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Foreword by Zach W. Hall
Preface
Contributors
1 Neuroanatomical Methods
2 Imaging
3 Cellular and Developmental Neuroscience
4 Gene Cloning, Expression, and Mutagenesis
5 Molecular Neuroscience
6 Neurophysiology
7 Neurochemistry/Neuropharmacology
8 Behavioral Neuroscience
9 Preclinical Models of Neurologic and Psychiatric Disorders
Appendices
1 Neuroanatomical Methods
1.1 Basic Neuroanatomical Methods
1.2 Immunohistochemical Localization of Neurochemicals
1.3 Hybridization Histochemistry of Neural Transcripts
1.4 Receptor Binding Techniques
1.5 Transneuronal Circuit Analysis With Pseudorabies Viruses
1.6 Metabolic Mapping
1.7 Targeted Toxins
1.8 Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ
Hybridization (catFISH)
1.9 Detection of DNA Damage in Tissue Sections by In Situ Nick Translation
2 Imaging
2.1 Theory and Application of Fluorescence Microscopy
2.2 Basic Confocal Microscopy
2.3 Imaging Nervous System Activity
2.4 Electronic Imaging in Neuroscience
2.5 Loading Neurons with Dextran-Conjugated Calcium Indicators in Intact Nervous
Tissue (New, July 2002)
3 Cellular and Developmental
Neuroscience
3.1 Culture of Neuroepithelial Stem Cells
3.2 Long-Term Culture of Hippocampal Neurons
3.3 Culture of Substantia Nigra Neurons
3.4 Isolation and Purification of Primary Oligodendrocyte Precursors
3.5 Isolation and Purification of Primary Rodent Astrocytes
3.6 Differentiation of Embryonic Stem Cells
3.7 Immortalizing Central Nervous System Cells
3.8 Identification of Neural Programmed Cell Death Through the Detection of
DNA Fragmentation In Situ and by PCR
3.9 Quantitative Analysis of In Vivo Cell Proliferation
3.10 Experimental Transplantation in the Embryonic, Neonatal, and Adult Mammalian
Brain
3.11 CaPO4 Transfection of DNA into Cultured Neurons
3.12 Oligodendrocyte Purification by Immunomagnetic Separation and Percoll Gradients
3.13 Isolation and Generation of Oligodendrocytes by Immunopanning
3.14 Generation and Utilization of Phosphorylation State-Specific Antibodies
3.15 Neuronal Transfection Using Particle-Mediated Gene Transfer
3.16 Gene Expression in Transgenic Mice Using Neural Promoters
3.17 Isolation and Purification of Primary Schwann Cells
4 Gene Cloning, Expression, and Mutagenesis
4.1 Homology Cloning of cDNA or Genomic DNA
4.2 PCR Cloning of Neural Gene Products
4.3 Expression Cloning of Neural Genes Using Xenopus Oocytes
4.4 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
4.5 Overview of Neural Gene Expression
4.6 Selection of Transfected Mammalian Cells
4.7 Transient Expression of Proteins Using COS Cells
4.8 cDNA Expression Cloning in Mammalian Cells
4.9 Protein Structure-Function Relationships
4.10 Oligonucleotide-Directed Mutagenesis Without Phenotypic Selection
4.11 Directed Mutagenesis Using the Polymerase Chain Reaction
4.12 Overview of Gene Delivery into Cells Using HSV-1-Based Vectors
4.13 Generation of High-Titer Defective HSV-1 Vectors
4.14 Gene Delivery Using Helper-Virus-Free HSV-1 Amplicon Vectors
4.15 The Substituted-Cysteine Accessibility Method (SCAM) to Elucidate Membrane
Protein Structure
4.16 Protein Expression in the Drosophila Schneider 2 Cell System
4.17 Production of Recombinant Adeno-Associated Viral Vectors and Use in In
Vitro and In Vivo Administration
4.18 Overview of the Baculovirus Expression System
4.19 Protein Expression in the Baculovirus System
