Preface to the Series
Preface
Contents
Contributors
Part I: New Approaches to Study Glutamatergic Synapses Between Neurons
Chapter 1: Visualization of Glutamatergic Neurotransmission in Diverse Model Organisms with Genetically Encoded Indicators
1 Introduction
2 Discovery of Glutamate as a Neurotransmitter
3 Key Aspects of Glutamatergic Signaling
4 Glutamate Detection Methods
5 What Are the Properties of an Ideal Glutamate Sensor?
6 iGluSnFR3
7 Protocols for iGluSnFR Imaging in Several Preparations and Model Organisms
7.1 Bacteria (Escherichia coli)
7.2 Tissue Culture Cells
7.3 Brain Slices
7.4 Worm (Caenorhabditis elegans)
7.5 Fly (Drosophila melanogaster)
7.6 Zebrafish (Danio rerio)
7.7 Rodent (Mouse, Mus musculus)
8 In Vivo Functional Imaging of iGluSnFR3 Using Two-Photon Laser Scanning Microscopy
9 Future Optimization of iGluSnFR
9.1 Temporal Resolution
9.2 Saturation of Activation Kinetics
9.3 Sensitivity and Dynamic Range
9.4 Photostability
9.5 Nanoscopic Localization
9.6 Fluorescent Protein Scaffold
9.7 Cooperative Behavior
10 Conclusion and Outlook
References
Chapter 2: Surface Glutamate Receptor Nanoscale Organization with Super-Resolution Microscopy (dSTORM)
1 Introduction
2 Materials
2.1 Primary Rat Neuronal Cultures
2.2 Immunocytochemistry
2.3 Data Acquisition
2.4 Data Analysis
3 Methods
3.1 Primary Rat Neuronal Cultures
3.2 Immunocytochemistry
3.3 dSTORM
3.4 High-Resolution Image Generation
3.5 Segmentation of Super-Resolved Glutamate Receptor Clusters
4 Conclusions
References
Chapter 3: Ligand-Directed Chemical Labeling for Visualizing and Analyzing AMPA Receptors in Neurons
1 Introduction
2 Preparation of Cell Cultures
2.1 Preparation of Coverslips, Dishes, and Plates for Neuronal Cultures
2.2 Primary Culture of Cortical Neurons
2.3 Cultured Hippocampal Neurons
2.4 Potential Problems and Troubleshooting of Neuronal Culture
3 Chemicals
3.1 Chemical Reagents for Direct-Probe (One-Step) Labeling
3.2 Chemical Reagents for Two-Step Labeling
3.3 Potential Problems and Troubleshooting of Chemicals
4 Protocols for Chemical Labeling of AMPARs by Direct Fluorophore Labeling
4.1 Biochemical Analysis
4.2 Fluorescent Visualization
4.3 Potential Problems and Troubleshooting of Direct Fluorophore Labeling Using CAM2 Reagents
5 Protocols for Chemical Labeling of AMPARs by Two-Step Labeling for Biochemical Analyses
5.1 Biochemical Analysis
5.1.1 Labeling of Surface AMPARs
5.1.2 Labeling of Intracellular (or Internalized) AMPARs
5.1.3 Labeling of Both Surface and Intracellular AMPARs
5.1.4 Labeling of Recycled AMPARs
5.2 Fluorescent Visualization
5.3 Potential Problems and Troubleshooting of Two-Step Labeling Using CAM2 Reagents
6 Comparison of One-Step and Two-Step Labeling and Their Applicability for AMPAR Studies
7 Conclusion
References
Chapter 4: Live FRET-FLIM Imaging to Study Metabotropic Signaling via the NMDA Receptor
1 Introduction
1.1 Fluorescence Lifetime Imaging (FLIM) to Measure FΓΆrster Resonance Energy Transfer (FRET)
1.2 FRET-FLIM to Monitor Conformational Movement in the NMDARcd
