Carter / Shieh Guide to Research Techniques in Neuroscience

2. Auflage 2015
ISBN: 978-0-12-800597-2
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 418 Seiten

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 '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 is an Associate Professor of Biology at Williams College where he teaches courses in neuroscience and physiology. His research program focuses on how the brain regulates food intake and sleep and is funded by grants from the National Institutes of Health and National Science Foundation. In addition to primary scientific publications, he is also the author of Designing Science Presentations (Academic Press). He is a recipient of the Walter Gores Award for Excellence in Teaching from Stanford University and the Nelson Bushnell Prize for Excellence in Teaching and Writing from Williams College.

Autoren/Hrsg.

Carter, Matt

Shieh, Jennifer C.

Inhaltsverzeichnis

1;Front Cover;1
2;Guide to Research Techniques in Neuroscience;4
3;Copyright;5
4;Contents;6
5;Foreword to the Second Edition;14
6;Foreword to the First Edition;16
7;Preface to the Second Edition;18
8;Preface to the First Edition;20
9;Introduction;24
9.1;LEVELS OF INVESTIGATION;25
9.2;METHODS OF STUDYING THE NERVOUS SYSTEM;26
9.3;UNDERSTANDING TECHNIQUES IN NEUROSCIENCE;29
10;Chapter 1 - Whole-Brain Imaging;30
10.1;STRUCTURAL BRAIN IMAGING TECHNIQUES;31
10.2;FUNCTIONAL BRAIN IMAGING TECHNIQUES;43
10.3;FUNCTIONAL IMAGING EXPERIMENTAL DESIGN AND ANALYSIS;53
10.4;CONCLUSION;64
10.5;SUGGESTED READING AND REFERENCES;66
10.6;Books;66
10.7;Review Articles;66
10.8;Primary Research Articles—Interesting Examples from the Literature;66
10.9;Protocols;67
10.10;Websites;67
11;Chapter 2 - Animal Behavior;68
11.1;CONSIDERATIONS FOR CHOOSING AND PERFORMING A BEHAVIORAL ASSAY;69
11.2;RODENT BEHAVIORAL PARADIGMS;73
11.3;DROSOPHILA BEHAVIORAL PARADIGMS;90
11.4;CAENORHABDITIS ELEGANS BEHAVIORAL PARADIGMS;94
11.5;NONHUMAN PRIMATE BEHAVIORAL PARADIGMS;96
11.6;CONCLUSION;98
11.7;SUGGESTED READING AND REFERENCES;98
11.8;Books and Review Articles;98
11.9;Primary Research Articles—Papers First Describing Standard Behavioral Assays;98
11.10;Primary Research Articles—Interesting Examples from the Literature;99
11.11;Protocols;99
12;Chapter 3 - Stereotaxic Surgeries and In Vivo Techniques;102
12.1;STEREOTAXIC SURGERIES;103
12.2;IMPLANTS FOR LONG-TERM ACCESS TO THE BRAIN;109
12.3;MANIPULATING NEURAL ACTIVITY IN VIVO;115
12.4;CONCLUSION;115
12.5;SUGGESTED READING AND REFERENCES;115
12.6;Books;115
12.7;Review Articles;115
12.8;Primary Research Articles—Interesting Examples from the Literature;116
12.9;Protocols;116
13;Chapter 4 - Electrophysiology;118
13.1;A BRIEF REVIEW OF THE ELECTRICAL PROPERTIES OF NEURONS;119
13.2;THE ELECTROPHYSIOLOGY RIG;121
13.3;CATEGORIES OF ELECTROPHYSIOLOGY RECORDINGS;122
13.4;ELECTROPHYSIOLOGY TISSUE PREPARATIONS;137
13.5;MANIPULATING NEURAL ACTIVITY DURING ELECTROPHYSIOLOGY EXPERIMENTS;142
13.6;CONCLUSION;143
13.7;SUGGESTED READING AND REFERENCES;143
13.8;Books;143
13.9;Review Articles;143
13.10;Primary Research Articles—Interesting Examples from the Literature;143
13.11;Protocols;144
14;Chapter 5 - Microscopy;146
14.1;ESSENTIAL PRINCIPLES OF MICROSCOPY;147
14.2;LIGHT MICROSCOPY;155
14.3;FLUORESCENCE MICROSCOPY;157
14.4;ELECTRON MICROSCOPY;162
14.5;PREPARING AND INTERPRETING MICROSCOPY DATA;165
14.6;CONCLUSION;168
14.7;SUGGESTED READING AND REFERENCES;172
14.8;Books;172
14.9;Review Articles;172
14.10;Primary Research Articles—Interesting Examples from the Literature;172
14.11;Protocols;173
14.12;Websites;173
15;Chapter 6 - Visualizing Neural Structure;174
15.1;TISSUE PREPARATION;175
15.2;VISUALIZING MORPHOLOGY;179
15.3;VISUALIZING GENE AND PROTEIN EXPRESSION;185
15.4;VISUALIZING CIRCUITRY;191
15.5;CONCLUSION;194
15.6;SUGGESTED READING AND REFERENCES;194
15.7;Books;194
15.8;Review Articles;194
15.9;Primary Research Articles—Interesting Examples from the Literature;195
15.10;Protocols;195
