Receptor and ion channel detection in the brain : methods and protocols

Preface to the Series
Preface
Contents
Contributors
Part I: Molecular Techniques
	Chapter 1: Production of High-Quality Antibodies for the Study of Receptors and Ion Channels
		1 Introduction
		2 Materials
			2.1 Preparation of Peptides for Immunization and Affinity Purification
				2.1.1 Preparation of cDNA Fragments
				2.1.2 Plasmid Construction
				2.1.3 GST Fusion Proteins and GST-Free Peptides
				2.1.4 Synthetic Peptides
				2.1.5 Other Methods Worth Trying
			2.2 Immunization
			2.3 Serum Preparation
			2.4 Affinity Purification
		3 Methods
			3.1 Preparation of Peptides for Immunization and Affinity Purification
				3.1.1 Preparation of cDNA Fragments
				3.1.2 Plasmid Construction
				3.1.3 GST Fusion Proteins and GST-Free Peptides (Fig. 2)
				3.1.4 Synthetic Peptides
				3.1.5 Other Methods Worth Trying
			3.2 Immunization
			3.3 Serum Preparation
			3.4 Affinity Purification
			3.5 Specificity Tests
				3.5.1 Normal Tissue Samples
				3.5.2 Mammalian Cell Transfection
				3.5.3 Knockdown Cells and Tissues
				3.5.4 Preabsorption Test
				3.5.5 Comparison with In Situ Hybridization
				3.5.6 Gene Knockout Animals
		4 Notes
		References
	Chapter 2: Co-Immunoprecipitation from Brain
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Preparation of Total Brain Membranes
			3.2 Co-immunoprecipitation
			3.3 SDS-PAGE
			3.4 Immunoblotting
		4 Notes
		5 Concluding Remarks
		References
	Chapter 3: Subsynaptic Membrane Fractionation
		1 Introduction
		2 Materials
			2.1 Make Fresh Solutions Every Time Before Performing the Isolation
			2.2 Pre-, Post-, and Extrasynaptic Purification
			2.3 To Validate the Efficiency of the Fractionation, It Is Necessary to Perform a Western Blot Analysis for Different Markers
		3 Notes
		References
	Chapter 4: Quantitative Analysis of Cell Surface Expressed, Intracellular, and Internalized Neurotransmitter Receptor Populati...
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Buffers and Chemicals
			2.3 Laboratory Consumables
		3 Methods
			3.1 Preparation of Brain Slices
			3.2 Treatment of Brain Slices
			3.3 Cell Surface Biotinylation
			3.4 Investigation of Receptor Internalization from the Cell Surface
			3.5 Homogenization of Slices and Solubilization of Membrane Proteins
			3.6 Separation of Biotinylated and Nonbiotinylated Proteins
			3.7 Immunochemical Analysis of Proteins in Biotinylated and Nonbiotinylated Fractions
		4 Notes
		References
	Chapter 5: Single Nanoparticle Tracking of Surface Ion Channels and Receptors in Brain Cells
		1 Introduction
		2 Materials
			2.1 Media and Buffers
				2.1.1 Organotypic Slices Dissection Medium
				2.1.2 Acute Slices Dissection Medium
				2.1.3 HEPES-Based aCSF
				2.1.4 Classical aCSF
				2.1.5 Internal Solution
			2.2 Dissociated Neuronal Cultures
			2.3 Quantum Dot Labeling
			2.4 Imaging Setup
		3 Methods
			3.1 Transfections, Single-Cell and in Utero Electroporation
			3.2 Quantum Dot Labeling
			3.3 Imaging
				3.3.1 Dissociated Neuronal Cultures
				3.3.2 Organotypic and Acute Slices
			3.4 Image Analysis
		4 Notes
		References
	Chapter 6: Radioligand Binding Detection of Receptors in Brain Membranes
		1 Introduction
		2 Materials
			2.1 Buffers
				2.1.1 Receptor Binding Assay
				2.1.2 Functional ([35S]GTPγS) Binding Assay
			2.2 Drugs and Reagents (See Note 8)
			2.3 Radioligand (See Note 9)
				2.3.1 Receptor Binding Assay
				2.3.2 Functional ([35S]GTPγS) Binding Assay
				2.3.3 Calculations to Prepare Radioligand Concentrations
			2.4 Tissue Dissection
			2.5 Membrane Sample Preparation
			2.6 Other Materials and Apparatuses
		3 Methods
			3.1 Receptor Binding Assays
				3.1.1 Membrane Fraction Preparation for Receptor Binding
				3.1.2 Receptor Binding Saturation Assay
				3.1.3 Receptor Binding Competition Assay
			3.2 Functional Binding Assays ([35S]GTPγS Binding)
				3.2.1 Membrane Fraction Preparation for [35S]GTPγS Binding Assay
				3.2.2 Functional Binding Assay ([35S]GTPγS Binding)
			3.3 Protein Determination
			3.4 Data Analysis and Graphical Representation
