Cerebral Cortex Development: Methods and Protocols (Methods in Molecular Biology, 2794)

Preface
Contents
Contributors
Chapter 1: Overview: Research on the Genetic Architecture of the Developing Cerebral Cortex in Norms and Diseases
	1 Introduction
	2 From a Single Gene to Integrated Functional Genomic Analysis
	3 From Nonhuman Models to Human Brain Organoids
	4 Big Datasets and Computer-Driven Neuroscience
	5 Unraveling the Underlying Mechanisms of Neuropsychiatric Disorders Will Lead Us to Precision Medicine and a Biological Under...
	References
Chapter 2: In Situ Hybridization of Brain Slices
	1 Introduction
	2 Materials
		2.1 Probes, Kit, and Equipment
		2.2 Solutions
	3 Methods
		3.1 Preparation of Brain Slices and FFPE Blocks
		3.2 Prepare Slides
		3.3 Pretreat the Samples
		3.4 Prepare the Materials
		3.5 Run the Assay
		3.6 Evaluate the Results
	4 Notes
	References
Chapter 3: Immunohistochemistry of Brain Tissues
	1 Introduction
	2 Materials
		2.1 Solutions
		2.2 Heat-Induced Antigen Retrieval (HIAR)
		2.3 Antibodies
	3 Methods
		3.1 Perfusion Fixation and Process for Paraffin Embedding
		3.2 Slide Preparation, Deparaffinization, and Rehydration
		3.3 HIAR (See Note 17)
		3.4 Inactivation of Endogenous Peroxidase
		3.5 Blocking Nonspecific Binding Sites
		3.6 Primary and Secondary Antibody Application
		3.7 Chromogen Application
		3.8 Counterstaining, Dehydration, and Mounting
		3.9 Automated Immunostainer as another Option for IHC (See Note 28)
	4 Notes
	References
Chapter 4: Two-Photon FRET/FLIM Imaging of Cerebral Neurons
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Cell Culture and Transfection
		2.3 Cell Lysate
		2.4 Fluorospectrometric Assay
		2.5 Organotypic Slice Culture and Transfection
		2.6 Two-Photon FRET Imaging
		2.7 Two-Photon FLIM Imaging by Photoactivation
	3 Methods
		3.1 HEK293T Cell Culture and Transfection
		3.2 HEK293T Cell Lysate
		3.3 In Vitro Fluorospectrometric Assays for  FRET
		3.4 Organotypic Slice Culture and Transfection
		3.5 Two-Photon FRET Neuron Imaging
		3.6 In Vitro Two-Photon FLIM Imaging
		3.7 Two-Photon FLIM Neuron Imaging
	4 Notes
	References
Chapter 5: Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System
	1 Introduction
	2 Materials
		2.1 Fixation
		2.2 Instruments for Perfusion Fixation (In Case of Mouse)
		2.3 Immunoelectron Microscopy
		2.4 Instruments for Preembedding Immunoreaction
		2.5 Semithin and Ultrathin Sectioning
	3 Methods
		3.1 Fixation (In Case of Perfusion Fixation of Mice)
		3.2 Preembedding Method
			3.2.1 Sectioning with Vibrating Microtome
			3.2.2 Immunohistochemical Procedures (In Case of a Peroxidase Reaction)
			3.2.3 Postfixation in Osmium Tetroxide
			3.2.4 Dehydration and Embedding in Resin
			3.2.5 LM Observation and Excising of Region of Interest for  EM
			3.2.6 Trimming the Sections for Ultramicrotome
			3.2.7 Semithin Sectioning
			3.2.8 Ultrathin Sectioning
			3.2.9 Staining of Ultrathin Sections
		3.3 Postembedding Method (In Case of Freeze-Substituted with Lowicryl)
			3.3.1 Sectioning with Vibrating Microtome
			3.3.2 Cryoprotection
			3.3.3 Trimming of 500 μm Cubic Sections of Tissue
			3.3.4 Plunge-Freezing in Liquid Nitrogen (Metal Contact Method, MM80)
			3.3.5 Freeze-Substitution (Automatic Freeze Substitution System)
			3.3.6 Ultrathin Sectioning
			3.3.7 Immunogold Labeling (See Note 19)
			3.3.8 Negative Staining
		3.4 Observation
	4 Notes
	References
Chapter 6: Mouse Brain Tissue Preparation for Scanning Electron Microscopy
	1 Introduction
	2 Materials
		2.1 Solutions
	3 Methods
		3.1 Perfusion Fixation
		3.2 Dehydration
		3.3 Freezing
		3.4 Cracking
		3.5 Freeze-Drying
		3.6 Fixation
	4 Notes
	References
Chapter 7: Simple Method for the Preparation of Postsynaptic Density Fraction from Mouse Brain
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 8: Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules
	1 Introduction
	2 Materials
		2.1 Kinesin Expression and Lysate Collection Materials
		2.2 Taxol-Stabilized Microtubule Preparation and Storage Materials
		2.3 Septin Expression and Purification Materials
		2.4 Kinesin Motility Assay and Septin Coating Materials
	3 Methods
		3.1 Kinesin Expression and Lysate Collection
		3.2 Microtubule Polymerization
		3.3 Septin Expression and Purification
		3.4 Motility Assay Conditions and Septin Coating
	4 Notes
	References
Chapter 9: BioID Analysis of Actin-Binding Proteins
	1 Introduction
	2 Materials
		2.1 Generation of LifeAct-BioID
		2.2 Cell Growth and Treatment
		2.3 Pulldown Using Streptavidin Magnetic Beads
		2.4 Western Blotting with Fluorescent Streptavidin and Actin
	3 Methods
		3.1 LifeAct-TurboID Construct
		3.2 Transient Transfection into HEK293T Cells and Biotin Labeling After Disruption of Actin Polymerization
		3.3 Isolation of Biotinylated Proteins Using Magnetic Streptavidin Beads
		3.4 Western Blotting with Fluorescent Streptavidin and Actin Antibody
	4 Notes
	References
Chapter 10: Proteomic Analyses of the Mouse Brain
	1 Introduction
	2 Materials
		2.1 Protein Extraction from the Mouse Brain
		2.2 Protein Fractionation by SDS-PAGE
		2.3 Gel Excision and Protein Digestion
		2.4 Peptide Extraction
		2.5 LC-MS/MS Analysis
	3 Methods
		3.1 Protein Extraction from the Mouse Brain
		3.2 Protein Fractionation by SDS-PAGE (See Note 11)
		3.3 Gel Excision and Protein Digestion
		3.4 Peptide Extraction
		3.5 LC-MS/MS Analysis and Data Processing
	4 Notes
	References
Chapter 11: Generation of iPSCs Using Sendai Virus Vectors
	1 Introduction
	2 Materials
		2.1 Solutions and Reagents for Inducing iPSCs from Human DP Cells Using SeV Vectors
		2.2 SeV Vector Removal and Detection Reagents
	3 Methods
		3.1 Preparation of Dental Pulp Cells
			3.1.1 Thawing Cryopreserved Dental Pulp Cells
			3.1.2 Passaging Dental Pulp Cells
		3.2 Preparation of SNL Feeder Cells
			3.2.1 Thawing Cryopreserved SNL Feeder Cells
			3.2.2 Passaging SNL Cells
			3.2.3 Mitomycin C Treatment of SNL Feeder Cells
			3.2.4 Using Cryopreserved Mitomycin C-Treated SNL Feeder Cells
		3.3 Induction of iPSCs from Dental Pulp Cells
			3.3.1 Preparation of Dental Pulp Cells
				Day 0
			3.3.2 Sendai Virus Infection
				Day 1
				Day 2
				After Day 3
		3.4 Picking and Passaging Primary iPS Cell-Like Colonies
		3.5 Confirmation of SeV Vector Infection and Elimination (See Note 28)
		3.6 Passage of iPSCs
		3.7 Expansion of iPSCs
		3.8 Preservation of iPSCs
		3.9 Thawing Frozen iPSCs
	4 Notes
	References
Chapter 12: Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders
	1 Introduction
	2 Materials
	3 Methods
		3.1 iPSC Culture and Passaging
			3.1.1 Preparation of Matrigel-Coated Six-Well Plates
			3.1.2 Preparation of mTeSR1 Complete Medium
			3.1.3 Thawing hiPSCs
			3.1.4 Passaging hiPSCs
		3.2 Neural Progenitor Cell (NPC) Differentiation, Passaging, and Freezing
			3.2.1 NPC Differentiation
			3.2.2 NPC Passaging
			3.2.3 NPC Freezing
		3.3 Preparation of UNA Lentivirus for Neuron Differentiation
			3.3.1 HEK293T Cell Seeding for Transfection
			3.3.2 HEK293T Cell Transfection
			3.3.3 Lentivirus Collection and Purification
			3.3.4 Virus Titration
		3.4 Lentiviral Transduction of  NPCs
		3.5 Neuronal Differentiation and Maturation
			3.5.1 Neuronal Differentiation
			3.5.2 Neuronal Splitting and Maturation
	4 Notes
	References
Chapter 13: Generating Homogeneous Brain Organoids from Human iPSCs
	1 Introduction
	2 Materials
	3 Methods
		3.1 iPSC Culture and Passaging
		3.2 iPSC Freezing
		3.3 iPSC Thawing
		3.4 Generation of Brain Organoids
			3.4.1 EB Formation
			3.4.2 Neural Induction
			3.4.3 Matrigel Embedding and Removal
			3.4.4 Screening and Maintaining
			3.4.5 Immunostaining of Brain Organoids
	4 Notes
	References
Chapter 14: Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex
	1 Introduction
