Guide to Research Techniques in Neuroscience

 
 
Academic Press
  • 2. Auflage
  • |
  • erschienen am 27. Februar 2015
  • |
  • 418 Seiten
 
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978-0-12-800597-2 (ISBN)
 

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, 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
  • Englisch
  • Saint Louis
  • |
  • USA
Elsevier Science
  • 20,79 MB
978-0-12-800597-2 (9780128005972)
0128005971 (0128005971)
weitere Ausgaben werden ermittelt
1 - Front Cover [Seite 1]
2 - Guide to Research Techniques in Neuroscience [Seite 4]
3 - Copyright [Seite 5]
4 - Contents [Seite 6]
5 - Foreword to the Second Edition [Seite 14]
6 - Foreword to the First Edition [Seite 16]
7 - Preface to the Second Edition [Seite 18]
8 - Preface to the First Edition [Seite 20]
9 - Introduction [Seite 24]
9.1 - LEVELS OF INVESTIGATION [Seite 25]
9.2 - METHODS OF STUDYING THE NERVOUS SYSTEM [Seite 26]
9.3 - UNDERSTANDING TECHNIQUES IN NEUROSCIENCE [Seite 29]
10 - Chapter 1 - Whole-Brain Imaging [Seite 30]
10.1 - STRUCTURAL BRAIN IMAGING TECHNIQUES [Seite 31]
10.2 - FUNCTIONAL BRAIN IMAGING TECHNIQUES [Seite 43]
10.3 - FUNCTIONAL IMAGING EXPERIMENTAL DESIGN AND ANALYSIS [Seite 53]
10.4 - CONCLUSION [Seite 64]
10.5 - SUGGESTED READING AND REFERENCES [Seite 66]
10.6 - Books [Seite 66]
10.7 - Review Articles [Seite 66]
10.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 66]
10.9 - Protocols [Seite 67]
10.10 - Websites [Seite 67]
11 - Chapter 2 - Animal Behavior [Seite 68]
11.1 - CONSIDERATIONS FOR CHOOSING AND PERFORMING A BEHAVIORAL ASSAY [Seite 69]
11.2 - RODENT BEHAVIORAL PARADIGMS [Seite 73]
11.3 - DROSOPHILA BEHAVIORAL PARADIGMS [Seite 90]
11.4 - CAENORHABDITIS ELEGANS BEHAVIORAL PARADIGMS [Seite 94]
11.5 - NONHUMAN PRIMATE BEHAVIORAL PARADIGMS [Seite 96]
11.6 - CONCLUSION [Seite 98]
11.7 - SUGGESTED READING AND REFERENCES [Seite 98]
11.8 - Books and Review Articles [Seite 98]
11.9 - Primary Research Articles-Papers First Describing Standard Behavioral Assays [Seite 98]
11.10 - Primary Research Articles-Interesting Examples from the Literature [Seite 99]
11.11 - Protocols [Seite 99]
12 - Chapter 3 - Stereotaxic Surgeries and In Vivo Techniques [Seite 102]
12.1 - STEREOTAXIC SURGERIES [Seite 103]
12.2 - IMPLANTS FOR LONG-TERM ACCESS TO THE BRAIN [Seite 109]
12.3 - MANIPULATING NEURAL ACTIVITY IN VIVO [Seite 115]
12.4 - CONCLUSION [Seite 115]
12.5 - SUGGESTED READING AND REFERENCES [Seite 115]
12.6 - Books [Seite 115]
12.7 - Review Articles [Seite 115]
12.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 116]
12.9 - Protocols [Seite 116]
13 - Chapter 4 - Electrophysiology [Seite 118]
13.1 - A BRIEF REVIEW OF THE ELECTRICAL PROPERTIES OF NEURONS [Seite 119]
13.2 - THE ELECTROPHYSIOLOGY RIG [Seite 121]
13.3 - CATEGORIES OF ELECTROPHYSIOLOGY RECORDINGS [Seite 122]
13.4 - ELECTROPHYSIOLOGY TISSUE PREPARATIONS [Seite 137]
13.5 - MANIPULATING NEURAL ACTIVITY DURING ELECTROPHYSIOLOGY EXPERIMENTS [Seite 142]
13.6 - CONCLUSION [Seite 143]
13.7 - SUGGESTED READING AND REFERENCES [Seite 143]
13.8 - Books [Seite 143]
13.9 - Review Articles [Seite 143]
