Current Protocols in Neuroscience

What's New in Current Protocols in Neuroscience
Supplement 36, August 2006

UNIT 2.10 Whole-Mount Immunohistochemistry of the Brain (New)

UNIT 3.19 Rodent Sensory Neuron Culture and Analysis (New)

UNIT 9.23A Binge Eating in Rats Produced by Combining Dieting with Stress (New)

UNIT 9.23B Binge Eating in Rats with Limited Access to Vegetable Shortening (New)

UNIT 9.23C Sugar Bingeing in Rats (New)

UNIT 9.23D Characterization of Energy Expenditure in Rodents by Indirect Calorimetry (New)

Current Protocols in Neuroscience is a one-stop resource for finding and adapting the best models and methods for all types of neuroscience experiments.

Updated every three months in all formats, CPNS is constantly evolving to keep pace with the very latest discoveries and developments. A year of these quarterly updates is included in the initial CPNS purchase price. That's 570 pages of new (60%) and revised (40%) content on average every year since the initial publication of the work in October 1997! Presently three volumes in its looseleaf print version, CPNS...

* brings together hundreds of core techniques in all branches of neuroscience from top scientists around the world.
* draws from molecular neurobiology, neurophysiology, neuroanatomy, neuropharmacology, and behavioral neuroscience.
* includes both in vitro and in vivo models, prepared, edited, and tailored especially for neuroscience research.

Edited by: Charles Gerfen (Bethesda, Maryland); Andrew Holmes (Bethesda, Maryland); Michael Rogawski (Bethesda, Maryland); David Sibley (Bethesda, Maryland); Phil Skolnick (DOV Pharmaceuticals); Susan Wray (Bethesda, Maryland); Past Editor: Jacqueline Crawley; Ron McKay

Series Editor: Gwen Taylor


Chapter 1 Neuroanatomical Methods
Introduction
Unit 1.1 Basic Neuroanatomical Methods
Unit 1.2 Immunohistochemical Localization of Proteins in the Nervous System
Unit 1.3 Hybridization Histochemistry of Neural Transcripts
Unit 1.4 Receptor Binding Techniques
Unit 1.5 Transneuronal Circuit Analysis With Pseudorabies Viruses
Unit 1.6 Metabolic Mapping
Unit 1.7 Targeted Toxins
Unit 1.8 Cellular Compartment Analysis of Temporal Activity by Fluorescence In Situ Hybridization (catFISH)
Unit 1.9 Detection of DNA Damage in Tissue Sections by In Situ Nick Translation
Unit 1.10 Quantitative In Situ Hybridization for the Study of Gene Expression at the Regional and Cellular Levels
Unit 1.11 Counting Cells in Sectioned Material: A Suite of Techniques, Tools, and Tips
Unit 1.12 Intracellular and Juxtacellular Staining with Biocytin
Unit 1.13 Quantified Assessment of Terminal Density and Innervation

Chapter 2 Imaging
Introduction
Unit 2.1 Theory and Application of Fluorescence Microscopy
Unit 2.2 Basic Confocal Microscopy
Unit 2.3 Imaging Nervous System Activity
Unit 2.4 Electronic Imaging in Neuroscience
Unit 2.5 Loading Neurons with Dextran-Conjugated Calcium Indicators in Intact Nervous Tissue
Unit 2.6 Use of FM1-43 and Other Derivatives to Investigate Neuronal Function
Unit 2.7 Multidisciplinary Approaches for Characterizing Synaptic Vesicle Proteins
Unit 2.8 3-D Reconstruction of Neurons from Multichannel Confocal Laser Scanning Image Series
Unit 2.9 Two-Photon Imaging in Live Rodents
Unit 2.10 Whole-Mount Immunohistochemistry of the Brain

Chapter 3 Cellular and Developmental Neuroscience
Introduction
Unit 3.1 Culture of Neuroepithelial Stem Cells
Unit 3.2 Long-Term Culture of Hippocampal Neurons
Unit 3.3 Culture of Substantia Nigra Neurons
Unit 3.4 Isolation and Purification of Primary Oligodendrocyte Precursors
Unit 3.5 Isolation and Purification of Primary Rodent Astrocytes
Unit 3.6 Differentiation of Embryonic Stem Cells
Unit 3.7 Immortalizing Central Nervous System Cells
Unit 3.8 Identification of Neural Programmed Cell Death Through the Detection of DNA Fragmentation In Situ and by PCR
Unit 3.9 Quantitative Analysis of In Vivo Cell Proliferation
Unit 3.10 Experimental Transplantation in the Embryonic, Neonatal, and Adult Mammalian Brain
Unit 3.11 Calcium Phosphate Transfection of DNA into Neurons in Primary Culture
Unit 3.12 Purification of Oligodendrocytes and Their Progenitors Using Immunomagnetic Separation and Percoll Gradient Centrifugation
Unit 3.13 Isolation and Generation of Oligodendrocytes by Immunopanning
Unit 3.14 Generation and Utilization of Phosphorylation State—Specific Antibodies to Investigate Signaling Pathways
Unit 3.15 Neuronal Transfection Using Particle-Mediated Gene Transfer
Unit 3.16 Gene Expression in Transgenic Mice Using Neural Promoters
Unit 3.17 Isolation and Purification of Primary Schwann Cells
Unit 3.18 Fluorescence-Based Sorting of Neural Stem Cells and Progenitors
Unit 3.19 Rodent Sensory Neuron Culture and Analysis

