study Data analysis boxes in each chapter help with data interpretation and offer guidelines on how best to represent results Walk-through boxes guide readers step-by-step through experiments

Front Cover 1
Guide to Research Techniques in Neuroscience 4
Copyright 5
Contents 6
Foreword to the Second Edition 14
Foreword to the First Edition 16
Preface to the Second Edition 18
Preface to the First Edition 20
Introduction 24
LEVELS OF INVESTIGATION 25
METHODS OF STUDYING THE NERVOUS SYSTEM 26
UNDERSTANDING TECHNIQUES IN NEUROSCIENCE 29
Chapter 1 - Whole-Brain Imaging 30
STRUCTURAL BRAIN IMAGING TECHNIQUES 31
FUNCTIONAL BRAIN IMAGING TECHNIQUES 43
FUNCTIONAL IMAGING EXPERIMENTAL DESIGN AND ANALYSIS 53
CONCLUSION 64
SUGGESTED READING AND REFERENCES 66
Books 66
Review Articles 66
Primary Research Articles—Interesting Examples from the Literature 66
Protocols 67
Websites 67
Chapter 2 - Animal Behavior 68
CONSIDERATIONS FOR CHOOSING AND PERFORMING A BEHAVIORAL ASSAY 69
RODENT BEHAVIORAL PARADIGMS 73
DROSOPHILA BEHAVIORAL PARADIGMS 90
CAENORHABDITIS ELEGANS BEHAVIORAL PARADIGMS 94
NONHUMAN PRIMATE BEHAVIORAL PARADIGMS 96
CONCLUSION 98
SUGGESTED READING AND REFERENCES 98
Books and Review Articles 98
Primary Research Articles—Papers First Describing Standard Behavioral Assays 98
Primary Research Articles—Interesting Examples from the Literature 99
Protocols 99
Chapter 3 - Stereotaxic Surgeries and In Vivo Techniques 102
STEREOTAXIC SURGERIES 103
IMPLANTS FOR LONG-TERM ACCESS TO THE BRAIN 109
MANIPULATING NEURAL ACTIVITY IN VIVO 115
CONCLUSION 115
SUGGESTED READING AND REFERENCES 115
Books 115
Review Articles 115
Primary Research Articles—Interesting Examples from the Literature 116
Protocols 116
Chapter 4 - Electrophysiology 118
A BRIEF REVIEW OF THE ELECTRICAL PROPERTIES OF NEURONS 119
THE ELECTROPHYSIOLOGY RIG 121
CATEGORIES OF ELECTROPHYSIOLOGY RECORDINGS 122
ELECTROPHYSIOLOGY TISSUE PREPARATIONS 137
MANIPULATING NEURAL ACTIVITY DURING ELECTROPHYSIOLOGY EXPERIMENTS 142
CONCLUSION 143
SUGGESTED READING AND REFERENCES 143
Books 143
Review Articles 143
Primary Research Articles—Interesting Examples from the Literature 143
Protocols 144
Chapter 5 - Microscopy 146
ESSENTIAL PRINCIPLES OF MICROSCOPY 147
LIGHT MICROSCOPY 155
FLUORESCENCE MICROSCOPY 157
ELECTRON MICROSCOPY 162
PREPARING AND INTERPRETING MICROSCOPY DATA 165
CONCLUSION 168
SUGGESTED READING AND REFERENCES 172
Books 172
Review Articles 172
Primary Research Articles—Interesting Examples from the Literature 172
Protocols 173
Websites 173
Chapter 6 - Visualizing Neural Structure 174
TISSUE PREPARATION 175
VISUALIZING MORPHOLOGY 179
VISUALIZING GENE AND PROTEIN EXPRESSION 185
VISUALIZING CIRCUITRY 191
CONCLUSION 194
SUGGESTED READING AND REFERENCES 194
Books 194
Review Articles 194
Primary Research Articles—Interesting Examples from the Literature 195
Protocols 195
Websites 195
Chapter 7 - Visualizing Neural Function 196
STATIC MARKERS OF ACTIVITY 197
VISUALIZING NEURAL ACTIVITY 200
VISUALIZING PROTEIN FUNCTION 206
CONCLUSION 210
SUGGESTED READING AND REFERENCES 210
Books 210
Review Articles 210
