Preface
Contents
Contributors
Chapter 1: Real-Time Quantitative PCR and Fluorescence In Situ Hybridization for Subcellular Localization of miRNAs in Neurons
1 Introduction
2 Materials
2.1 Isolation of Total RNA from Cultured Neurons or Brain Tissue
2.2 Isolation of microRNA Fraction from Cultured Neurons or Brain Tissue
2.3 MicroRNA Reverse Transcription
2.4 Quantitative Real-Time PCR (qRT-PCR) Profiling of microRNAs
2.5 MiRNA Fluorescence In Situ Hybridization
3 Methods
3.1 MicroRNA Expression Analysis
3.1.1 Isolation of Total RNA from Cultured Neurons or Brain Tissue
3.1.2 Isolation of microRNA Fraction from Cultured Neurons or Brain Tissue
3.1.3 microRNA Reverse Transcription
3.1.4 Real-Time Quantitative PCR (RT-qPCR) Profiling of microRNAs
3.1.5 Analysis of qRT-PCR Data
3.1.6 miRNA Fluorescence In Situ Hybridization
3.1.7 Fixation of Neurons
3.1.8 Pre-hybridization and In Situ Hybridization
3.1.9 Detection of Hybridization
4 Notes
References
Chapter 2: Live-Imaging of Axonal Cargoes in Drosophila Brain Explants Using Confocal Microscopy
1 Introduction
2 Materials
2.1 Animal Collection
2.2 Imaging Chamber Preparation
2.3 Dissection of Pupal Brains
2.4 Mounting of Brains in the Imaging Chamber
2.5 Brain Imaging
3 Methods
3.1 Animal Collection
3.2 Imaging Chamber Preparation (See Note 3)
3.3 Dissection of Pupal Brains
3.4 Mounting of Brains in the Imaging Chamber
3.5 Brain Imaging
4 Notes
References
Chapter 3: Whole Endosome Recording of Vesicular Neurotransmitter Transporter Currents
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Electrophysiology
2.3 Buffers
3 Methods
3.1 General Timeline
3.2 Cell Culture
3.2.1 Passing Cells
3.2.2 Plating Cells for Transfection
3.2.3 Transfect Cells
3.2.4 Plating Cells for Recording
3.2.5 Coating Coverslips
3.3 Electrophysiology
3.3.1 Making Agar Bridges
3.3.2 Microelectrode Bleaching
3.3.3 Preparing Microforge
3.3.4 Shaping Pipettes
3.3.5 Preparing Electrophysiology Rig
3.3.6 Releasing Endosomes
3.3.7 Patching Endosomes
4 Notes
References
Chapter 4: Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons
1 Introduction
2 Materials
2.1 Molecular Biology
2.2 Production of Lentiviruses
2.3 Neuronal Cultures
2.4 Fluorescence Microscope Setup (Fig. 1)
3 Methods
3.1 Production of Lentiviral Vector Encoding a pH Sensor for Synaptic Vesicle Lumen
3.2 Preparation of Cultured Neurons Expressing Vesicular pH Probe
3.3 Estimation of the pKa and Hill Coefficient of pH Probes In Situ
3.4 Estimation of Vesicular pH
4 Notes
References
Chapter 5: Investigation of Synaptic Vesicle Proteins in Rat Brain Tissue Using Real-Time qPCR
1 Introduction
2 Materials
2.1 Extraction of RNA and Protein with PARIS Kit
2.2 RNA Quality and Quantity Check
2.3 cDNA Synthesis
2.4 Real-Time qPCR
3 Methods
3.1 Collection of Brain Tissue
3.2 Homogenization of Brain Tissue
3.3 Extraction of RNA and Protein from the Brain Samples
3.4 cDNA Synthesis
3.5 Real-Time qPCR
3.6 Primer Design
3.7 Normalization
4 Notes
References
Chapter 6: Mass Synaptometry: Applying Mass Cytometry to Single Synapse Analysis
1 Introduction
2 Materials
2.1 Reagents and Disposables (See Note 1)
2.2 Equipment
2.3 Suggested Data Analysis Tools
2.4 Brain Tissue/Biological Material
3 Methods
3.1 Synaptosome Preparation
3.2 Modular Design and Implementation of Synaptic Antibody Panel
3.2.1 Selecting the Antibody Panel
3.2.2 Optimizing Antibodies