				3.4.1 Analysis of Receptor Binding Assays
					Analysis of Saturation Binding  Data
					Analysis of Competition Binding  Data
				3.4.2 Analysis of [35S]GTPγS Binding Assays
		4 Notes
		References
	Chapter 7: Recombinant Alphavirus-Mediated Expression of Ion Channels and Receptors in the Brain
		1 Introduction
		2 Materials
		3 Methods
			3.1 Subcloning into SFV and SIN Vectors
			3.2 DNA Linearization
			3.3 In Vitro Transcription
			3.4 Electroporation of RNA into BHK Cells
			3.5 Lipid-Mediated Transfection of  RNA
			3.6 Harvest of Recombinant Viral Particles
			3.7 Activation of Recombinant SFV Particles Generated with pSFV-Helper2
			3.8 Virus Titer Determination
			3.9 Gene Expression Evaluation
			3.10 Primary Neurons in Culture
			3.11 Virus Stock Purification and Concentration
				3.11.1 Ultracentrifugation
				3.11.2 Centriprep Concentration
				3.11.3 Affinity Chromatography Concentration
			3.12 Cultured Hippocampal Slices
				3.12.1 Micropipette Preparation
				3.12.2 Assembly of the Virus Injection Setup
				3.12.3 Micropipette Loading
				3.12.4 Virus Injection
			3.13 In Vivo Delivery of Alphavirus Vectors
		4 Notes
		References
	Chapter 8: Time-Resolved Fluorescence Resonance Energy Transfer Using Fluorescent Ligands to Study Native G Protein-Coupled Re...
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Preparation of Purified Striatal Membranes
		4 Notes
		References
	Chapter 9: Study of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity ...
		1 Introduction
			1.1 The Value of Proximity Ligation Assay in Understanding the Heteroreceptor Complexes and Their Allosteric Receptor-Receptor...
			1.2 The In Situ PLA: Principle of the Assay
		2 Materials and Buffers
		3 Assay Protocol
		4 Advantages and Disadvantages of the PLA Method
		References
	Chapter 10: Amplified Luminescent Proximity Homogeneous Assay (Alpha)-Based Technique to Detect GPCR Oligomers in Human Postmo...
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Human Brain Samples
			2.3 Buffers and Reagents
			2.4 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Membrane Preparation
			3.2 Alpha Technology
		4 Data Analysis
		5 Notes
		References
Part II: Neuroanatomical Techniques
	Chapter 11: Fluorescent In Situ Hybridization for Sensitive and Specific Labeling
		1 Introduction
			1.1 Overview of Procedures
		2 Materials
			2.1 Preparation and PCR Amplification of the Target cDNA and Plasmid Construction
			2.2 In Vitro Transcription
			2.3 Preparing Brain Samples, Prehybridization, and Hybridization
			2.4 Immunohistochemical Detection of Probes
			2.5 Equipment (Key Equipment Is Shown in Fig. 1)
			2.6 Reagent Setup
		3 Methods
			3.1 Preparing Riboprobes
				3.1.1 Preparation of the Target cDNA
				3.1.2 PCR Amplification of the Target cDNA
				3.1.3 Plasmid Construction
				3.1.4 Linearization of Plasmid DNA Carrying cDNA of Interest
				3.1.5 In Vitro Transcription
			3.2 Preparing Brain Samples
				3.2.1 Fresh-Frozen Sections
				3.2.2 Microslicer Sections of Perfusion-Fixed Brain
			3.3 Prehybridization
			3.4 Hybridization
			3.5 Posthybridization Wash
			3.6 Blocking
			3.7 Immunohistochemical Detection of the First Probe
			3.8 Immunohistochemical Detection of the Second Probe
		4 Options
			4.1 Triple FISH
				4.1.1 Immunohisto chemical Detection of the Third Probe
			4.2 Counterstaining
			4.3 Combination with Immunofluorescence
			4.4 Combination with Retrograde Tracer Labeling
		5 Notes
		References
	Chapter 12: Autoradiographic Visualization of G Protein-Coupled Receptors in Brain
		1 Introduction
		2 Materials
			2.1 5-HT1A Receptor Autoradiography
				2.1.1 Reagents
				2.1.2 Buffers
			2.2 [35S]GTPγS Binding in Sections or Functional Autoradiography
				2.2.1 Reagents
				2.2.2 Buffers
			2.3 Other Materials and Apparatuses
		3 Methods
			3.1 Receptor Autoradiography
				3.1.1 Tissue Preparation
				3.1.2 Assay Protocol (See Note 9)
			3.2 [35S]GTPγS Binding in Sections or Functional Autoradiography
				3.2.1 Tissue Preparation
				3.2.2 Assay Protocol (See Note 9)