Primary Research Articles—Interesting Examples from the Literature 211
Protocols 212
Chapter 8 - Manipulating Neural Activity 214
EXPERIMENTAL LOGIC OF MANIPULATING NEURAL ACTIVITY 215
PHYSICAL MANIPULATION 216
ELECTRICAL MANIPULATION 218
PHARMACOLOGICAL MANIPULATION 218
GENETIC MANIPULATION 220
PHARMACOGENETIC MANIPULATION 221
OPTOGENETIC MANIPULATION 222
NEUROMODULATION TECHNIQUES USED IN HUMANS 226
CONCLUSION 226
SUGGESTED READING AND REFERENCES 229
Books 229
Review Articles 229
Primary Research Articles—Interesting Examples from the Literature 229
Protocols 230
Websites 230
Chapter 9 - Identifying Genes and Proteins of Interest 232
A BRIEF REVIEW OF GENES AND PROTEINS 233
GENETIC SCREENS 238
IN SILICO SCREENS 243
MOLECULAR SCREENS 243
CONCLUSION 245
SUGGESTED READING AND REFERENCES 246
Books 246
Review Articles 246
Primary Research Articles—Interesting Examples from the Literature 246
Protocols 246
Websites 247
Chapter 10 - Molecular Cloning and Recombinant DNA Technology 248
ISOLATING DNA FRAGMENTS 249
CLONING DNA 256
IDENTIFYING DNA 260
CONCLUSION 265
SUGGESTED READING AND REFERENCES 265
Books 265
Review Articles 266
Primary Research Articles—Interesting Examples from the Literature 266
Protocols 266
Websites 266
Chapter 11 - Gene Delivery Strategies 268
PHYSICAL GENE DELIVERY 268
CHEMICAL GENE DELIVERY 273
VIRAL GENE DELIVERY 276
CONCLUSION 279
SUGGESTED READING AND REFERENCES 280
Books 280
Review Articles 280
Primary Research Articles—Interesting Examples from the Literature 281
Protocols 281
Websites 281
Chapter 12 - Making and Using Transgenic Organisms 282
TRANSGENES 283
THE TRANSGENIC CONSTRUCT 287
BINARY TRANSGENIC SYSTEMS 290
MAKING TRANSGENIC ORGANISMS 294
CONCLUSION 298
SUGGESTED READING AND REFERENCES 299
Books 299
Review Articles 299
Primary Research Articles—Interesting Examples from the Literature 299
Protocols 300
Websites 300
Chapter 13 - Manipulating Endogenous Genes 302
CLASSICAL GENE TARGETING METHODS 303
NUCLEASE-BASED GENE TARGETING METHODS 313
DISRUPTING GENE PRODUCTS 316
CONCLUSION 320
SUGGESTED READING AND REFERENCES 320
Books 320
Review Articles 321
Primary Research Articles—Interesting Examples from the Literature 321
Protocols 321
Chapter 14 - Cell Culture Techniques 324
CELL CULTURE EQUIPMENT AND REAGENTS 325
IMMORTALIZED CELL LINES 327
PRIMARY CELL AND TISSUE CULTURE 330
STEM CELL CULTURES 333
MANIPULATING CELLS IN CULTURE 336
CONCLUSION 337
SUGGESTED READING AND REFERENCES 338
Books 338
Review Articles 338
Primary Research Articles—Interesting Examples from the Literature 338
Protocols 339
Websites 339
Chapter 15 - Biochemical Assays and Intracellular Signaling 340
INTRODUCTION TO SIGNAL TRANSDUCTION AND INTRACELLULAR SIGNALING 342
FUNDAMENTAL TOOLS USED TO STUDY PROTEINS 342
INVESTIGATING PROTEIN EXPRESSION 349
INVESTIGATING PROTEIN–PROTEIN INTERACTIONS 356
INVESTIGATING POSTTRANSLATIONAL MODIFICATIONS 360
INVESTIGATING PROTEIN–DNA INTERACTIONS 366
CONCLUSION 371
SUGGESTED READING AND REFERENCES 371
Books 371
Review Articles 372
Primary Research Articles—Interesting Examples from the Literature 372
Protocols 372
Websites 372
Glossary 374
Index 406