15.11;Websites;195
16;Chapter 7 - Visualizing Neural Function;196
16.1;STATIC MARKERS OF ACTIVITY;197
16.2;VISUALIZING NEURAL ACTIVITY;200
16.3;VISUALIZING PROTEIN FUNCTION;206
16.4;CONCLUSION;210
16.5;SUGGESTED READING AND REFERENCES;210
16.6;Books;210
16.7;Review Articles;210
16.8;Primary Research Articles—Interesting Examples from the Literature;211
16.9;Protocols;212
17;Chapter 8 - Manipulating Neural Activity;214
17.1;EXPERIMENTAL LOGIC OF MANIPULATING NEURAL ACTIVITY;215
17.2;PHYSICAL MANIPULATION;216
17.3;ELECTRICAL MANIPULATION;218
17.4;PHARMACOLOGICAL MANIPULATION;218
17.5;GENETIC MANIPULATION;220
17.6;PHARMACOGENETIC MANIPULATION;221
17.7;OPTOGENETIC MANIPULATION;222
17.8;NEUROMODULATION TECHNIQUES USED IN HUMANS;226
17.9;CONCLUSION;226
17.10;SUGGESTED READING AND REFERENCES;229
17.11;Books;229
17.12;Review Articles;229
17.13;Primary Research Articles—Interesting Examples from the Literature;229
17.14;Protocols;230
17.15;Websites;230
18;Chapter 9 - Identifying Genes and Proteins of Interest;232
18.1;A BRIEF REVIEW OF GENES AND PROTEINS;233
18.2;GENETIC SCREENS;238
18.3;IN SILICO SCREENS;243
18.4;MOLECULAR SCREENS;243
18.5;CONCLUSION;245
18.6;SUGGESTED READING AND REFERENCES;246
18.7;Books;246
18.8;Review Articles;246
18.9;Primary Research Articles—Interesting Examples from the Literature;246
18.10;Protocols;246
18.11;Websites;247
19;Chapter 10 - Molecular Cloning and Recombinant DNA Technology;248
19.1;ISOLATING DNA FRAGMENTS;249
19.2;CLONING DNA;256
19.3;IDENTIFYING DNA;260
19.4;CONCLUSION;265
19.5;SUGGESTED READING AND REFERENCES;265
19.6;Books;265
19.7;Review Articles;266
19.8;Primary Research Articles—Interesting Examples from the Literature;266
19.9;Protocols;266
19.10;Websites;266
20;Chapter 11 - Gene Delivery Strategies;268
20.1;PHYSICAL GENE DELIVERY;268
20.2;CHEMICAL GENE DELIVERY;273
20.3;VIRAL GENE DELIVERY;276
20.4;CONCLUSION;279
20.5;SUGGESTED READING AND REFERENCES;280
20.6;Books;280
20.7;Review Articles;280
20.8;Primary Research Articles—Interesting Examples from the Literature;281
20.9;Protocols;281
20.10;Websites;281
21;Chapter 12 - Making and Using Transgenic Organisms;282
21.1;TRANSGENES;283
21.2;THE TRANSGENIC CONSTRUCT;287
21.3;BINARY TRANSGENIC SYSTEMS;290
21.4;MAKING TRANSGENIC ORGANISMS;294
21.5;CONCLUSION;298
21.6;SUGGESTED READING AND REFERENCES;299
21.7;Books;299
21.8;Review Articles;299
21.9;Primary Research Articles—Interesting Examples from the Literature;299
21.10;Protocols;300
21.11;Websites;300
22;Chapter 13 - Manipulating Endogenous Genes;302
22.1;CLASSICAL GENE TARGETING METHODS;303
22.2;NUCLEASE-BASED GENE TARGETING METHODS;313
22.3;DISRUPTING GENE PRODUCTS;316
22.4;CONCLUSION;320
22.5;SUGGESTED READING AND REFERENCES;320
22.6;Books;320
22.7;Review Articles;321
22.8;Primary Research Articles—Interesting Examples from the Literature;321
22.9;Protocols;321
23;Chapter 14 - Cell Culture Techniques;324
23.1;CELL CULTURE EQUIPMENT AND REAGENTS;325
23.2;IMMORTALIZED CELL LINES;327
23.3;PRIMARY CELL AND TISSUE CULTURE;330
23.4;STEM CELL CULTURES;333
23.5;MANIPULATING CELLS IN CULTURE;336
23.6;CONCLUSION;337
23.7;SUGGESTED READING AND REFERENCES;338
23.8;Books;338
23.9;Review Articles;338
23.10;Primary Research Articles—Interesting Examples from the Literature;338
23.11;Protocols;339
23.12;Websites;339
24;Chapter 15 - Biochemical Assays and Intracellular Signaling;340
24.1;INTRODUCTION TO SIGNAL TRANSDUCTION AND INTRACELLULAR SIGNALING;342
24.2;FUNDAMENTAL TOOLS USED TO STUDY PROTEINS;342
24.3;INVESTIGATING PROTEIN EXPRESSION;349
24.4;INVESTIGATING PROTEIN–PROTEIN INTERACTIONS;356
24.5;INVESTIGATING POSTTRANSLATIONAL MODIFICATIONS;360
24.6;INVESTIGATING PROTEIN–DNA INTERACTIONS;366
24.7;CONCLUSION;371
24.8;SUGGESTED READING AND REFERENCES;371
24.9;Books;371
24.10;Review Articles;372
24.11;Primary Research Articles—Interesting Examples from the Literature;372
24.12;Protocols;372
24.13;Websites;372
25;Glossary;374
26;Index;406

Leseproben

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...