Chapter 4 Gene Cloning, Expression, and Mutagenesis
Introduction
Unit 4.1 Homology Cloning of cDNA or Genomic DNA
Unit 4.2 PCR Cloning of Neural Gene Products
Unit 4.3 Expression Cloning of Neural Genes Using Xenopus laevis Oocytes
Unit 4.4 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
Unit 4.5 Overview of Neural Gene Expression
Unit 4.6 Selection of Transfected Mammalian Cells
Unit 4.7 Transient Expression of Proteins Using COS Cells
Unit 4.8 cDNA Expression Cloning in Mammalian Cells
Unit 4.9 Mutagenesis Approaches for Elucidation of Protein Structure-Function Relationships
Unit 4.10 Oligonucleotide-Directed Mutagenesis Without Phenotypic Selection
Unit 4.11 Directed Mutagenesis Using the Polymerase Chain Reaction
Unit 4.12 Overview of Gene Delivery into Cells Using HSV-1-Based Vectors
Unit 4.13 Generation of High-Titer Defective HSV-1 Vectors
Unit 4.14 Gene Delivery Using Helper Virus—Free HSV-1 Amplicon Vectors
Unit 4.15 The Substituted-Cysteine Accessibility Method (SCAM) to Elucidate Membrane Protein Structure
Unit 4.16 Protein Expression in the Drosophila Schneider 2 Cell System
Unit 4.17 Production of Recombinant Adeno-Associated Viral Vectors and Use for In Vitro and In Vivo Administration
Unit 4.18 Overview of the Baculovirus Expression System
Unit 4.19 Protein Expression in the Baculovirus System
Unit 4.20 Calcium Flux Assay in Xenopus Oocytes
Unit 4.21 Production of High-Titer Lentiviral Vectors
Unit 4.22 Alphavirus-Mediated Gene Transfer into Neurons
Unit 4.23 Gene Transfer into Neural Cells In Vitro Using Adenoviral Vectors
Unit 4.24 Gene Transfer into Rat Brain Using Adenoviral Vectors
Unit 4.25 Overview of Nucleic Acid Arrays
Unit 4.26 Preparation of mRNA for Expression Monitoring
Unit 4.27 cDNA Library Construction from Single Cells
Unit 4.28 Gene Expression Analysis Using cDNA Microarrays
Unit 4.29 Overview of Gene Targeting by Homologous Recombination
Unit 4.30 Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Chapter 5 Molecular Neuroscience
Introduction
Unit 5.1 RNA Analysis by Nuclease Protection
Unit 5.2 Differential Display of mRNA by PCR
Unit 5.3 Analysis of mRNA Populations from Single Live and Fixed Cells of the Central Nervous System
Unit 5.4 Reducing Gene Expression in the Brain via Antisense Methods
Unit 5.5 Production of Polyclonal Antisera
Unit 5.6 Production of Antipeptide Antisera
Unit 5.7 Production of Antisera Using Fusion Proteins
Unit 5.8 Epitope Tagging of Recombinant Proteins
Unit 5.9 Overview of Membrane Protein Solubilization
Unit 5.10 Metal-Chelate Affinity Chromatography
Unit 5.11 Immunoaffinity Chromatography
Unit 5.12 The Use of Phage Display in Neurobiology
Unit 5.13 Fluorescent In Situ Transcription in Cells and Tissues
Unit 5.14 Detection of Protein Phosphorylation in Tissues and Cells
Unit 5.15 Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
Unit 5.16 Green Fluorescent Protein in the Study of Neuronal Signaling Pathways
Unit 5.17 Analysis of RNA by Northern and Slot-Blot Hybridization
Unit 5.18 Using Phage Display in Neurobiology
Unit 5.19 Immunoblotting and Immunodetection
Unit 5.20 An Overview on the Generation of BAC Transgenic Mice for Neuroscience Research
Unit 5.21 Modification of Bacterial Artificial Chromosomes (BACs) and Preparation of Intact BAC DNA for Generation of Transgenic Mice
Unit 5.22 Imaging Protein-Protein Interactions by Fluorescence Resonance Energy Transfer (FRET) Microscopy
Unit 5.23 Monitoring Protein-Protein Interactions in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)
Unit 5.24 Immunoprecipitation
Unit 5.25 Detection of Protein-Protein Interactions by Coprecipitation