13.10 - Primary Research Articles-Interesting Examples from the Literature [Seite 143]
13.11 - Protocols [Seite 144]
14 - Chapter 5 - Microscopy [Seite 146]
14.1 - ESSENTIAL PRINCIPLES OF MICROSCOPY [Seite 147]
14.2 - LIGHT MICROSCOPY [Seite 155]
14.3 - FLUORESCENCE MICROSCOPY [Seite 157]
14.4 - ELECTRON MICROSCOPY [Seite 162]
14.5 - PREPARING AND INTERPRETING MICROSCOPY DATA [Seite 165]
14.6 - CONCLUSION [Seite 168]
14.7 - SUGGESTED READING AND REFERENCES [Seite 172]
14.8 - Books [Seite 172]
14.9 - Review Articles [Seite 172]
14.10 - Primary Research Articles-Interesting Examples from the Literature [Seite 172]
14.11 - Protocols [Seite 173]
14.12 - Websites [Seite 173]
15 - Chapter 6 - Visualizing Neural Structure [Seite 174]
15.1 - TISSUE PREPARATION [Seite 175]
15.2 - VISUALIZING MORPHOLOGY [Seite 179]
15.3 - VISUALIZING GENE AND PROTEIN EXPRESSION [Seite 185]
15.4 - VISUALIZING CIRCUITRY [Seite 191]
15.5 - CONCLUSION [Seite 194]
15.6 - SUGGESTED READING AND REFERENCES [Seite 194]
15.7 - Books [Seite 194]
15.8 - Review Articles [Seite 194]
15.9 - Primary Research Articles-Interesting Examples from the Literature [Seite 195]
15.10 - Protocols [Seite 195]
15.11 - Websites [Seite 195]
16 - Chapter 7 - Visualizing Neural Function [Seite 196]
16.1 - STATIC MARKERS OF ACTIVITY [Seite 197]
16.2 - VISUALIZING NEURAL ACTIVITY [Seite 200]
16.3 - VISUALIZING PROTEIN FUNCTION [Seite 206]
16.4 - CONCLUSION [Seite 210]
16.5 - SUGGESTED READING AND REFERENCES [Seite 210]
16.6 - Books [Seite 210]
16.7 - Review Articles [Seite 210]
16.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 211]
16.9 - Protocols [Seite 212]
17 - Chapter 8 - Manipulating Neural Activity [Seite 214]
17.1 - EXPERIMENTAL LOGIC OF MANIPULATING NEURAL ACTIVITY [Seite 215]
17.2 - PHYSICAL MANIPULATION [Seite 216]
17.3 - ELECTRICAL MANIPULATION [Seite 218]
17.4 - PHARMACOLOGICAL MANIPULATION [Seite 218]
17.5 - GENETIC MANIPULATION [Seite 220]
17.6 - PHARMACOGENETIC MANIPULATION [Seite 221]
17.7 - OPTOGENETIC MANIPULATION [Seite 222]
17.8 - NEUROMODULATION TECHNIQUES USED IN HUMANS [Seite 226]
17.9 - CONCLUSION [Seite 226]
17.10 - SUGGESTED READING AND REFERENCES [Seite 229]
17.11 - Books [Seite 229]
17.12 - Review Articles [Seite 229]
17.13 - Primary Research Articles-Interesting Examples from the Literature [Seite 229]
17.14 - Protocols [Seite 230]
17.15 - Websites [Seite 230]
18 - Chapter 9 - Identifying Genes and Proteins of Interest [Seite 232]
18.1 - A BRIEF REVIEW OF GENES AND PROTEINS [Seite 233]
18.2 - GENETIC SCREENS [Seite 238]
18.3 - IN SILICO SCREENS [Seite 243]
18.4 - MOLECULAR SCREENS [Seite 243]
18.5 - CONCLUSION [Seite 245]
18.6 - SUGGESTED READING AND REFERENCES [Seite 246]
18.7 - Books [Seite 246]
18.8 - Review Articles [Seite 246]
18.9 - Primary Research Articles-Interesting Examples from the Literature [Seite 246]
18.10 - Protocols [Seite 246]
18.11 - Websites [Seite 247]
19 - Chapter 10 - Molecular Cloning and Recombinant DNA Technology [Seite 248]
19.1 - ISOLATING DNA FRAGMENTS [Seite 249]
19.2 - CLONING DNA [Seite 256]
19.3 - IDENTIFYING DNA [Seite 260]
19.4 - CONCLUSION [Seite 265]
