Guide to Research Techniques in Neuroscience

Front Cover
Guide to Research Techniques in Neuroscience
Guide to Research Techniques in Neuroscience
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Contents
Preface
Introduction
LEVELS OF INVESTIGATION
APPROACHES TO STUDYING THE NERVOUS SYSTEM
Examining Case Studies
Screens
Description
Manipulation
UNDERSTANDING TECHNIQUES IN NEUROSCIENCE
1 - Noninvasive Brain Imaging
STRUCTURAL BRAIN IMAGING TECHNIQUES
Cerebral Angiography
Computerized Tomography
Magnetic Resonance Imaging
The Electromagnetic Basis of MRI Technology
Generating an Image
Selecting a “Slice” of Brain to Image
Diffusion Magnetic Resonance Imaging
FUNCTIONAL BRAIN IMAGING TECHNIQUES
Functional Magnetic Resonance Imaging
Positron Emission Tomography
Single-Proton Emission Computerized Tomography
Electroencephalography
Magnetoencephalography
Optical Imaging
FUNCTIONAL IMAGING EXPERIMENTAL DESIGN AND ANALYSIS
Planning the Experiment
Practical Considerations
Experimental Paradigm of a Functional Imaging Experiment
Task Paradigms
Pilot Experiments
Planning an Experiment that Uses Human Subjects
Conducting the Experiment
Working With Human Subjects
Data Acquisition
Manipulating Neural Activity During an Experiment
Postexperimental Data Analysis
Data Analysis
Preparation of Figures
CONCLUSION
SUGGESTED READING AND REFERENCES
2 - Animal Behavior
CONSIDERATIONS FOR CHOOSING AND PERFORMING A BEHAVIORAL ASSAY
Choosing an Appropriate Model Organism
Choosing an Appropriate Behavioral Paradigm
Variability in Individuals
Using Animal Behavior as a Model for Human Behavior
RODENT BEHAVIORAL PARADIGMS
Locomotor Activity
Running Wheel
Homecage Activity
Open Field Test
Motor Coordination and Balance
Rotarod
Footprint Pattern Assay
Hanging Wire Assay
Vertical Pole Test
Sensory Function
Visual Cliff Assay
Startle Response Assay
Taste/Flavor Assays
Olfaction Assays
Nociception
Tail Flick Assay
Hargreaves Assay
Hot Plate Assay
Von Frey Assay
Formalin Assay
Spatial Learning and Memory
Morris Water Maze
Barnes Maze
Radial Arm Maze
Virtual Reality Mazes
Nonspatial Learning and Memory
Novel Object Recognition
Delayed Match to Sample/Nonmatch to Sample Task
Attention and Impulsivity
Go/No-go Task
Five Choice Serial Reaction Time Task
Reward-Related Behaviors
Self-Administration
Conditioned Place Preference/Avoidance Assay
Real-Time Place Preference Assay
Social Behaviors
Resident–Intruder Assay
Social Approach/Avoidance
Anxiety
Open Field Test
Elevated Plus Maze
Defensive Marble Burying
Geller-Seifter Conflict Test
Depression
Forced Swim Test (Porsolt Test)
Tail Suspension Assay
Sucrose Preference Test
DROSOPHILA BEHAVIORAL PARADIGMS
Locomotor Behavior
Flight
Sensory Function
Vision Assays
Olfaction Assays
Taste Assays
Learning and Memory
Social Behaviors
Courtship
Aggression
C. ELEGANS BEHAVIORAL PARADIGMS
Locomotor Behavior
Sensory Behavior
Mechanosensation
Thermosensation
Chemosensation
NONHUMAN PRIMATE BEHAVIORAL PARADIGMS
CONCLUSION
SUGGESTED READING AND REFERENCES
3 - Stereotaxic Surgeries
DETERMINING COORDINATES OF BRAIN REGIONS
STEREOTAXIC SURGERIES IN RODENTS
Creating a Sterile Environment
Anesthesia
Positioning a Rodent on a Stereotaxic Instrument
Accessing the Brain
Penetrating the Brain and Attaching Implants
Sealable Chambers
Cannulae
Electrophysiology Implants
Optical Implants
Finishing the Surgery
STEREOTAXIC SURGERIES IN NONHUMAN PRIMATES
CONCLUSION
SUGGESTED READING AND REFERENCES
4 - Electrophysiology
A BRIEF REVIEW OF THE ELECTRICAL PROPERTIES OF NEURONS
