Current Protocols in Neuroscience
Table of Contents

Foreword by Zach W. Hall
Preface
Contributors

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
1.10 Quantitative In Situ Hybridization for the Study of Gene Expression at the Regional and Cellular Levels (New, April 2003)

1.11 Counting Cells in Sectioned Material: A Suite of Techniques, Tools, and Tips
12 Intracellular and Juxtacellular Staining with Biocytin
1.13 Quantified Assessment of Terminal Density and Innervation

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
2.6 Use of FM1-43 and Other Derivatives to Investigate Neuronal Function
2.7 Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins

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 
4.28 Gene Expression Analysis Using cDNA Microarrays
4.29 Gene Targeting by Homologous Recombination 
4.30 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

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
5.19 Immunoblotting and Immunodetection

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 
6.16 Chronic Recording of Extracellular Neuronal Activity in Behaving Animals Learning 
6.17 Imaging Nervous System Activity with Voltage- Sensitive Dyes  
6.18 Recording in the Cerebellar Slice
6.19 Patch-Clamp Recording from Neuronal Dendrites

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 
8.5D Social Learning of Food Preferences in Rodents: Rapid Appetitive 
8.5E Assessment of Sustained and Divided Attention in Rats
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 
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 
8.16 Mouse Social Recognition and Preference
8.17 Assessement of Murine Startle Reactivity, Prepulse Inhibition, and Habituation
8.18 Assessment of Developmental Milestones in Rodents

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
9.12 Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in Treating Alcoholism and Studying the Neuropharmacological Bases of Ethanol-Seeking Behaviors in Rats 
9.13 Models of Amyotrophic Lateral Sclerosis
9.14 Models of Neuropathic Pain in the Rat
9.15 Place Preference Test in Rodents 
9.16 Inducing Photochemical Cortical Lesions in Rat Brain
9.17 Measurement of Panic-Like Responses Following Intravenous Infusion of Sodium Lactate in Panic-Prone Rats
9.18 Animal Models of Painful Diabetic Neuropathy: The STZ Rat Model

Appendix 1 Molecular Biology Techniques
1A Molecular Biology References
1B Optimization of Transfection
1C Calcium Phosphate Transfection
1D Transfection Using DEAE-Dextran
1E Transfection by Electroporation
1F Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents
1G Purification and Concentration of DNA from Aqueous Solutions
1H Preparation of Genomic DNA from Mammalian Tissue
1I Preparation of RNA from Tissues and Cells
1J Preparation of Bacterial Plasmid DNA
1K Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
1L Introduction of Plasmid DNA into Cells
1M Digestion of DNA with Restriction Endonucleases
1N Agarose Gel Electrophoresis
Appendix 2 Stock Solutions, Equipment, and Laboratory Guidelines
2A Common Stock Solutions, Buffers, and Media
2B Standard Laboratory Equipment
Appendix 3 Commonly Used Techniques
3A Preparation of an Iodinated Radioligand
3B Techniques for Mammalian Cell Tissue Culture
Appendix 4 Animal Techniques
4A High Precision Stereotaxic Surgery in Mice
4B Mouse and Rat Anesthesia and Analgesia
4C Animal Health Assurance
4D Handling and Restraint
4E Animal Identification
Appendix 5 Databases
5A Internet Resources for Genomic, Bioinformatics, and Medical Genetics Information
5B Databases and Web Sites for Neurogenetics
SUPPLIERS APPENDIX
Selected Suppliers of Reagents and Equipment

Index