			3.3 Quantification of Autoradiogram
				3.3.1 Scanning the Films
				3.3.2 Autoradiograms Quantification
		4 Notes
		References
	Chapter 13: Localization of Neurotransmitter Receptor and Ion Channel Proteins in Unfixed Brains Using In Situ Immunoblotting
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Buffers and Chemicals
			2.3 Laboratory Consumables
		3 Methods
			3.1 Preparation of Brain Sections for Histoblots
			3.2 Transfer of Proteins from Brain Sections onto Nitrocellulose Membranes
			3.3 Preparation of Histoblots for Immunolabeling
			3.4 Immunolabeling
			3.5 Scanning of Histoblots and Quantitative Analysis of Captured Images
		4 Notes
		References
	Chapter 14: Immunohistochemistry for Ion Channels and Their Interacting Molecules: Tips for Improving Antibody Accessibility
		1 Introduction
		2 Materials
			2.1 Immunohistochemistry with Paraffin Sections
				2.1.1 Fixation
				2.1.2 Preparation of Paraffin Sections
				2.1.3 Antigen-Retrieval and Antigen-Exposing Treatments
				2.1.4 Immunohistochemical Staining
			2.2 Immuno@histochemistry with Fresh Frozen Sections
		3 Methods
			3.1 Immunohistochemistry with Paraffin Sections
				3.1.1 Fixation
				3.1.2 Preparation of Paraffin Sections
				3.1.3 Antigen-Retrieval and Antigen-Exposing Treatments
				3.1.4 Immunohistochemical Staining
			3.2 Immunofluorescence with Fresh Frozen Sections
				3.2.1 Preparation of Fresh Frozen Sections
				3.2.2 Fixation of Sections and Immunohistochemical Staining
		References
	Chapter 15: Localization of GFP-Tagged Proteins Under the Electron Microscope
		1 Introduction
			1.1 Examples of the Application of GFP in Biology
		2 Materials
			2.1 Buffers and Solutions
			2.2 Labware and Other Equipment
		3 Methods
			3.1 GFP Preembedding Immunogold Labeling (Fig. 3)
			3.2 Correlative Light-Electron Microscopy for Cell Culture Study
		4 Notes
		References
	Chapter 16: Tyramide Signal Amplification for Immunoelectron Microscopy
		1 Introduction
		2 Materials
			2.1 Buffers and Solutions
			2.2 Labware and Other Equipment
		3 Methods
			3.1 Pre-embedding TSA Labeling
			3.2 Immunogold Labeling
		4 Notes
		References
	Chapter 17: Pre-Embedding Methods for the Localization of Receptors and Ion Channels
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Tissue and Fixation
			2.3 Sectioning of the Brain
			2.4 Membrane Permeabilization
				2.4.1 Triton X-100
				2.4.2 Freeze-Thaw
			2.5 Pre-embedding Immunohistochemistry
			2.6 Processing Sections for Electron Microscopy
			2.7 Ultrathin Sectioning
			2.8 Staining of Ultrathin Sections
		3 Methods
			3.1 Tissue and Fixation
			3.2 Sectioning of the Brain
			3.3 Membrane Permeabilization
				3.3.1 Freeze-Thaw
				3.3.2 Triton X-100
			3.4 Pre-embedding Immunoperoxidase
			3.5 Pre-embedding Immunogold
			3.6 Double Labeling Pre-embedding HRP-Immunogold
			3.7 Processing Sections for Electron Microscopy
			3.8 Ultrathin Sectioning
			3.9 Staining of Ultrathin Sections
		4 Notes
		References
	Chapter 18: Post-Embedding Immunohistochemistry in the Localization of Receptors and Ion Channels
		1 Introduction
		2 Materials
			2.1 Buffers
			2.2 Chemical Fixation
			2.3 Brain Sectioning
			2.4 Cryofixation
			2.5 Freeze-Substitution
			2.6 Processing Sections for Electron Microscopy
			2.7 Coating Slides with Gelatine and Semithin Sectioning
			2.8 Ultrathin Sectioning of Lowicryl-Embedded Tissue
			2.9 Immunolabeling Semithin Sections: Post-embedding  HRP
			2.10 Immunolabeling Ultrathin Sections: Post-embedding Immunogold
			2.11 Staining of Ultrathin Sections
		3 Methods
			3.1 Tissue and Fixation
			3.2 Sectioning of the Brain
			3.3 Cryofixation
			3.4 Freeze-Substitution
			3.5 Processing Sections for Electron Microscopy
			3.6 Coating Slides with Gelatine and Semithin Sectioning
			3.7 Ultrathin Sectioning
			3.8 Post-embedding on Semithin Sections
			3.9 Post-embedding on Ultrathin Sections
			3.10 Staining of Ultrathin Sections
			3.11 Controls
		4 Notes
		References
	Chapter 19: High-Resolution Localization and Quantitation of Membrane Proteins by SDS-Digested Freeze-Fracture Replica Labelin...