4.20 Calcium Flux Assay in Xenopus Oocytes
4.21 Production of High-Titer Lentiviral Vectors
4.22 Alphavirus-Mediated Gene Transfer into Neurons
4.23 Gene Transfer into Neural Cells In Vitro Using Adenoviral Vectors
4.24 Gene Transfer into Rat Brain Using Adenoviral Vectors
4.25 Overview of Nucleic Acid Arrays
4.26 Preparation of mRNA for Expression Monitoring
4.27 cDNA Library Construction from Single Cells (New, April 2002)
4.28 Gene Expression Analysis Using cDNA Microarrays (New, July 2002)
5 Molecular Neuroscience
5.1 RNA Analysis by Nuclease Protection
5.2 Differential Display of mRNA By PCR
5.3 Analysis of mRNA Populations from Single Live and Fixed Cells of the CNS
5.4 Reducing Gene Expression in The Brain Via Antisense Methods
5.5 Production of Polyclonal Antisera
5.6 Production of Antipeptide Antisera
5.7 Production of Antisera Using Fusion Proteins
5.8 Epitope Tagging of Recombinant Proteins
5.9 Overview of Membrane Protein Solubilization
5.10 Metal-Chelate Affinity Chromatography
5.11 Immunoaffinity Chromatography
5.12 The Use of Phage Display in Neurobiology
5.13 Fluorescent In Situ Transcription (FIST) in Cells and Tissues
5.14 Detection of Protein Phosphorylation in Tissues and Cells
5.15 Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics
In Vivo
5.16 Green Fluorescent Protein in the Study of Neuronal Signaling Pathways
5.17 Analysis of RNA by Northern and Slot-Blot Hybridization
5.18 Using Phage Display in Neurobiology
6 Neurophysiology
6.1 The Electrophysiology Setup
6.2 Principles of Signal Conditioning
6.3 Fabrication of Patch Pipets
6.4 Preparation of Hippocampal Brain Slices
6.5 Acute Isolation of Neurons from the Mature Mammalian Central Nervous System
6.6 Whole-Cell Voltage Clamp Recording
6.7 Patch-Pipet Recording in Brain Slices
6.8 Single-Channel Recording
6.9 Rapid Solution Application Methods
6.10 Recording and Analyzing Synaptic Currents and Synaptic Potentials
6.11 Preparation and Maintenance of Organotypic Slice Cultures of CNS
6.12 Voltage Clamp Recordings from Xenopus Oocytes
6.13 Synaptic Plasticity in the Hippocampal Slice Preparation
6.14 Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at
Single Cells
6.15 Preparation and Maintenance of Organotypic Cultures for Multi-Electrode
Array Recordings (New, April 2002)
7 Neurochemistry/Neuropharmacology
7.1 Overview of Microdialysis
7.2 Microdialysis in Rodents
7.3 Microdialysis in Nonhuman Primates
7.4 Detection and Quantification of Neurotransmitters in Dialysates
7.5 Analyzing Radioligand Binding Data
7.6 Saturation Assays of Radioligand Binding to Receptors and Their Allosteric
Modulatory Sites
7.7 Saturation Analysis of Ligand Binding Using a Centrifugation Procedure
7.8 Ligand Characterization Using Microphysiometry
7.9 Uptake and Release of Neurotransmitters
7.10 Chloride Movement in Neuronal Preparation
7.11 Cation Movement in Primary Cultures
7.12 Signal Transduction: cAMP and Inositol Phosphates
7.13 Measurement of NO and NO Synthase
7.14 Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic
Microsensor
7.15 Scintillation Proximity Assay
7.16 Tissue Peripheral Type Benzodiazepine Binding Sites as a Tool for the Detection
and Quantification of CNS Injury
7.17 Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues
7.18 Assessment of Cell Viability in Primary Neuronal Cultures
7.19 In Vivo Measurement of Blood-Brain Barrier Permeability
7.20 Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes
in Tissue Homogenates: ELISA Assay of Amyloid b-Peptides
8 Behavioral Neuroscience
8.1 Locomotor Behavior
8.2 Sexual and Reproductive Behaviors
8.3 Animal Tests of Anxiety
8.4 Application of Experimental Stressors in Laboratory Rodents
8.5A Assessment of Spatial Memory Using the Radial Arm and Morris Water Mazes
8.5B Assessment of Spatial Memory Using the T Maze
8.5C Cued and Contextual Fear Conditioning in Mice
8.6A Overview of Methodological Approaches to the Study of Ingestive Behavior
8.6B Basic Measures of Food Intake
8.6C Direct, Continuous Behavioral Analysis of Drug Action on Feeding
8.6D Sham Feeding in Rats with Chronic, Reversible Gastic Fistulas
8.6E Conditioned Flavor Aversions: Drug-Induced Suppression of Food Intake
8.6F Conditioned Flavor Preferences: Postingestive Reinforcement by Nutrients
8.6G Macronutrient Selection in Experimental Animals
8.7 Measurement of Startle Response, Prepulse Inhibition, and Habituation
8.8 Measurement of Rodent Stereotyped Behavior
8.9 Models of Nociception: Hot-Plate, Tail-Flick, and Formalin Tests in Rodents
8.10A Rodent Models of Depression: Forced Swimming and Tail Suspension Behavioral
Despair Tests in Rats and Mice
8.10B Rodent Models of Depression: Learned Helplessness Using a Triadic Design
in Rats
8.10C Rodent Models of Depression: Learned Helplessness Induced in Mice
8.11A Fear-Potentiated Startle in Rats
8.11B Fear-Potentiated Startle in Mice (New, April 2002)
8.12 Motor Coordination and Balance in Rodents
8.13 Latent Inhibition
8.14 Ultrasonic Vocalizations in Rat and Mouse Pups
8.15 Parental Behaviors in Rats and Mice
9 Preclinical Models of Neurologic and Psychiatric Disorders
9.1 Dural Inflammation Model of Migraine Pain
9.2 Traumatic Brain Injury in the Rat Using the Fluid-Percussion Model
9.3 Preclinical Models of Hepatic Encephalopathy
9.4 Preclinical Models of Parkinson's Disease
9.5 Rodent Models of Global Cerebral Ischemia
9.6 Rodent Models of Focal Cerebral Ischemia
9.7 Experimental Autoimmune Encephalomyelitis
9.8 A Murine Model of Retroviral Encephalopathy
9.9 Animal Models of Retroviral Encephalopathies: Feline Model
9.10 Overview of Rodent Models for Obesity Research
9.11 Ethanol Self-Administration in Mice (New, April 2002)
9.12 Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in
Treating Alcoholism and Studying the Neuropharmacological Bases of Ethanol-Seeking
Behaviors in Rats (New, April 2002)
9.13 Models of Amyotrophic Lateral Sclerosis (New, July 2002)
Appendices
1 Commonly Used Techniques
Molecular Biology Techniques
Optimization of Transfection
Calcium Phosphate Transfection
Transfection Using DEAE-Dextran
Transfection by Electroporation
Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
Purification and Concentration of DNA from Aqueous Solutions
Preparation of Genomic DNA from Mammalian Tissue
Preparation of RNA from Tissue and Cells
Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
Preparation of Bacterial Plasmid DNA
Introduction of Plasmid DNA into Cells
Digestion of DNA with Restriction Endonucleases
Agarose Gel Electrophoresis
2 Laboratory Guidelines, Equipment, Stock Solutions
Commonly Used Reagents
3 Commonly Used Techniques
Preparation of an Iodinated Radioligand
Techniques for Mammalian Cell Tissue Culture
4 Animal Techniques
High Precision Stereotaxic Surgery in Mice
Mouse and Rat Anesthesia and Analgesia
Animal Health Assurance
Index