1.3 FRET-FLIM to Study Interactions Between the NMDAR and Signaling Proteins
2 Materials
2.1 Primary Hippocampal Neurons
2.2 Two-Photon Fluorescence Lifetime Microscope
2.3 Electrophysiology Equipment to Infuse Antibodies into Neurons
3 Methods
3.1 Preparation of Primary Hippocampal Neurons
3.2 Expression of Fluorescently Tagged NMDAR Subunits and/or Other NMDAR Interacting Proteins
3.3 Live Fluorescence Lifetime Imaging
3.4 Infusion of an Antibody Binding to GluN1 C-terminal Domain via a Patch Pipette to Block NMDAR Metabotropic Function
3.5 Data Analysis
4 Conclusions
4.1 Limitations
4.2 Other Applications of FRET-FLIM in Neuroscience and Future Perspectives
5 Notes
References
Chapter 5: Quantitative Analysis of Single Glutamatergic Vesicles in the Brain
1 Introduction: Monitoring Exocytosis Release and Quantifying Glutamate in Synaptic Vesicles
1.1 High-Speed Amperometry Records Single Vesicle Exocytosis
1.2 Ultrafast Amperometric Biosensor for Detection of Glutamate Exocytosis
1.3 Ultrafast Amperometric Detection of Glutamate Exocytosis in Brain Slices and Cells
2 Materials
2.1 Chemicals and Materials
2.2 Fabrication of Enzyme-Coated AuNP-Modified Carbon Fiber Microelectrode Glutamate Sensor
2.2.1 CFE Fabrication
2.2.2 AuNP Deposition onto the CFE Surface
2.2.3 Determining the Surface Area of AuNPs at the CFE Surface
2.2.4 Enzyme Coating of AuNP-Functionalized CFEs
2.3 Sensor Testing Before Amperometry Measurement in Brain Slice and of Glutamate-Filled Liposomes
2.4 Preparing Glutamate-Filled Liposomes for Glutamate Biosensor Calibration
2.4.1 Synthesis of Glutamate-Filled Liposomes
2.4.2 Freeze-Thawing of Liposomes
2.4.3 Liposome Extrusion
2.4.4 Separation of Liposomes and Glutamate Bulk Solution Using Spin Microcolumns
2.4.5 Measuring the Glutamate Liposome Size
3 Methods
3.1 Using a Glutamate Biosensor to Record Individual Glutamate Exocytosis Events in Brain Slice
3.2 Amperometric Data Analysis to Characterize Exocytosis Activity and Fusion Pore-Regulated Glutamate Release
3.3 Quantification of Quantal Glutamate Release and Synaptic Vesicle Quantal Size
3.3.1 Amperometric Glutamate Sensor Recording of Glutamate Release from Single Glutamate-Filled Liposomes
3.3.2 Using the Calibration Curve to Quantify Glutamate Release during Exocytosis
4 Conclusions
5 Notes
References
Chapter 6: Imaging Microtubule Network in Rodent Giant Glutamatergic Presynaptic Terminals
1 Introduction
2 Materials
2.1 Solutions
2.2 Chemicals
2.3 Equipment
3 Methods
3.1 Preparation of Biological Samples
3.1.1 Brainstem Slice
3.1.2 Primary Dissociated Culture
3.1.3 SiR-Tubulin Labeling of Calyceal Synapses for Live Confocal Imaging and Quantification of Microtubule Depolymerization
3.1.4 Immunolabeling of Microtubules in Fixed Samples for Confocal Imaging and Quantification of Microtubule Network in Calyce...
3.1.5 Immunolabeling of Microtubules in Fixed Samples for STED Imaging of Synaptic Vesicles and Microtubules in Calyceal Termi...
4 Notes
References
Chapter 7: Photooxidation of Genetically Encoded MiniSOG-Fused VGLUT2 for Identification of Glutamatergic Synapses by Transmis...