3.2.3 Antibody Conjugation
3.3 Staining Synaptosomes for Mass Synaptometry
3.4 Synaptosome Acquisition Parameters and Processing
3.4.1 Acquisition Setting for Synaptosomes
3.4.2 Post-Acquisition Normalization and Gating
3.5 Advanced Visualization and Analysis Tools for High-Dimensional Mass Synaptometry Datasets
4 Notes
References
Chapter 7: A Guide to Analysis of Relative Synaptic Protein Abundance by Quantitative Fluorescent Western Blotting
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 SDS Polyacrylamide Gel Electrophoresis
2.3 Electrotransfer of Proteins
2.4 Other Supplies Required
3 Methods
3.1 Sample Preparation
3.2 SDS Polyacrylamide Gel Electrophoresis
3.3 Electrotransfer of Proteins
4 Notes
References
Chapter 8: Synaptosomes and Metamodulation of Receptors
1 An Historical Overview on Synaptosomes and their Use in the Study of Neurotransmission
2 ``Metamodulation´´ of Release-Regulating Presynaptic Receptors
3 Metamodulation of Presynaptic Release-Regulating Receptors: The Case of Presynaptic Release-Regulating NMDA Receptors
3.1 Metamodulation Involving Ionotropic Receptors
3.2 Metamodulation Involving Metabotropic Receptors
3.3 Metamodulation Involving Transmitter Transporter
3.4 Metamodulation and Presynaptic Receptors Trafficking
4 Concluding Remark
References
Chapter 9: A Novel Method to Monitor GABA Loading into Synaptic Vesicles by Combining Patch Pipette Perfusion and Intracellula...
1 Introduction
2 Materials
2.1 Slice Preparation
2.2 Electrophysiological Recording (Simultaneous Presynaptic and Postsynaptic Recordings)
2.3 Pipette Perfusion
2.4 DPNI-GABA Uncaging
3 Methods
3.1 Slice Preparation
3.2 Electrophysiological Recording (Pair Recording)
3.3 Pipette Perfusion
3.4 DPNI-GABA Uncaging
4 Notes
References
Chapter 10: Rapid Isolation of Functional Synaptic Vesicles from Tissues Through Cryogrinding, Ultracentrifugation, and Size E...
1 Introduction
2 Materials
2.1 Sucrose Beads
2.2 Cryogrinding
2.3 Centrifugation
2.4 Fast Protein Liquid Chromatography (FPLC)
3 Methods
3.1 Preparation of Sucrose Beads
3.2 Cryogrinding
3.3 Warming of the Solution
3.4 Low-Speed Centrifugation
3.5 High-Speed Centrifugation
3.6 Fast Protein Liquid Chromatography (FPLC) (See Note 12)
3.7 Characterization of the Different Portion During Isolation Procedure
4 Notes
References
Chapter 11: Isolation of Synaptic Vesicles from Mammalian Brain
1 Introduction
2 Materials
2.1 Equipment
2.2 Tools for Brain Dissection
2.3 Tools for Tissue Fractionation
2.4 Controlled-Pore Glass (CPG) Size Exclusion Chromatography
2.5 Solutions
3 Methods
3.1 Extracting and Dissecting the Brain
3.2 Isolation of Synaptosomes
3.3 Purification of the Synaptic Vesicles
4 Notes
References
Chapter 12: Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime ...
1 Introduction
1.1 Mathematical Background of Fluorescence Correlation and Cross-Correlation
1.2 Fluorescence Lifetime Analysis and FRET
1.3 Technical Realization
2 Materials
2.1 Building a FCCS Setup
2.1.1 General Equipment
2.1.2 Excitation Path (See Note 6)
2.1.3 Detection
2.2 Fine Adjustment of the APDs
2.3 Calibration of the Focal Volume
3 Methods
3.1 Building a FCCS Setup
3.2 Fine Adjusting the APDs
3.3 Calibration of the FCS Setup
4 Notes
References
Chapter 13: Fluorescence Lifetime and Cross-correlation Spectroscopy for Observing Membrane Fusion of Liposome Models Containi...
