Neurobiology: Methods and Protocols (Methods in Molecular Biology, 2746)

Preface
Contents
Contributors
Chapter 1: Plate-Based Assays for the Characterization of Mitochondrial and Cellular Phenotypes
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Equipment
		2.3 Reagents and  Kits
		2.4 Mitochondrial Staining Solutions
		2.5 NAD/NADH Detection Reagents
		2.6 AMPK and TORC1 Activity Assay Reagents
	3 Methods
		3.1 Mitochondrial Mass, Mitochondrial Membrane Potential, and Reactive Oxygen Species Measurements in Dictyostelium Cells
		3.2 Mitochondrial Mass, Mitochondrial Membrane Potential, and Reactive Oxygen Species Measurements in Lymphoblastoid Cell Lines
		3.3 Total Cellular NAD Assay
		3.4 AMPK and TORC1 Activity Assays for Human LCLs
	4 Notes
	References
Chapter 2: Behavioral Tests for Associative Learning in Caenorhabditis elegans
	1 Introduction
		1.1 Background
		1.2 C. elegans as a Model System for Learning and Memory Studies
		1.3 Types of Associative Learning Assays Using C. elegans
			1.3.1 Butanone Appetitive Learning
			1.3.2 Salt Aversive Learning
		1.4 C. elegans Maintenance for Associative Learning Assays
	2 Materials
		2.1 Buffers and Solutions
		2.2 Agar Plates
		2.3 Equipment
	3 Methods
		3.1 C. elegans Food Preparation
		3.2 Pouring Agar Plates
		3.3 C. elegans Maintenance
			3.3.1 Picking
			3.3.2 Chunking
		3.4 Butanone Appetitive Learning Assay
			3.4.1 Transferring Worms from Agar Plates (The ``Washing´´ Step)
			3.4.2 Starving Worms in Solution
			3.4.3 Conditioning Worms on Plates
			3.4.4 Butanone Chemotaxis Assays
		3.5 Salt Aversive Learning Assay
			3.5.1 Transferring Worms from Agar Plates (The ``Washing´´ Step)
			3.5.2 Conditioning Worms in Solution
			3.5.3 Salt Chemotaxis Assays
	4 Notes
	References
Chapter 3: Mechanical Ablation of Larval Zebrafish Spinal Cord
	1 Introduction
	2 Materials
		2.1 Tungsten Dissecting Needles
		2.2 Larval Mounting
		2.3 Larval Injuries
	3 Methods
		3.1 Preparation of Tungsten Dissecting Needles
		3.2 Larval Mounting
		3.3 Larval Injury
	4 Notes
	References
Chapter 4: High-Throughput Multiplex Immunohistochemistry of Glioma Organoids
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 3D Invasion Assay
		2.3 Spheroid Microarray
		2.4 Multiplex Immunohistochemistry
		2.5 Equipment
	3 Methods
		3.1 Cell Culture
		3.2 3D Invasion Assay
		3.3 Spheroid Microarray
			3.3.1 Generating the HistoGel Microarray/Grid
			3.3.2 Fixing and Embedding the Spheroids
		3.4 Multiplex Immunohistochemistry
	4 Notes
	References
Chapter 5: The Bilateral Carotid Artery Stenosis (BCAS) Model of Vascular Dementia
	1 Introduction
	2 Materials
	3 Methods
	4 Conclusion
	5 Notes
	References
Chapter 6: In Situ Hybridization to Characterize Neurulation and Midbrain-Hindbrain Boundary Formation in Zebrafish
	1 Introduction
	2 Materials
		2.1 Topoisomerase Cloning
		2.2 Embryo Preparation
		2.3 RNA Probe Transcription
	3 Methods
		3.1 PCR Amplification and Cloning Preparation
		3.2 Topoisomerase Cloning
		3.3 Selecting Positive Clones
		3.4 Riboprobe Synthesis
	4 Notes
	References
Chapter 7: Administering a Behavioral Test Battery in Rodents
	1 Introduction
	2 Materials
		2.1 General
		2.2 Elevated Plus Maze
		2.3 Open Field
		2.4 Novel Object Recognition Test
		2.5 Y-Maze Spatial Recognition Memory
		2.6 Social Interaction Test
		2.7 Forced Swim Test
		2.8 Fear Conditioning Test
		2.9 Locomotor (Hyper)Activity
	3 Methods
		3.1 General
		3.2 Elevated Plus Maze
		3.3 Open Field
		3.4 Novel Object Recognition Test
		3.5 Y-Maze Short-Term Spatial Recognition Memory
		3.6 Social Interaction Test
		3.7 Forced Swim Test
		3.8 Fear Conditioning  Test
		3.9 Locomotor (Hyper)Activity
	4 Notes
	References
Chapter 8: A Simple Method for Quantifying Larval Locomotion in Drosophila melanogaster
	1 Introduction
	2 Materials
		2.1 Recommended Equipment
		2.2 Solutions and Chemical Components
	3 Methods
		3.1 Preparation of Food Plates
		3.2 Larval Collection and Video Acquisition
		3.3 Video Processing and Data Collection
	4 Notes
	References
Chapter 9: Neural Stem/Progenitor Cell (NSPC) Extraction and Culture
	1 Introduction
	2 Materials
		2.1 NSPC Isolation and Culture
		2.2 Neurosphere Differentiation
		2.3 Retroviral Transduction
	3 Methods
		3.1 Embryonic NSPC Isolation
		3.2 NSPC Culture
