Translational Research Methods in Neurodevelopmental Disorders (Neuromethods, 185)

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Using Lentiviral shRNA Delivery to Knock Down Proteins in Cultured Neurons and In Vivo
	1 Introduction
		1.1 Overview of the Protocol
	2 Materials
		2.1 Cloning of shRNA Oligos into pSUPER-neo-GFP
		2.2 Cloning of H1-shRNA Cassette into pXLG3-PX-GFP-WPRE
		2.3 Production of Lentivirus
		2.4 Concentration of Lentivirus
	3 Methods
		3.1 Design of shRNA Sequence and Cloning into pSUPER-neo-GFP
		3.2 Cloning of the H1-shRNA Cassette into pXLG3-PX-GFP-WPRE
		3.3 Preparation of Lentivirus for Use on Cultured Neurons
			3.3.1 Preparation of Transfection Mixture
			3.3.2 Harvesting Lentivirus
			3.3.3 Adding the Virus to Primary Neurons
		3.4 Preparation of Lentivirus for In Vivo Delivery
			3.4.1 Estimation of Virus Titer
			3.4.2 Extensions of the Protocol: Cloning of Overexpression or ``Rescue´´ Constructs
		3.5 Overexpression Constructs
		3.6 Rescue Constructs
	4 Notes
	References
Chapter 2: Synaptosomal Preparation and Its Application in Preclinical Studies
	1 Introduction
		1.1 Synaptogenesis and Synapses
		1.2 Pre- and Postsynaptic sides of synapses
	2 Materials
	3 Methods
		3.1 Synaptosomal Preparation
			3.1.1 Extraction of Crude Synaptosomes
			3.1.2 Different Methods to Purify Synaptosomes
			3.1.3 Purification of Specific Synaptosomal Populations Using Fluorescence-Activated Synaptosome Sorting (FASS)
			3.1.4 Study of the Presynaptic Release of Neurotransmitters by Measuring Vesicle Release of FM1-43
	4 Applications of Synaptosome Preparation to Preclinical Studies
		4.1 Pre-, Post-, and Extrasynaptic Protein Purification
		4.2 Use of Synaptosomes in Neurological Disorder Studies
	5 Pros and Cons on the Use of Synaptosomes
	6 Notes
	7 Summary
	References
Chapter 3: Assessing the Nanoscale Organization of Excitatory and Inhibitory Synapses Using Recombinant Probes to Visualize En...
	1 Introduction
	2 Materials
		2.1 Materials and Reagents
		2.2 Equipment and Software
	3 Methods
		3.1 Primary Cultures of Mouse Cortical Neurons
			3.1.1 Preparation of Primary Cultures of Cortical Neurons
			3.1.2 Transfection of Primary Cortical Neurons
			3.1.3 Immunocytochemistry for Confocal and STED Microscopy
		3.2 Cortex-Directed In Utero Electroporation
			3.2.1 Preparation
			3.2.2 Surgery (Laparatomy + Injection)
			3.2.3 Stitching and Post-surgery  Care
			3.2.4 Sample Preparation for Confocal and STED Microscopy
		3.3 Imaging Acquisition and Processing
			3.3.1 Confocal
			3.3.2 STED
			3.3.3 Deconvolution
	4 Notes
	References
Chapter 4: Agonist-Induced Functional Analysis and Cell Sorting (aiFACS) to Select and Characterize Interneurons During Brain ...