		1 Introduction
		2 Materials
			2.1 Buffers and Stock Solutions
			2.2 Tissue Fixation
			2.3 Slice Preparation and Cryoprotection
			2.4 High-Pressure Freezing
			2.5 Fracturing and Replication
			2.6 SDS Solubilization
			2.7 Immunogold Labeling
		3 Methods
			3.1 Fixation and Tissue Preparation
			3.2 Brain Slice Preparation
			3.3 Cryoprotection
			3.4 High-Pressure Freezing
			3.5 Freeze-Fracture Replication
			3.6 SDS Solubilization
			3.7 Immunolabeling
			3.8 Sampling and Quantitative Analysis of Immunogold Particles
		4 Notes
		5 Concluding Remarks
		References
	Chapter 20: Pre-Embedding Immunostaining of Brain Tissue and Three-Dimensional Imaging with FIB-SEM
		1 Introduction
		2 Materials
			2.1 Solutions and Buffers
			2.2 Anesthesia, Fixation and Tissue Preparation
			2.3 Tissue Permeabilization
			2.4 Immunocytochemistry
			2.5 Postfixation and Osmication
			2.6 En Bloc Staining with Uranyl Acetate and Dehydration
			2.7 Flat-Embedding in Araldite
			2.8 Semithin Sectioning
			2.9 Preparation of the Specimen Block for FIB-SEM
		3 Methods
			3.1 Fixation
				3.1.1 Intracardial Perfusion (Rodents)
				3.1.2 Immersion Fixation (Biopsies and Autopsies)
			3.2 Vibratome Sectioning
			3.3 Tissue Permeabilization (Freeze-Thaw)
			3.4 Pre-Embedding Immunocytochemistry
			3.5 Postfixation and Osmication
			3.6 En Bloc Uranyl Acetate Staining and Dehydration
			3.7 Flat Embedding in Araldite
			3.8 Semithin Sectioning
			3.9 Preparation of the Block for FIB-SEM Imaging
			3.10 FIB-SEM Imaging
			3.11 Serial Section Alignment and Analysis
		4 Notes
		References
	Chapter 21: Application of Virus Vectors for Anterograde Tract-Tracing and Single-Neuron Labeling Studies
		1 Introduction
		2 Materials
			2.1 Preparation of Sindbis Virus Particles
				2.1.1 In Vitro Transcription
				2.1.2 Preparation of Cells
				2.1.3 Electroporation of Cells
				2.1.4 Concentration of Sindbis Virus Particles
			2.2 Injection
			2.3 Fixation
			2.4 Sectioning
			2.5 Fluorescent Nissl-like Staining
			2.6 Immunoperoxidase Staining
			2.7 Reconstruction of Dendrites and Axons
			2.8 Counterstaining with Cresyl Violet
			2.9 Preparation of Lentivirus Particles
				2.9.1 Production of Lentivirus Vector Under Serum-Free Conditions
				2.9.2 Concentration of Lentivirus Particles
		3 Methods
			3.1 Production and Concentration of Sindbis Virus Vector
				3.1.1 In Vitro Transcription
				3.1.2 Preparation of Cells
				3.1.3 Electroporation of Cells
				3.1.4 Concentration of Sindbis Virus Particles
			3.2 Injection
			3.3 Fixation
			3.4 Sectioning
			3.5 Fluorescent Nissl-like Staining
			3.6 Immunoperoxidase Staining
				3.6.1 ABC Method for the Sections Containing the Cell Body and Dendrites
				3.6.2 ABC Method with BT-GO Amplification for the Remaining Serial Sections
			3.7 Reconstruction of Dendrites and Axons
			3.8 Counterstaining with Cresyl Violet
			3.9 Preparation of Lentivirus Vector
				3.9.1 Production of Lentivirus Vector Under Serum-Free Conditions
				3.9.2 Concentration of Lentivirus Particles
		4 Notes
		References
	Chapter 22: Efficient Labeling of Neurons and Identification of Postsynaptic Sites Using Adeno-Associated Virus Vector