Chapter 6 Neurophysiology
Introduction
Unit 6.1 The Electrophysiology Setup
Unit 6.2 Principles of Signal Conditioning
Unit 6.3 Fabrication of Patch Pipets
Unit 6.4 Preparation of Hippocampal Brain Slices
Unit 6.5 Acute Isolation of Neurons from the Mature Mammalian Central Nervous System
Unit 6.6 Whole-Cell Voltage Clamp Recording
Unit 6.7 Patch-Pipet Recording in Brain Slices
Unit 6.8 Single-Channel Recording
Unit 6.9 Rapid Solution Application Methods
Unit 6.10 Recording and Analyzing Synaptic Currents and Synaptic Potentials
Unit 6.11 Preparation and Maintenance of Organotypic Slice Cultures of CNS Tissue
Unit 6.12 Voltage Clamp Recordings from Xenopus Oocytes
Unit 6.13 Synaptic Plasticity in the Hippocampal Slice Preparation
Unit 6.14 Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at Single Cells
Unit 6.15 Preparation and Maintenance of Organotypic Cultures for Multi-Electrode Array Recordings
Unit 6.16 Chronic Recording of Extracellular Neuronal Activity in Behaving Animals
Unit 6.17 Imaging Nervous System Activity with Voltage-Sensitive Dyes
Unit 6.18 Recording in the Cerebellar Slice
Unit 6.19 Patch-Clamp Recording from Neuronal Dendrites

Chapter 7 Neurochemistry/Neuropharmacology
Introduction
Unit 7.1 Overview of Microdialysis
Unit 7.2 Microdialysis in Rodents
Unit 7.3 Microdialysis in Nonhuman Primates
Unit 7.4 Detection and Quantification of Neurotransmitters in Dialysates
Unit 7.5 Analyzing Radioligand Binding Data
Unit 7.6 Saturation Assays of Radioligand Binding to Receptors and Their Allosteric Modulatory Sites
Unit 7.7 Saturation Analysis of Ligand Binding Using a Centrifugation Procedure
Unit 7.8 Ligand Characterization Using Microphysiometry
Unit 7.9 Uptake and Release of Neurotransmitters
Unit 7.10 Measurement of Chloride Movement in Neuronal Preparations
Unit 7.11 Measurement of Cation Movement in Primary Cultures Using Fluorescent Dyes
Unit 7.12 Measurement of Second Messengers in Signal Transduction: cAMP and Inositol Phosphates
Unit 7.13 Measurement of NO and NO Synthase
Unit 7.14 Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic Microsensor
Unit 7.15 Scintillation Proximity Assay
Unit 7.16 Peripheral Type Benzodiazepine Binding Sites as a Tool for the Detection and Quantification of CNS Injury
Unit 7.17 Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues
Unit 7.18 Assessment of Cell Viability in Primary Neuronal Cultures
Unit 7.19 In Vivo Measurement of Blood-Brain Barrier Permeability
Unit 7.20 Methods for Sample Preparation for Direct Immunoassay Measurement of Analytes in Tissue Homogenates: ELISA Assay of Amyloid -Peptides

