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
	B
	C
	D
	E
	F
	G
	H
	I
	J
	K
	L
	M
	N
	O
	P
	Q
	R
	S
	T
	U
	V
	W
	X
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