	1 Introduction
	2 Material
		2.1 Animal Handling and  Care
	3 Methods
		3.1 Brain Preparation
		3.2 Neuron Dissociation and Enrichment
		3.3 Neuron Labeling
		3.4 Pipeline aiFACS Sorting
		3.5 Gating Strategy
		3.6 Validation
	4 Notes
	References
Chapter 5: A CRISPR/Cas9-Based Toolkit to Test Gain- and Loss-of-Gene Function in Brain Organoids
	1 Introduction
		1.1 Modelling Normal Brain Development and Neurodevelopmental Diseases with Stem Cell-Based Models
		1.2 CRISPR/Cas9 to Generate Gain and Loss of Function Embryonic Stem Cell Lines
		1.3 Of Mice and  Men
	2 Materials
		2.1 ESC Culture
		2.2 Plasmids for CRISPR/Cas9
			2.2.1 Plasmids for CRISPRi
			2.2.2 Plasmids for CRISPRa SAM (All from Feng Zhang)
			2.2.3 Plasmids for CRISPR Editing (iSTOP)
		2.3 Brain Organoids
		2.4 Molecular Biology
		2.5 Antibodies
		2.6 Other Materials
	3 Methods
		3.1 Generation of Stable CRISPRi (dCas9-KRAB-MeCP2) ESC Lines by PiggyBac Transposition
		3.2 Generation of Stable CRISPRa (SAM) ESC Lines by Lentiviral Transduction
		3.3 Selection of sgRNAs and Generation of Stable CRISPRa and CRISPRi ESC Lines Targeting the Gene of Interest
		3.4 Generation of CRISPR-Edited iSTOP ESC Lines
		3.5 Generation of Brain Organoids from mESCs
	4 Notes
	References
Chapter 6: Assessment of Adult Mouse Brain Neuroanatomical Phenotypes Using Quantitative and Precision Histology
	1 Introduction
	2 Materials and Instruments
		2.1 Brain Dissection, Fixation, and Paraffin Inclusion
			2.1.1 Mice
			2.1.2 Consumables
			2.1.3 Devices
		2.2 Brain Sectioning
			2.2.1 Consumables
			2.2.2 Devices
		2.3 Luxol-Nissl Staining
			2.3.1 Consumables
			2.3.2 Devices
		2.4 Whole Slide and Image Processing
			2.4.1 Consumables
			2.4.2 Devices
	3 Methods
		3.1 Brain Harvest, Fixation, and Paraffin-Embedding
		3.2 Brain Sectioning
		3.3 Staining with Luxol Fast Blue and Cresyl Violet
		3.4 Whole Slide Scanning and Image Processing
		3.5 Image Analysis
	4 Notes
	References
Chapter 7: Leveraging Microelectrode Array Technology for Phenotyping Stem Cell-Derived Neurodevelopmental Disease Models
	1 Introduction
	2 Materials
		2.1 Culture Preparation
		2.2 MEA Plate Coating and Seeding Neurons
		2.3 MEA Recording and Data Acquisition
		2.4 Data Processing and Analysis (Filtering and Extraction)
	3 Methods
		3.1 MEA as a Model for Physiology
			3.1.1 Healthy Development of Neuronal Network Activity on MEA
			3.1.2 Receptor-Specific Effect on Neuronal Network Activity Patterns on MEA
		3.2 MEA as a Model for Pathology
		3.3 MEA as a Model for Toxicity and Drug Screening
			3.3.1 MEAs as a Model for Seizure Liability
			3.3.2 From Seizure Induction to (Adverse) Drug Screening
			3.3.3 Patient-Specific Drug Screening
	4 Notes and Conclusion
	References
Chapter 8: Humanized Chimeric Mouse Models to Study Human Neural Development and Pathogenesis of Brain Diseases
	1 Introduction
	2 Materials
		2.1 Media
	3 Methods
		3.1 Neuronal Progenitor Cells (NPC) from Human iPSC Lines
		3.2 NPC Labeling and Amplification
		3.3 Grafting into Mouse Neonates
		3.4 In Utero Grafting of Mouse Embryos
			3.4.1 Material and Equipment Settings
			3.4.2 In Utero Surgery Procedures
		3.5 Grafting into the Dentate Gyrus of Adult Mouse Brain
		3.6 Immunofluorescence Labeling
		3.7 Representative Data
	4 Notes
	References
Chapter 9: In Vivo Manipulations to Correct for Behavioral Phenotypes in Cognitive Disorder Mouse Models
	1 Introduction
		1.1 Generating the Working Hypothesis and Choosing In Vivo Functional and Corrective Manipulations
		1.2 Designing the Experimental Protocol
			1.2.1 Choosing the Timing for In Vivo Manipulations
			1.2.2 Designing the Control Groups for In Vivo Manipulations
		1.3 Two Examples for Corrective Strategies in CD Mouse Models
			1.3.1 Correcting an Inhibition/Excitation Imbalance in Amygdala in Il1rapl1 Deficiency