Introduction

The human mind has been studied for thousands of years, but the human brain, as well as the brains of other species, has only been studied for about a century. Only 150 years ago, the ability to study the nervous systems of humans and other animals was limited to direct observation and by examining the effects of brain damage in people and other organisms. With the advent of histology came the ability to visualize and differentiate between neurons based on morphology. The great neuroscientist Santiago Ramón y Cajal used a method called Golgi staining to visualize the morphology and architecture of neurons and their circuits throughout the brain. Cajal used the Golgi stain to propel the field of neuroscience into its modern state.

Indeed, in the history of neuroscience, each leap forward in knowledge has been based on a leap forward in techniques and technology. Just as Ramón y Cajal used Golgi staining to greatly advance our understanding of the structure of the nervous system, scientists throughout the twentieth century used more and more advanced techniques to contribute to our understanding of the function of the nervous system: Eccles, Hodgkin, and Huxley used intracellular recording technology to investigate the ionic basis of membrane potentials; Hubel and Wiesel used extracellular recording technology to investigate how information is processed and recorded in the visual system; Neher and Sakmann used patch-clamp technology to investigate the physiology of single-ion channels. In the latter half of the twentieth century, the explosion of molecular biology techniques and methods of genetically manipulating model organisms allowed neuroscientists to study individual genes, proteins, and cell types. Technology has progressed so far in the past 100 years that the Golgi stain itself seems to have been reinvented through powerful technologies (Chapter 12) that allow investigators to turn specific neurons different colors to further investigate the structure and connectivity of the nervous system. In fact, technologies are developing at such a rapid pace that in 2013, U.S. President Barack Obama announced the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative to double federal funding for new techniques and technologies to accelerate our understanding of the nervous system.

The modern neuroscientist now has hundreds of techniques that can be used to answer specific scientific questions. This book contains 15 chapters that provide an overview of the most commonly used techniques. Although there are dozens of techniques that seem very different at first glance, many of them attempt to study the nervous system in the same way. For example, transcranial magnetic stimulation (Chapter 1), physical lesions (Chapter 8), optogenetic inhibition (Chapter 8), and genetic knockdown or knockouts (Chapter 13) are all attempts to test the effect of a loss-of-function of some aspect of the nervous system on another aspect of the nervous system. For each level of investigation (whole brains to individual genes), research strategies can be similar in concept even if the techniques used are very different in practice.

Levels of Investigation

Something immediately obvious to all students of neuroscience is that the nervous system is exceptionally complicated and can be examined at multiple levels of investigation. The basic functional unit of the nervous system is the neuron. The human brain is composed of approximately 100 billion neurons that are connected into circuits via approximately 100 trillion synapses. Neural circuits are organized into anatomical structures and larger networks of neurons that can integrate information across modalities from many different parts of the brain. These networks process sensory information from the external and internal environment and provide the neural basis of cognition—learning, memory, perception, decision-making, emotion, and other higher-order processes. The final output of the nervous system is a behavior composed of a coordinated motor action. This behavior can be either extremely simple, such as a motor reflex, or incredibly complicated, such as dancing, typing, or playing a musical instrument. Behavior is usually defined not just by what an organism does, but what it chooses to do. Therefore, except in rare circumstances of lesion or disease, cognition and behavior are inseparably linked, and in animals other than humans, behavior is used as a readout of animal cognition.