	2 Materials
		2.1 Brain Dissection
		2.2 Coating Dishes
		2.3 Gene Transfection and Culture
		2.4 Obtaining the Cell Lysate
	3 Methods
		3.1 Coating Dishes and Preparation for Culture
		3.2 Neuron Isolation
		3.3 DNA Transfection
		3.4 Culture and Preparation of Cell Lysates
	4 Notes
	References
Chapter 15: Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons
	1 Introduction
	2 Materials
		2.1 Coating Coverslips
		2.2 Fixation and Permeabilization
		2.3 Blocking and Antibody Reaction
		2.4 Mounting and Observation
	3 Methods
		3.1 Coating Coverslips and Culturing Neurons
		3.2 Fix the Neurons
		3.3 Permeabilization
		3.4 Blocking and Antibody Reaction
		3.5 Fixing Coverslips on the Slide Glass and Analyzing
	4 Notes
	References
Chapter 16: Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in th...
	1 Introduction
	2 Materials
		2.1 Plasmid Preparation
		2.2 Microcapillary Preparation
		2.3 Surgery Preparation
		2.4 Animal Preparation
		2.5 Dissection for Fixation of Brains
		2.6 Brain Sample Preparation for Cryostat
		2.7 Cryostat for Generating Brain Slices
		2.8 Immunohistochemistry (IHC)
	3 Methods
		3.1 Preparation
		3.2 Anesthesia
		3.3 Surgery
		3.4 Postsurgery
		3.5 Dissection for Fixation of Brains
		3.6 Brain Sample Preparation for Cryostat
		3.7 Cryostat for Generating Brain Slices
		3.8 IHC
	4 Notes
	References
Chapter 17: Live Imaging of Migrating Neurons and Glial Progenitors Visualized by in Utero Electroporation
	1 Introduction
	2 Materials
		2.1 In Utero Electroporation
		2.2 Slice Culture
	3 Methods
		3.1 In Utero Electroporation
		3.2 Slice Preparation
		3.3 Time-Lapse Observation
	4 Notes
	References
Chapter 18: Ca2+ Imaging in Immature Cortical Neurons
	1 Introduction
	2 Materials
		2.1 Brain Slice Cutting Solution
		2.2 Slice Culture Medium
		2.3 Plasmids and Anesthetics Used for IUE
	3 Methods
		3.1 IUE and Slice Preparation
		3.2 Ca2+ Imaging
		3.3 Quantitative Analysis of Ca2+ Transients
	4 Notes
	References
Chapter 19: In Vitro Patch-Clamp
	1 Introduction
	2 Materials
		2.1 Extracellular Solutions
		2.2 Slicing
		2.3 Electrodes and Intracellular Solutions
		2.4 Electrophysiology Rig SetUp
	3 Methods
		3.1 Acute Brain Slice Preparation Using a Tissue Slicer
		3.2 Manufacturing Electrodes
		3.3 Patching Cells
		3.4 Recording Action Potentials
		3.5 Recording Ion Channel Activity
		3.6 Recording Synaptic Activity
	4 Notes
	References
Chapter 20: In Vivo Whole-Cell Recording from the Mouse Brain
	1 Introduction
	2 Materials
		2.1 Animals
		2.2 Head-Post Implantation
		2.3 Habituation for Head-Fixation
		2.4 Craniotomy and Durotomy
		2.5 Recording
	3 Methods
		3.1 Surgery for Head-Post Implantation
		3.2 Habituation
		3.3 Craniotomy
		3.4 Recording
	4 Notes
	References
Chapter 21: In Vivo Brain Microdialysis
	1 Introduction
	2 Materials
		2.1 Stereotaxic Surgery
		2.2 In Vivo Microdialysis
	3 Methods
		3.1 Stereotaxic Surgery for Guide Cannula Implantation
		3.2 In Vivo Microdialysis in Freely Moving Mice
			3.2.1 Construction of the Microdialysis Circuit
			3.2.2 Preparation of the Microdialysis Probe and Connection to the Circuit
			3.2.3 Insertion of the Probe into the Mouse Brain and Initiation of Sampling
	4 Notes
	References
Chapter 22: CT Scan Data Analysis in Malformations of Cortical Development
	1 Introduction
	2 Materials
		2.1 CT Scan Machine
	3 Methods
		3.1 Scanning of the Patient
		3.2 CT Scan Images
		3.3 CT Data Analysis
	4 Notes
		4.1 Concluding Remarks
	References
Chapter 23: MRI Data Analysis in Malformations of Cortical Development
	1 Introduction
	2 Materials
		2.1 Magnetic Resonance Scanner
	3 Methods
		3.1 Imaging Acquisition
		3.2 Brain MRI Protocol for Studying MCD
		3.3 Data Analysis and Clinical Implications
	4 Notes
	5 Conclusions
	References
Chapter 24: Genomic Copy Number Analysis Using Droplet Digital PCR: A Simple Method with EvaGreen Single-Color Fluorescent Des...