1 Introduction
2 Materials
2.1 Reagents (see Note 1)
2.2 Equipment
2.2.1 For Transfection of Primary Neuron Cultures
2.2.2 For In Vivo Experiment: Stereotaxic Surgery, Perfusion Fixation, and Vibratome Sectioning
2.2.3 Photooxidation
2.2.4 TEM
2.2.5 Serial Block Face Scanning EM
3 Methods
3.1 Transfection of Primary Neuron Cultures and Fixation
3.2 Stereotaxic Surgery and Perfusion Fixation of Mice
3.3 Photooxidation of Fixed Primary Neurons
3.4 Photooxidation of Fixed Brain Tissue
3.5 Transmission Electron Microscopy (TEM)
3.6 Serial Block-Face Scanning Electron Microscopy (SBEM)
4 Notes
5 Conclusions
References
Part II: New Approaches to Study Role of Astrocytes at Neuronal Synapses
Chapter 8: Chemogenetic Approaches to Study Astrocytes at Glutamatergic Synapses
1 Introduction
2 Methods
2.1 Viral Construct
2.2 Viral Delivery
2.3 Designer Drug
2.4 Visualization of DREADDs
2.5 Long-Term Synaptic Potentiation
2.6 Other Approaches to Study Chemogenetic Modulation of Astrocytes
3 Conclusion
References
Chapter 9: Studying the Role of Astrocytes at Synapses Using Single-Cell Transcriptomics
1 Introduction: The Role of Astrocytes at the Synapse
1.1 Insights into Astrocyte Function from Gene Expression Profiling
2 Materials
2.1 Tissue Dissociation and Astrocyte Isolation
2.2 Library Preparation and Sequencing
2.3 Computational Analysis
2.4 Cell-Type Identification Methods and Expression Quantification
3 Methods
3.1 Tissue Dissociation and Astrocyte Isolation
3.1.1 Tissue Extraction and Dissociation into a Single-Cell Solution
3.1.2 Antibody-Labeling and Isolation of Astrocytes Using FACS
3.2 Library Preparation and Sequencing
3.2.1 Library Preparation: Reverse Transcription and Template Switching
3.2.2 Library Preparation: Tagmentation and Indexing
3.2.3 Sequencing
3.3 Computational Analysis
3.3.1 Read Mapping/Alignment, Quality Control, and Gene/Transcript Quantification
3.4 Cell-Type Identification Methods
3.4.1 Quality Control
3.4.2 Data Normalization
3.4.3 Dimensionality Reduction
3.4.4 Clustering
3.5 Expression Quantification Methods
3.5.1 Differential Gene Expression Analysis
3.5.2 Functional Analysis
4 Conclusion
5 Notes
References
Chapter 10: Analysis of Synaptic Glutamate Clearance as a Possible Indicator of Synaptic Health in the Degenerating Rodent Bra...
1 Introduction
2 Methods and Experimental Paradigms
2.1 Analysis of Individual Synapses: Tests That Can be Performed in Acute Brain Slices from Adult Mice to Identify Specific Ty...
2.1.1 The Necessity to Investigate the Synapses In Situ and at an Appropriate Age
2.1.2 Selection of the Appropriate Sensor for the Given Type of Synapse
2.1.3 Requirements for Sequential Testing of Different Synapses Within One Slice
2.1.4 Extraction of Suitable Indicators for Release and Clearance
2.1.5 Characterization of PT-Type as Opposed to IT-Type Corticostriatal Synapses
2.1.6 Any Evidence for Saturation of Astrocytic Glutamate Uptake in Normal Mice?
2.2 Single-Synapse Analysis in Transgenic Mice
2.2.1 mHTT-Related Alterations in the State of Individual Astrocytes, as Revealed by Sodium Imaging of Glutamate Uptake with S...