19.5 - SUGGESTED READING AND REFERENCES [Seite 265]
19.6 - Books [Seite 265]
19.7 - Review Articles [Seite 266]
19.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 266]
19.9 - Protocols [Seite 266]
19.10 - Websites [Seite 266]
20 - Chapter 11 - Gene Delivery Strategies [Seite 268]
20.1 - PHYSICAL GENE DELIVERY [Seite 268]
20.2 - CHEMICAL GENE DELIVERY [Seite 273]
20.3 - VIRAL GENE DELIVERY [Seite 276]
20.4 - CONCLUSION [Seite 279]
20.5 - SUGGESTED READING AND REFERENCES [Seite 280]
20.6 - Books [Seite 280]
20.7 - Review Articles [Seite 280]
20.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 281]
20.9 - Protocols [Seite 281]
20.10 - Websites [Seite 281]
21 - Chapter 12 - Making and Using Transgenic Organisms [Seite 282]
21.1 - TRANSGENES [Seite 283]
21.2 - THE TRANSGENIC CONSTRUCT [Seite 287]
21.3 - BINARY TRANSGENIC SYSTEMS [Seite 290]
21.4 - MAKING TRANSGENIC ORGANISMS [Seite 294]
21.5 - CONCLUSION [Seite 298]
21.6 - SUGGESTED READING AND REFERENCES [Seite 299]
21.7 - Books [Seite 299]
21.8 - Review Articles [Seite 299]
21.9 - Primary Research Articles-Interesting Examples from the Literature [Seite 299]
21.10 - Protocols [Seite 300]
21.11 - Websites [Seite 300]
22 - Chapter 13 - Manipulating Endogenous Genes [Seite 302]
22.1 - CLASSICAL GENE TARGETING METHODS [Seite 303]
22.2 - NUCLEASE-BASED GENE TARGETING METHODS [Seite 313]
22.3 - DISRUPTING GENE PRODUCTS [Seite 316]
22.4 - CONCLUSION [Seite 320]
22.5 - SUGGESTED READING AND REFERENCES [Seite 320]
22.6 - Books [Seite 320]
22.7 - Review Articles [Seite 321]
22.8 - Primary Research Articles-Interesting Examples from the Literature [Seite 321]
22.9 - Protocols [Seite 321]
23 - Chapter 14 - Cell Culture Techniques [Seite 324]
23.1 - CELL CULTURE EQUIPMENT AND REAGENTS [Seite 325]
23.2 - IMMORTALIZED CELL LINES [Seite 327]
23.3 - PRIMARY CELL AND TISSUE CULTURE [Seite 330]
23.4 - STEM CELL CULTURES [Seite 333]
23.5 - MANIPULATING CELLS IN CULTURE [Seite 336]
23.6 - CONCLUSION [Seite 337]
23.7 - SUGGESTED READING AND REFERENCES [Seite 338]
23.8 - Books [Seite 338]
23.9 - Review Articles [Seite 338]
23.10 - Primary Research Articles-Interesting Examples from the Literature [Seite 338]
23.11 - Protocols [Seite 339]
23.12 - Websites [Seite 339]
24 - Chapter 15 - Biochemical Assays and Intracellular Signaling [Seite 340]
24.1 - INTRODUCTION TO SIGNAL TRANSDUCTION AND INTRACELLULAR SIGNALING [Seite 342]
24.2 - FUNDAMENTAL TOOLS USED TO STUDY PROTEINS [Seite 342]
24.3 - INVESTIGATING PROTEIN EXPRESSION [Seite 349]
24.4 - INVESTIGATING PROTEIN-PROTEIN INTERACTIONS [Seite 356]
24.5 - INVESTIGATING POSTTRANSLATIONAL MODIFICATIONS [Seite 360]
24.6 - INVESTIGATING PROTEIN-DNA INTERACTIONS [Seite 366]
24.7 - CONCLUSION [Seite 371]
24.8 - SUGGESTED READING AND REFERENCES [Seite 371]
24.9 - Books [Seite 371]
24.10 - Review Articles [Seite 372]
24.11 - Primary Research Articles-Interesting Examples from the Literature [Seite 372]
24.12 - Protocols [Seite 372]
24.13 - Websites [Seite 372]
25 - Glossary [Seite 374]
26 - Index [Seite 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...

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