THE ELECTROPHYSIOLOGY RIG
CATEGORIES OF ELECTROPHYSIOLOGY RECORDINGS
Extracellular Recording
Intracellular Recording
Patch Clamp Techniques
ELECTROPHYSIOLOGY TISSUE PREPARATIONS
In Vitro Recordings
Heterologous Expression Systems
Primary Cultures
Slice Cultures
In Vivo Recordings
Acute Versus Chronic Recordings
Anesthetized Versus Awake Animals
CONCLUSION
SUGGESTED READING AND REFERENCES
5 - Microscopy
ESSENTIAL PRINCIPLES OF MICROSCOPY
Fundamental Parameters in Microscopy
The Design of a Compound Microscope
The Design of a Stereomicroscope
LIGHT MICROSCOPY
FLUORESCENCE MICROSCOPY
Epifluorescent Microscopy
Confocal Microscopy
Two-Photon Microscopy
Light Sheet Fluorescence Microscopy
Total Internal Reflection Fluorescence Microscopy
ELECTRON MICROSCOPY
Transmission Electron Microscopy
Scanning Electron Microscopy
Electron Tomography
PREPARING AND INTERPRETING MICROSCOPY DATA
Image Processing
Interpreting Images
CONCLUSION
SUGGESTED READING AND REFERENCES
6 - Visualizing Nervous System Structure
TISSUE PREPARATION
Fixation
Embedding
Sectioning
Tissue Clearing
VISUALIZING MORPHOLOGY
Cell Body Stains
Fiber Stains
Golgi Stain
Intracellular and Juxtacellular Labeling
VISUALIZING GENE AND PROTEIN EXPRESSION
In Situ Hybridization
Immunohistochemistry
Enzymatic Histochemistry
Reporter Proteins
VISUALIZING CIRCUITRY
Anterograde and Retrograde Tracers
Transsynaptic Labeling
Viruses
Plant Lectins
CONCLUSION
SUGGESTED READING AND REFERENCES
7 - Visualizing and Measuring Neural Dynamics
STATIC MARKERS OF ACTIVITY
Assaying Neural Activity in Fixed Tissue
Assaying Cellular Function in Fixed Tissue
Assaying Cell Proliferation with Thymidine Analogs
CLASSICAL METHODS OF MEASURING EXTRACELLULAR NEUROCHEMISTRY IN VIVO
Microdialysis
Voltammetry and Amperometry
BIOSENSORS FOR VISUALIZING NEURAL ACTIVITY
Biosensors for Imaging Calcium Dynamics
Genetically Encoded Calcium Indicators
Calcium Indicator Dyes
Biosensors for Imaging Membrane Voltage
Genetically Encoded Voltage Indicators
Voltage-Sensitive Dyes
BIOSENSORS FOR VISUALIZING NEUROTRANSMISSION
Biosensors for Imaging Presynaptic Vesicle Release
FM Dyes
pH-Sensitive Fluorescent Proteins
Biosensors for Imaging Neurotransmitter/Neuromodulator Signaling
Neurotransmitter Signaling
Neuromodulator Signaling
STRATEGIES FOR VISUALIZING AND MEASURING ACTIVITY FROM FLUORESCENT BIOSENSORS
Fiber Photometry
Microendoscopy
Cranial Windows
IMAGE PROCESSING IN VISUALIZATION EXPERIMENTS
CONCLUSION
SUGGESTED READING AND REFERENCES
8 - Manipulating Neural Activity
EXPERIMENTAL LOGIC OF MANIPULATING NEURAL ACTIVITY
PHYSICAL MANIPULATION
Ablation
Thermal Cooling
ELECTRICAL MANIPULATION
PHARMACOLOGICAL MANIPULATION
GENETIC MANIPULATION
CHEMOGENETIC MANIPULATION
OPTOGENETIC MANIPULATION
Optogenetic Transgenes
Optogenetic Stimulation
Optogenetic Inhibition
Methods of Delivering Light to the Nervous System
Strategies for Studying Neural Circuits Using Optogenetics
NEUROMODULATION TECHNIQUES USED IN HUMANS
CONCLUSION
SUGGESTED READING AND REFERENCES
9 - Identifying Genes and Proteins of Interest
HOW GENES ENCODE FOR PROTEINS
The Central Dogma of Molecular Biology
DNA
Transcription
Translation
GENETIC SCREENS
Forward Genetic Screen
Reverse Genetic Screen
IN SILICO SCREENS
BASIC LOCAL ALIGNMENT SEARCH TOOL (BLAST)
Ensembl
MOLECULAR SCREENS
RNA Sequencing
CRISPR-Cas9 Screens
CONCLUSION
SUGGESTED READING AND REFERENCES
10 - Molecular Cloning and Recombinant DNA Technology
ISOLATING DNA FRAGMENTS
Restriction Enzymes
Polymerase Chain Reaction
Standard PCR