		3.3 Neurosphere Dissociation and Passaging
		3.4 Cumulative Cell Number Assay
		3.5 Neurosphere Survival Assay
		3.6 Clonal Density Assay to Measure Neurosphere Formation Independent of ``In Vitro Niche´´
		3.7 Single-Cell Assay to Determine Neurosphere Forming Potential
		3.8 Differentiation Assay to Quantitate Neurogenic Potential
		3.9 Freezing Neurospheres
		3.10 Thawing Neurospheres
		3.11 Retroviral Transduction
	4 Notes
	References
Chapter 10: Testing Prepulse Inhibition of Acoustic Startle in Rodents
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 11: Syngeneic Mouse Model of Glioblastoma: Intracranial Implantation of GL261 Cells
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Surgery Room Equipment
		2.3 Consumables and Surgical Tools
	3 Methods
		3.1 Preparation of GL261 Cells
		3.2 Intracranial Implantation of GL261 Cells
	4 Notes
	References
Chapter 12: Measurement of Murine Neuromuscular Function Using the In Situ Preparation
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Mouse Hindlimb
		3.2 Determination of Optimal Length (Lo) of the Muscle
		3.3 Determination of Maximal Isometric Force (Po) Using a Force-Frequency Relationship
		3.4 Dissection of TA Muscle
		3.5 Post-function Calculations
	4 Notes
	References
Chapter 13: Cryosectioning and Immunohistochemistry Using Frozen Adult Murine Brain Neural Tissue
	1 Introduction
	2 Materials
	3 Methods
		3.1 Cardiac Perfusion
		3.2 Brain Extraction
		3.3 Cryosectioning
		3.4 Immunohistochemistry
	4 Notes
	References
Chapter 14: Phenotypic Analysis of Early Neurogenesis in a Mouse Chimeric Embryo and Stem Cell-Based Neuruloid Model
	1 Introduction
	2 Materials
		2.1 Choice of Embryonic Stem Cells
		2.2 Mice
		2.3 Cell Culture
		2.4 Mice Dissection and Microinjection
		2.5 Immunofluorescence Imaging
	3 Methods
		3.1 Generating Neuruloids
		3.2 Generating Chimeras
		3.3 Analyzing mESC Contribution to Chimeric Embryos
		3.4 Protein Expression by Immunofluorescence and Visualization by Imaging
	4 Notes
	References
Chapter 15: Active Induction of a Multiple Sclerosis-Like Disease in Common Laboratory Mouse Strains
	1 Introduction
	2 Materials
		2.1 Mouse Strains
		2.2 Stock Reagents
	3 Methods
		3.1 Preparation of Reagents for EAE Induction
		3.2 3.2 Final Concentrations of Components/Mouse
		3.3 Preparation of Reagents for Sham Induction
		3.4 EAE Induction
		3.5 Generation of a Chronic-Progressive EAE Variant in the C57BL/6J Mouse with MOG35-55 Peptide (Fig. 7) (See Notes 13-15)
		3.6 Generation of a Chronic-Relapsing EAE Variant in the NOD/ShiLtJ Mouse with MOG35-55 Peptide (Fig. 8) (See Notes 16 and 17)
		3.7 Generation of a Monophasic EAE Variant in the BALB/cJ Mouse with PLP180-199 Peptide (Fig. 9) (See Notes 18 and 19)
		3.8 Monitoring of Disease Progression and Care of Animals
	4 Notes
	References
Chapter 16: Immunohistochemical Analysis of the Drosophila Larval Neuromuscular Junction
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation for Dissection
		3.2 Tissue Dissection
		3.3A Standard Fixation
		3.3B Alternative Fixation (For Membrane Clustered Proteins)
		3.4 Immunostaining
		3.5 Mounting
	4 Notes
	References
Chapter 17: Neural Endophenotype Assessment in Zebrafish Larvae Using Optomotor and ZebraBox Locomotion Assessment
	1 Introduction
	2 Materials
		2.1 Optomotor Response
		2.2 Locomotor Response
	3 Methods
		3.1 Optomotor Response
			3.1.1 Zebrafish Preparation
			3.1.2 Arena Positioning
			3.1.3 Stimulus Presentation and Zebrafish Imaging
			3.1.4 Optomotor Response Image Processing
			Box 1 Example MATLAB Code for Extracting Larval Positions
			3.1.5 Optomotor Index Calculations
			3.1.6 Statistical Analysis of Optomotor Index
		3.2 ZebraBox Locomotor Response
			3.2.1 Experimental Locomotion Assay Setup
			3.2.2 ZebraBox Assay Analysis
	4 Notes
	References
Chapter 18: The Photothrombotic Model of Ischemic Stroke
	1 Introduction
	2 Materials
		2.1 Photosensitive Dye Preparation
		2.2 Surgical Instruments and Setup
	3 Methods
		3.1 Anesthesia and Surgical Preparation
		3.2 Surgery and Stereotaxic Instrument Setup
		3.3 Rose Bengal Injection and Induction of Photothrombosis
		3.4 Wound Closure and Recovery
		3.5 Limitations and Considerations
	4 Notes
	References
Corrections to: Neurobiology: Methods and Protocols
Index