1 Introduction
2 Material
2.1 Lipid Film
2.2 Formation of Proteoliposomes
2.3 FCCS Measurement
3 Methods
3.1 Lipid Film
3.2 Formation of Proteoliposomes
3.3 FCCS Measurement
4 Notes
References
Chapter 14: Synaptic Vesicle Pool Monitoring with Synapto-pHluorin
1 Introduction
2 Materials
2.1 Preparation of Primary Hippocampus Cultures from Newborn Rats
2.2 Transfection of Primary Hippocampus Cultures (for Three 12-Well Plates)
2.3 Fluorescence Live-Cell Imaging of Exocytosis
2.4 Data Analysis of Fluorescence Images
3 Methods
3.1 Preparation of Primary Hippocampus Cultures (Four 12-Well Plates)
3.2 Calcium-Phosphate Transfection of Primary Hippocampus Cultures
3.3 Fluorescence Live-Cell Imaging of Exocytosis
3.4 Analysis of Fluorescence Images
4 Notes
References
Chapter 15: Imaging Neuropeptide Release at Drosophila Neuromuscular Junction with a Genetically Engineered Neuropeptide Relea...
1 Introduction
2 Materials
2.1 Fly Strains
2.2 Experiment Reagents
2.3 Equipment
2.4 Software
3 Methods
3.1 Drosophila Larval NMJ Preparation
3.2 Electric Stimulation
3.3 Calcium Imaging
3.4 Data Analysis and Interpretation
3.5 Representative Results
4 Notes
References
Chapter 16: Imaging Synaptic Glutamate Release with Two-Photon Microscopy in Organotypic Slice Cultures
1 Introduction
2 Materials
2.1 Organotypic Slice Cultures
2.2 Single-Cell Electroporation
2.2.1 Electroporation Equipment
2.3 Functional Imaging of Synaptic Transmission
2.3.1 Imaging Setup
2.3.2 Electrophysiology Setup
3 Methods
3.1 Culture Preparation
3.2 Choice of GEGI Variant
3.3 Neuron Transfection
3.3.1 DNA and Plasmids Preparation
3.3.2 Electroporation
3.4 Functional Imaging of Synaptic Transmission
3.5 GEGI Signal Extraction
4 Notes
References
Chapter 17: Dynole 34-2 and Acrylo-Dyn 2-30, Novel Dynamin GTPase Chemical Biology Probes
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvents
2.3 Equipment
3 Methods
3.1 Dynole Compounds
3.1.1 Synthesis of 2-Cyano-N-octylacetamide (Compound 4, Dynole Active Intermediate)
3.1.2 Synthesis of 2-Cyano-N-Propylacetamide (Compound 7, Dynole Inactive Intermediate)
3.1.3 Synthesis of 1-(3-(Dimethylamino)propyl)-1H-indole-3-carbaldehyde (Compound 11, Dynole Common Intermediate)
3.1.4 Synthesis of (E)-2-Cyano-3-(1-(3-(dimethylamino)propyl)-1H-indol-3-yl)-N-octylacrylamide (Compound 2, Dynole Active)
3.2 Acrylo-Dyn Compounds
3.2.1 Synthesis of (Z)-2-(3,4-Dichlorophenyl)-3-(1-(3-(dimethylamino)propyl)-1H-indol-3-yl)acrylonitrile (Compound 12, Acrylo-...
Microwave Method
Batch Method
3.2.2 Synthesis of (Z)-2-(3,4-ichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (Compound 15, Acrylonitrile Inactive Intermediate)
3.2.3 Synthesis of (Z)-3-(1-Benzyl-1H-pyrrol-2-yl)-2-(3,4-dichlorophenyl)acrylonitrile (Compound 13, Acrylonitrile Inactive)
3.3 Biological Methods for Whole Cell Assay
4 Notes
References
Chapter 18: Synthesis of Phthaladyn-29 and Naphthalimide-10, GTP Site Directed Dynamin GTPase Inhibitors
1 Introduction
2 Materials
2.1 Reagents
2.2 Solvents
2.3 Equipment
3 Methods
3.1 Phthaladyn Compounds
3.1.1 Synthesis of 4-Chloro-2-(1,3-dioxo-1,3-dihydroisobenzofuran-5-carboxamido)benzoic Acid (Compound 6, Phthaladyn Intermedi...
3.1.2 Synthesis of 4-Chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic Acid (Compound 2, Phthal...
3.1.3 Synthesis of 4-Chloro-2-(1,3-dioxo-2-phenylisoindoline-5-carboxamido)benzoic Acid (3, Phthaladyn Inactive)
3.2 Naphthalimide Compounds
3.2.1 Synthesis of 2-(4-Aminobenzyl)-5-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (Compound 10, Naphthalimide Active)
Microwave Method
Batch Method
3.2.2 Synthesis of 2-(2-Hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (Compound 11, Naphthalimide Inactive)
Microwave Method
Batch Method
3.3 Biological Methods for Whole Cell Assay
4 Notes
References
Index