2.2.2 Experiments to Characterize the HD Phenotype of Glutamate Uptake
2.2.3 Imaging of Glutamate Clearance in Synapses Contacting Astrocytes with Genetically Altered Glutamate Uptake
2.2.4 Can Single-Synapse Data be Used to Estimate the Disease-Modifying Effect of Altered Gene Expression in Astrocytes?
3 Conclusions
References
Chapter 11: Computational Models of Astrocyte Function at Glutamatergic Synapses
1 Introduction
2 Signal Transmission at Tripartite Synapses
2.1 Model of Glutamate-Induced IP3 and Ca2+ Oscillations in Astrocytes
2.2 Model of Ca2+ Activity in an Astrocyte Connected to a Neuronal Network
2.3 Model of Signal Transmission at a Glutamatergic Tripartite Synapse
2.4 Discussion
3 Glutamate Uptake
3.1 Model of Glutamate Uptake in Relation to Glutamate Transporter Density
3.2 Model of Glutamate Uptake by Astrocytes and Its Effects on Postsynaptic Neuronal Excitability
3.3 A Spatial Model of Glutamate Uptake and Ca2+ and Na+ Signaling in PAP Microdomains
3.4 Discussion
4 Ion Homeostasis
4.1 Model of Astrocyte Ion Fluxes-Mediated ECS Shrinkage
4.2 Model of Potassium and Sodium Microdomains in Astrocytes
4.3 Model of Ca2+ Dynamics Mediated by Two Different Spatially Segregated Pathways
4.4 Discussion
5 Metabolism
5.1 Top-Down Model of the Compartmentalization of Metabolic Pathways at Tripartite Synapses
5.2 Model of Lactate and Glucose Levels in Neurons and Astrocytes During Visual Stimulation
5.3 Discussion
6 Structure-Function Coupling
6.1 ASTRO: A Tool to Simulate Astrocyte Activity in Realistic Astrocyte Ultrastructures at the Whole-Cell Level
6.2 A Multicompartmental Model of Ca2+ Activity in an Astrocyte
6.3 A Spatial Model of Ca2+ Activity in a Perisynaptic Astrocyte Process
6.4 Discussion
7 Astrocyte Networks
7.1 A Topologically Realistic Model of Astrocyte Networks
7.2 A Topologically Realistic Model of Neuron-Astrocyte Networks
7.3 A Network Model of GABA-Evoked Neuron-Astrocyte Communication
7.4 Discussion
8 Concluding Remarks
9 List of Resources
References
Part III: Technical Approaches to Study Fast Signaling at Neuron-Glia Synapses
Chapter 12: Electrophysiological Recordings of Oligodendroglia in Adult Mouse Brain Slices
1 Introduction
2 Materials
2.1 Animals
2.2 Buffer and Intracellular Solutions
2.2.1 N-Methyl-D-Glucamine (NMDG)-Based Solution
2.2.2 Artificial Cerebrospinal Fluid (aCSF) Solution
2.2.3 Intracellular Solutions
2.3 Material for Brain Slice Storage and Recordings
2.4 Whole-Cell Patch-Clamp Recordings
2.5 Photostimulation During Whole-Cell Recordings
3 Methods
3.1 Brain Slices
3.2 Whole-Cell Recording
3.3 OL Lineage Identification Hallmarks
3.3.1 Morphology
3.3.2 Inward Sodium Currents
3.4 Photostimulation of Neurons During OPC Whole-Cell Recordings
4 Notes
References
Chapter 13: Synaptic Integration at Neuron-OPC Synapses
1 Introduction
2 Electrophysiological and Pharmacological Approaches to Study Somatic Integration
2.1 Generating EPSC Waveform Template
2.2 Investigating the Recruitment of Voltage-Gated Ion Channels in Mock EPSP
2.3 Analyzing Mock EPSP Data across Different OPCs
2.4 Consideration of the Driving Force Alteration
3 Utilizing Two-Photon Glutamate Uncaging to Investigate Local Integration of Synaptic Inputs
3.1 Caged-Glutamate Compound and the Application to Acute Brain Slices
3.2 Laser Alignment and Calibration
3.3 Two-Photon Glutamate Uncaging in OPCs