Reverse Transcription PCR
Quantitative Real-Time PCR
Modifying DNA Sequences Using PCR
Isolation and Characterization of DNA Fragments Using Gel Electrophoresis
CLONING DNA
Vectors
Common Vector Features
Types of Vectors
Ligation
Transformation
Purifying DNA from Host Cells
IDENTIFYING DNA
DNA Sequencing
Nucleic Acid Hybridization Techniques
Southern Blot
Northern Blot
CONCLUSION
SUGGESTED READING AND REFERENCES
11 - Gene Delivery Strategies
PHYSICAL GENE DELIVERY
Microinjection
Electroporation
Biolistics
CHEMICAL GENE DELIVERY
Calcium Phosphate Transfection
Lipid Transfection
VIRAL GENE DELIVERY
Adeno-Associated Virus
Lentivirus
Canine Adenovirus
Rabies Virus
CONCLUSION
SUGGESTED READING AND REFERENCES
12 - Making and Using Genetically Modified Organisms
REASONS FOR ENGINEERING A GENETICALLY MODIFIED ORGANISM
Disrupting Endogenous Gene Function
Replacing Endogenous Genetic Material
Inserting a Functional Transgene
COMMONLY USED TRANSGENES
Reporter Genes
Transgenes Used to Measure Neural Activity
Transgenes Used to Manipulate Neural Activity
Transgenes Used to Silence Neural Activity
Transgenes Used to Ablate Neurons
USING PROMOTERS TO REGULATE GENE EXPRESSION
GENERAL STRATEGIES FOR GENOME MODIFICATION
Nonspecific Transgene Insertion
Classical DNA Targeting in Mice Using Homologous Recombination
DNA Targeting/Editing Using CRISPR/Cas9 Endonucleases
ENGINEERING GENETICALLY MODIFIED ORGANISMS
Making Genetically Engineered Mice
Making Genetically Engineered Flies
Making Genetically Modified Worms
Making Other Genetically Modified Organisms
BINARY EXPRESSION SYSTEMS
The Gal4/UAS System
The Cre/lox System
The Flp/FRT System
The Tet-off/Tet-on System
DISRUPTING GENE PRODUCTS
RNA Interference
Morpholinos
CONCLUSION
SUGGESTED READING AND REFERENCES
13 - Cell Culture Techniques
CELL CULTURE EQUIPMENT AND REAGENTS
Equipment
Culture Media
IMMORTALIZED CELL LINES
PRIMARY CELL AND TISSUE CULTURE
Slice Cultures
Explant Cultures
Dissociated Cell Cultures
STEM CELL CULTURES
Embryonic Stem Cells
Neural Stem Cells
Induced Pluripotent Stem Cells
Brain Organoids
MANIPULATING CELLS IN CULTURE
CONCLUSION
SUGGESTED READINGS AND REFERENCES
14 - Intracellular Signaling and Biochemical Assays
INTRODUCTION TO SIGNAL TRANSDUCTION AND INTRACELLULAR SIGNALING
FUNDAMENTAL TOOLS USED TO STUDY PROTEINS
Making and Using Antibodies
Purifying Proteins
Chromatography
Immunoprecipitation
INVESTIGATING PROTEIN EXPRESSION
Western Blot
Enzyme-Linked Immunosorbent Assay
Radioimmunoassay
Immunohistochemistry
Visualizing Protein Dynamics Using Fused Reporter Proteins
Assaying Protein Trafficking with Pulse-Chase Labeling
INVESTIGATING PROTEIN-PROTEIN INTERACTIONS
Co-immunoprecipitation
Protein Affinity Chromatography
Yeast Two-Hybrid Assay
Proximity Labeling to Identify Protein–Protein Interactions
Fluorescent Visualization of Protein–Protein Interactions
INVESTIGATING POSTTRANSLATIONAL MODIFICATIONS
Detection of PTMs
PTM-Specific Assays
INVESTIGATING PROTEIN–DNA INTERACTIONS
Electrophoretic Mobility Shift Assay
Chromatin Immunoprecipitation
Luciferase Assay
CONCLUSION
SUGGESTED READING AND REFERENCES
Glossary
Index
A
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D
E
F
G
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I
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K
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M
N
O
P
Q
R
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T
U
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W
X
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