Chapter 8 Behavioral Neuroscience
Introduction
Unit 8.1 Locomotor Behavior
Unit 8.2 Sexual and Reproductive Behaviors
Unit 8.3 Animal Tests of Anxiety
Unit 8.4 Application of Experimental Stressors in Laboratory Rodents
Unit 8.5A Assessment of Spatial Memory Using the Radial Arm Maze and Morris Water Maze
Unit 8.5B Assessment of Spatial Memory Using the T Maze
Unit 8.5C Cued and Contextual Fear Conditioning in Mice
Unit 8.5D Social Learning of Food Preferences in Rodents: Rapid Appetitive Learning
Unit 8.5E Assessment of Sustained and Divided Attention in Rats
Unit 8.5F Assessment of Learning and Memory Using the Autoshaping of Operant Responding in Mice
Unit 8.5G Social Transmission of Food Preference in Mice
Unit 8.5H Assaying Aspects of Attention and Impulse Control in Mice Using the 5-Choice Serial Reaction Time Task
Unit 8.6A Overview of Methodological Approaches to the Study of Ingestive Behavior
Unit 8.6B Basic Measures of Food Intake
Unit 8.6C Direct, Continuous Behavioral Analysis of Drug Action on Feeding
Unit 8.6D Sham Feeding in Rats with Chronic, Reversible Gastric Fistulas
Unit 8.6E Conditioned Flavor Aversions: Assessment of Drug-Induced Suppression of Food Intake
Unit 8.6F Conditioned Flavor Preferences: Evaluating Postingestive Reinforcement by Nutrients
Unit 8.6G Macronutrient Selection in Experimental Animals
Unit 8.7 Measurement of Startle Response, Prepulse Inhibition, and Habituation
Unit 8.8 Measurement of Rodent Stereotyped Behavior
Unit 8.9 Models of Nociception: Hot-Plate, Tail-Flick, and Formalin Tests in Rodents
Unit 8.10A Rodent Models of Depression: Forced Swimming and Tail Suspension Behavioral Despair Tests in Rats and Mice
Unit 8.10B Rodent Models of Depression: Learned Helplessness Using a Triadic Design in Rats
Unit 8.10C Rodent Models of Depression: Learned Helplessness Induced in Mice
Unit 8.11A Fear-Potentiated Startle in Rats
Unit 8.11B Fear-Potentiated Startle in Mice
Unit 8.12 Motor Coordination and Balance in Rodents
Unit 8.13 Latent Inhibition
Unit 8.14 Ultrasonic Vocalizations in Rat and Mouse Pups
Unit 8.15 Parental Behaviors in Rats and Mice
Unit 8.16 Mouse Social Recognition and Preference
Unit 8.17 Assessment of Murine Startle Reactivity, Prepulse Inhibition, and Habituation
Unit 8.18 Assessment of Developmental Milestones in Rodents
Unit 8.19 Defensive Responses to Predator Threat in the Rat and Mouse
Unit 8.20 Olfactometry with Mice
Unit 8.21A Overview of Methods for Assessing the Mouse Auditory System
Unit 8.21B Measurement of the Auditory Brainstem Response (ABR) to Study Auditory Sensitivity in Mice
Unit 8.21C Assessment of Cochlear Function in Mice: Distortion-Product Otoacoustic Emissions
Unit 8.21D Behavioral Assessment of Hearing in Mice—Conditioned Suppression

Chapter 9 Preclinical Models of Neurologic and Psychiatric Disorders
Introduction
Unit 9.1 Dural Inflammation Model of Migraine Pain
Unit 9.2 Traumatic Brain Injury in the Rat Using the Fluid-Percussion Model
Unit 9.3 Preclinical Models of Hepatic Encephalopathy
Unit 9.4 Preclinical Models of Parkinson's Disease
Unit 9.5 Rodent Models of Global Cerebral Ischemia
Unit 9.6 Rodent Models of Focal Cerebral Ischemia
Unit 9.7 Experimental Autoimmune Encephalomyelitis (EAE)
Unit 9.8 A Murine Model of Retroviral Encephalopathy
Unit 9.9 Animal Models of Retroviral Encephalopathies: Feline Model
Unit 9.10 Overview of Rodent Models for Obesity Research
Unit 9.11 Intravenous Self-Administration of Ethanol in Mice
Unit 9.12 Preclinical Models to Evaluate Potential Pharmacotherapeutic Agents in Treating Alcoholism and Studying the Neuropharmacological Bases of Ethanol-Seeking Behaviors in Rats
Unit 9.13 Models of Amyotrophic Lateral Sclerosis
Unit 9.14 Models of Neuropathic Pain in the Rat
Unit 9.15 Place Preference Test in Rodents
Unit 9.16 Inducing Photochemical Cortical Lesions in Rat Brain
Unit 9.17 Measurement of Panic-Like Responses Following Intravenous Infusion of Sodium Lactate in Panic-Prone Rats
Unit 9.18 Animal Models of Painful Diabetic Neuropathy: The STZ Rat Model
Unit 9.19 Chemoconvulsant Model of Chronic Spontaneous Seizures
Unit 9.20 Chronic Intravenous Drug Self-Administration in Rats and Mice
Unit 9.21 Intravenous Self-Administration Techniques in Monkeys
Unit 9.22 Overview on Rodent Models of Alzheimer's Disease
Unit 9.23A Binge Eating in Rats Produced by Combining Dieting with Stress
Unit 9.23B Binge Eating in Rats with Limited Access to Vegetable Shortening
Unit 9.23C Sugar Bingeing in Rats
Unit 9.23D Characterization of Energy Expenditure in Rodents by Indirect Calorimetry

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
4F Parenteral Injections
4G Blood Collection
4H Euthanasia

Appendix 5 Databases
5A Internet Resources for Genomic, Bioinformatics, and Medical Genetics Information
5B Databases and Web Sites for Neurogenetics

Suppliers
Selected Suppliers of Reagents and Equipment