3.4 Investigating the Summation of Local Synaptic Inputs Using Two-Photon Glutamate Uncaging
4 Combining Two-Photon Glutamate Uncaging with Two-Photon Ca2+ Imaging
4.1 Selection of Scanning Mode for Ca2+ Imaging
4.2 Performing Two-Photon Ca2+ Imaging and Glutamate Uncaging Simultaneously with Electrophysiological Recording
5 Computational Simulation of Synaptic Integration in OPCs
References
Chapter 14: Mapping Synaptic Inputs to Oligodendroglial Cells Using In Vivo Monosynaptic Viral Tracing
1 Introduction
2 Materials
2.1 Animal Preparations
2.2 Virus Stocks
2.3 Consumables for Surgery
2.4 Equipment for Surgery
3 Methods
3.1 Animal Preparations
3.2 Injection of Rabies Virus into the Brain
3.3 Tissue Analysis
3.4 Mapping the Synaptic Network
4 Notes
5 Troubleshooting
6 Conclusions
References
Chapter 15: In Vivo Viral Gene Delivery to Manipulate Functional Properties of AMPA Receptors in Oligodendrocyte Lineage Cells
1 Introduction
2 Generation of Retrovirus Carrying Modified GluA2 Subunit of AMPARs
2.1 Amplification of Plasmids for Retroviral System
2.2 Subcloning the Gene of Interest
2.3 Retroviral Production with Lipofectamine 2000
2.4 Estimation of the Viral Titer
3 Injection of Retrovirus into the Mouse Corpus Callosum
3.1 Required Equipment, Materials, and Experimental Animals
3.2 Experimental Procedures
4 Electrophysiology to Evaluate Virus-Induced Changes to Functional Properties of AMPARs in OPC
4.1 Required Equipment, Materials, and Experimental Animals
4.2 Experimental Procedures
4.2.1 Slice Preparation for Electrophysiology
4.2.2 Patch-Clamp Recordings
5 Evaluating the Changes in Proliferation and Differentiation of OPCs with Modified AMPARs
5.1 Required Equipment, Materials, and Experimental Animals
5.2 Experimental Procedures
5.2.1 In Vivo EdU Treatment
5.2.2 Immunohistochemistry
5.2.3 Image Acquisition
5.2.4 Cell Counting
6 Conclusion
References
Chapter 16: Studying Synaptic Integration of Glioma Cells into Neural Circuits
1 Introduction
2 Neuron-Glioma Coculture
2.1 Materials
2.1.1 Culture Media
2.1.2 Cultureware
2.1.3 Dissociation/Isolation Kits
2.1.4 Mice
2.1.5 Equipment
2.2 Methods
2.2.1 Preparation
2.2.2 Neuron Isolation
2.2.3 Neuron Culture
2.2.4 Neuron-Glioma Coculture
2.2.5 Experimental Paradigms
2.2.6 Coculture Fixation/Staining
2.3 Notes
3 Orthotopic Xenografting of Glioma Cells
3.1 Materials
3.1.1 Solutions for Cell Culture
3.1.2 Consumables for Cell Culture
3.1.3 Equipment for Cell Culture
3.1.4 Consumables for Surgery
3.1.5 Equipment for Surgery
3.1.6 Mice
3.2 Methods
3.2.1 Preparing Glioma Cells in a Biological Safety Cabinet
3.2.2 Stereotactic Injection of Glioma Cells into Mouse Brain
3.3 Notes
4 Acute Slice Preparation of Hippocampal Xenografts
4.1 Materials
4.1.1 Solutions
4.1.2 Equipment
4.2 Methods
4.3 Notes
5 Electrophysiological Recording of Postsynaptic Responses in Glioma Cells
5.1 Materials
5.1.1 Solutions
5.1.2 Equipment and Software
5.2 Methods
5.3 Notes
6 Two-Photon In Situ Calcium Imaging of Glioma Cells
6.1 Materials
6.1.1 Solutions
6.1.2 Equipment
6.1.3 Software
6.2 Methods
6.3 Notes
7 Immunoelectron Microscopy
7.1 Materials
7.1.1 Solutions
7.1.2 Equipment
7.1.3 Antibodies
7.2 Methods
7.2.1 Tissue Collection and Fixation
7.2.2 Sample Preparation
7.2.3 Immunohistochemistry
7.2.4 Imaging and Analysis of Neuron-Glioma Synaptic Structures
7.3 Notes
8 Conclusion
References
Index