Just as one can start with a neuron and scale up toward circuits, cognition, and behavior, a scientist can also scale down and examine the components that make up a neuron. A neuron is itself defined as having a cell body (soma), axon, and dendrites. These neuronal components contain subcellular specializations that make the neuron unique among other cell types. Specialized organelles in a neuron, such as vesicles containing neurotransmitters, provide the cell with the ability to signal to other neurons. Specialized cytoskeletal elements allow a neural process to extend great distances throughout the brain and body. Several proteins provide neurons with their intercellular signaling abilities and physiological characteristics. For example, biosynthetic enzymes produce neurotransmitters, while other proteins serve as receptors for these signaling molecules. One of the most important types of proteins in the nervous system form ion channels, the transmembrane structures that allow neurons to become electrically active under certain conditions. All of these proteins are the products of genes, the functional units of an organism’s genome. The human genome contains approximately 30,000 genes, with each neural subtype expressing its own subset of these genes.

The complexity of the nervous system is awesome in scope. It is amazing that a mutation in a single gene, such as a gene that codes for a transmembrane ion channel, can produce effects that alter the electrical properties of a neuron, in turn altering the normal firing patterns of a neural circuit and thus causing an abnormal behavior.

A neuroscientist can approach the study of the nervous system through any of these levels of organization. The 15 chapters of this book provide a guide to the types of experiments that can be performed at each level. However, irrespective of technique, the basic scientific approach one can use to study the nervous system is consistent from level to level, whether the subject is human cognition or axon guidance in cell culture.

Methods of Studying the Nervous System

There are four general methods of studying the nervous system:

1. Examining case studies—identifying interesting events that have occurred naturally and using these events to develop hypotheses that can be tested in future experiments

2. Screens—performing unbiased searches for anatomical structures, neurons, proteins, or genes that could play a role in a subject of interest

3. Description—using techniques that allow a scientist to observe the nervous system without manipulating any variables

4. Manipulation—testing hypotheses by determining the effect of an independent variable on a dependent variable

Each of these four methods is described in more detail below.

Examining Case Studies

A case study is an example of an event that happened to a subject (most often a human or group of humans) that demonstrates an important role for an aspect of the nervous system. The circumstances surrounding the event are usually nonrepeatable and cannot be precisely recreated in a laboratory setting. Such demonstrations are, therefore, not true experiments in that no variables are deliberately controlled by a scientist. However, these events can often reveal substantial information about an aspect of neural function that was previously unknown.

For example, consider the case of Phineas Gage, a railroad worker who was involved in an accident in 1848 that caused an iron rod to pass through his skull. The rod entered the left side of his face, passed just behind his left eye, and exited through the top of his head, completely lesioning his frontal lobes. This is an amazing event, not only because Gage survived (and lived for another 12 years), but also because it informed scientists about the function of the frontal lobe of the brain. The event allowed investigators to retrospectively ask the question: “What is the effect of removing the frontal lobe on consciousness and behavior?” According to Gage’s friends, family, and coworkers, he was “no longer Gage.” He retained the ability to learn, remember, sense, and perceive his environment, to execute motor functions, and to live a fairly normal life, but it seemed to people who knew him that his personality had changed completely. After the accident, Gage was described as impolite, erratic, unreliable, and offensive to others. He wound up losing his job at the railroad, not because of any physical or mental incapacity, but because he was simply so disrespectful and offensive that people could not stand to work with him.

This case study is not a true experiment; no scientist decided to test the removal of the frontal lobe on personality. But the incident, and others like it, allows neuroscientists to form hypotheses based on naturally occurring events. Because of Gage’s story, neuroscientists could hypothesize about the contribution of the frontal lobe to human personality. Future experiments could test these hypotheses on animal models (that share certain...

Erscheint lt. Verlag	27.2.2015
Sprache	englisch
Themenwelt	Medizin / Pharmazie ► Medizinische Fachgebiete ► Neurologie
	Naturwissenschaften ► Biologie ► Humanbiologie
	Naturwissenschaften ► Biologie ► Zoologie
ISBN-10	0-12-800597-1 / 0128005971
ISBN-13	978-0-12-800597-2 / 9780128005972

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