			1.3.2 Depressing PKA Hyperactivity in Prefrontal Cortex in Ophn1 Deficiency
	2 Equipment and Reagents
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 Animals and Ethics Statement
		3.2 Surgery Procedure
		3.3 Electrophysiological Recordings
		3.4 Synaptic Priming of Caudal Hippocampus to BLA Circuits In Vivo
		3.5 Cannula Drug Delivery into Dorsal Hippocampus and Medial PFC
	4 Behavioral Readouts and Analysis
		4.1 Contextual Fear Conditioning
		4.2 Y-Maze Spatial Working Memory (Delayed Spatial Alteration, DSA) Task
	5 Summary
		5.1 Synaptic Priming of cHPC to BLA Circuits In Vivo Restores Contextual Fear Expression Deficit in Il1rapl1 Deficiency
		5.2 Depressing PKA Hyperactivity in PFC In Vivo Restores Working Memory Deficit in Ophn1 Deficiency
	6 Notes
		6.1 Synaptic Priming of cHPC to BLA Circuits
		6.2 Depressing PKA Hyperactivity in PFC In Vivo
	References
Chapter 10: Assessing Dyadic Social Interactions in Rodent Models of Neurodevelopmental Disorders
	1 Introduction
	2 Materials
		2.1 Animals, Equipment, and Room Setup
	3 Methods
		3.1 Experimental Design
		3.2 Video Recording of Social Behavior in Rats and  Mice
		3.3 Procedures to Assess Social Play Behavior and Social Interaction in Rats and  Mice
		3.4 Scoring Social Play Behavior in Juvenile  Rats
		3.5 Scoring Social Interactions in Adult  Rats
		3.6 Scoring Social Play/Interactions in  Mice
		3.7 Data Analysis
	4 Automated Scoring of Social Behavior in Rodents: Benefits and Limitations
	5 Notes
	References
Chapter 11: Central and Peripheral Explorations by Metabolomics to Study Neurodevelopmental Disorders
	1 Introduction
	2 Materials
		2.1 Sample Collection, Treatment, and Storage
		2.2 Sample Metabolic Extraction and Analysis
		2.3 Data Processing
		2.4 Statistical Analyses
	3 Methods
		3.1 Sample Collection, Treatment, and Storage
			3.1.1 Maternal Matrices Collection
				3.1.1.0 Plasma
				3.1.1.0 Feces
				3.1.1.0 Urine
			3.1.2 Gestational and Fetal Matrices Collection
				3.1.2.0 Amniotic Fluid
				3.1.2.0 Placenta
				3.1.2.0 Fetal Brain
			3.1.3 Postnatal Offspring Matrices
				3.1.3.0 Feces and Urine
				3.1.3.0 Plasma
				3.1.3.0 Brain
		3.2 Sample Metabolic Extraction
			3.2.1 Plasma
			3.2.2 Feces
			3.2.3 Urine
			3.2.4 Amniotic Fluid
			3.2.5 Placenta
			3.2.6 Fetal Brain
			3.2.7 Brain
		3.3 Sample Analysis: LC-MS Parameters
		3.4 Data Processing
		3.5 Statistical Analyses
			3.5.1 Basic Information Related to the Number of Detected Metabolites
			3.5.2 Multivariate Analyses Using Principal Component Analysis
			3.5.3 Multivariate and Univariate Analyses to Better Characterize the Metabolic Differences Between Two Groups
			3.5.4 Pathway Analyses
			3.5.5 Correlation Analysis
	4 Notes, Major Source of Problems
	References
Chapter 12: Genetic Engineering of Nonhuman Primate Models for Studying Neurodevelopmental Disorders
	1 Introduction
		1.1 Nonhuman Primates as a Unique Model System in Biomedical Research
		1.2 Modeling Neurodevelopmental Disorders with NHP Models
		1.3 SHANK3 Mutation in NDDs and Engineering of Mutant Monkeys
		1.4 Genetic Engineering of NHPs and Recent Advance Through CRISPR-Cas9
	2 Materials
		2.1 Gene Editing: Design Guide RNA for Gene of Interest (GOI)
		2.2 Gene Editing: Examine Efficiency of Guide RNAs in Cultured Monkey Cells
		2.3 Gene Editing: CRISPR-Cas9-Mediated Editing of Monkey Embryos
		2.4 Gene Editing: Genotyping Newborn Monkeys from Biopsies
	3 Methods
		3.1 Behavioral Analysis: Home Cage Behavioral Scoring
		3.2 Behavioral Analysis: Activity Monitoring and Sleep Analysis
		3.3 Behavioral Analysis: Social Interaction and Exploration
		3.4 Behavioral Analysis: Eye-Tracking
		3.5 Behavioral Analysis: Wisconsin General Test Apparatus
	4 Notes
	References
Index