	1 Introduction
	2 Materials
	3 Methods
		3.1 PCR Primer Design
		3.2 Sample Preparation (Fig. 1, Table 3)
		3.3 Droplet Generation (Fig. 2, See Note 11)
		3.4 PCR (See Note 13)
		3.5 Setting Up QuantaSoft Software
		3.6 Droplet Reading (See Note 20)
		3.7 Data Analysis (See Note 22)
	4 Notes
	References
Chapter 25: Locomotor Assays in Drosophila Larvae and Adult Flies
	1 Introduction
		1.1 Crawling Assay Using Larvae
		1.2 Climbing Assay Using Adult Flies
	2 Materials
		2.1 Crawling Assay Using Larvae
		2.2 Climbing Assay Using Adult Flies
	3 Methods
		3.1 Crawling Assay Using Larvae
		3.2 Climbing Assay Using Adult Flies
	4 Notes
	References
Chapter 26: Analyses of Genetic Regulation of the Nervous System in the Nematode Caenorhabditis elegans
	1 Introduction
	2 Materials
	3 Methods
		3.1 Insertion of LoxP Sequences for Tissue-Specific Knockout by the Cre-LoxP Recombination
		3.2 Insertion of Degron Sequences for Temporal Protein Degradation by Auxin-Inducible Degron Protein Degradation System
		3.3 Micro-injection and Screening
	4 Notes
	References
Chapter 27: Analyses of Neural Circuits Governing Behavioral Plasticity in the Nematode Caenorhabditis elegans
	1 Introduction
	2 Materials
		2.1 Single-Worm Tracking Assay
		2.2 Multi-Worm Tracker Assay
		2.3 Recording and Analysis in Multi-Worm Tracker
	3 Methods
		3.1 Calcium Imaging and Single-Worm Tracking
		3.2 Behavioral Analysis Using the Multi-Worm Tracker (MWT)
		3.3 Generating a Temperature Gradient
	4 Notes
	References
Chapter 28: Social Behavior in Animal Models of Autism Spectrum Disorder
	1 Introduction
	2 Social Interaction Test
		2.1 Materials
			2.1.1 Animals
			2.1.2 Equipment (Fig. 1)
		2.2 Methods (Fig. 2)
			2.2.1 Preparation
			2.2.2 Habituation
			2.2.3 Test
			2.2.4 Analysis
	3 Three-Chamber Test
		3.1 Materials
			3.1.1 Animals
			3.1.2 Equipment (Fig. 4)
		3.2 Methods
			3.2.1 Preparation
			3.2.2 Habituation
			3.2.3 Test (Fig. 6)
				Phase 1 (Chamber 1: Empty; Chamber 3: Empty)
				Phase 2 (Chamber 1: Social Stimulus Mouse (1); Chamber 3: Empty)
				Phase 3 (Chamber 1: Social Stimulus Mouse (1); Chamber 3: Social Stimulus Mouse (2))
			3.2.4 Analysis (Fig. 7)
	4 Notes
	References
Chapter 29: Preparation of Healthy Single Neuron or Astrocyte Suspension from Adult Mouse Brain for RNA-seq
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Reagent Setup
	3 Methods
	4 Notes
	References
Index