		1 Introduction
		2 Materials
			2.1 Production and Purification of AAV Vector
				2.1.1 Plasmid Transfection
				2.1.2 Virus Extraction
				2.1.3 Virus Purification
				2.1.4 Virus Concentration
				2.1.5 Virus Titration
			2.2 Virus Injection
			2.3 Tissue Preparation
				2.3.1 Fixation
				2.3.2 Sectioning
				2.3.3 Fluorescent Immunostaining
		3 Methods
			3.1 Production and Purification of AAV Vector
				3.1.1 Plasmid Transfection
				3.1.2 Virus Extraction
				3.1.3 Virus Purification
				3.1.4 Virus Concentration
				3.1.5 Virus Titration
			3.2 Virus Injection
			3.3 Tissue Preparation
				3.3.1 Fixation
				3.3.2 Sectioning
				3.3.3 Fluorescent Immunostaining
		4 Notes
		References
	Chapter 23: Analysis of Synaptic Connections at the Electron Microscopic Level Using Sindbis Virus Vectors
		1 Introduction
		2 Materials
			2.1 Surgery for Virus Injection
			2.2 Fixation
			2.3 Sectioning on a Vibrating Microtome
			2.4 Immunohistochemical Staining for GFP
			2.5 Preparation for Ultrathin Sectioning
			2.6 Ultrathin Sectioning
			2.7 Post-embedding Immunohistochemistry for GABA
		3 Methods
			3.1 Virus Injection Surgery
			3.2 Fixation
			3.3 Sectioning of the Brain
			3.4 Immunohistochemistry for GFP
			3.5 Preparation for Ultrathin Sectioning
			3.6 Ultrathin Sectioning
			3.7 Post-embedding Immunohistochemistry for GABA
		4 Notes
		References
	Chapter 24: Morphological and Neurochemical Characterization of Electrophysiologically Identified Cells
		1 Introduction
		2 Material
			2.1 Fixation
				2.1.1 Paraformaldehyde
				2.1.2 Glutaraldehyde
				2.1.3 Picric Acid
				2.1.4 How to Make Fixative
				2.1.5 Preparation of Microwave for Rapid Microwave-Enhanced Fixation
			2.2 Resection of the Fixed Slice
				2.2.1 Resection Method
					Preparation of Tools (Fig. 4a)
					Preparation of Solution
					How to Resection the Slice
			2.3 Freeze and Stock
				2.3.1 Recipe of a Cryoprotectant Solution (30% Glycerol, 30% Ethylene Glycol, 0.04 M PBS)
				2.3.2 Store Sections in the Cryoprotectant Solution
			2.4 Immunohistochemical Staining
			2.5 Histological Staining
				2.5.1 ABC Complex
				2.5.2 Features of Nickel-Diaminobenzidine Tetrahydrochloride (DAB) Molecule
				2.5.3 How to Make the Nickel DAB Solution
			2.6 Osmium Fixation, Dehydration, Embedding
				2.6.1 Osmium Tetroxide: OsO4
				2.6.2 Uranyl Acetate
				2.6.3 Resin for Electron Microscopy (EM) Tissue Embedding
				2.6.4 How to Make Resin
					Epon 812 (TAAB, Aldermaston, U.K.)
					Durcupan ACM (Sigma-Aldrich, St. Louis, U.S.A.)
				2.6.5 How to Wash Glass Vials Contaminated with Epon
				2.6.6 Propylene Oxide
				2.6.7 Lead Aspartate
				2.6.8 How to Make Walton´s Lead Aspartate
				2.6.9 Microwave with Temperature Control (Fig. 8)
			2.7 Perfusion for In Vivo Recording
				2.7.1 Preparation of Solution
					Pre-fixative (100 ml)
					Fixative (100 ml)
		3 Methods
			3.1 Slice Fixation
			3.2 Slice Resection
			3.3 Freeze and Stock
			3.4 Neurochemical Characterization with Immunohistochemical Staining
			3.5 Histological Staining
			3.6 Osmium Postfixation, Dehydration, and Embedding
				3.6.1 Conventional Tissue Process for LM (Fig. 7)
				3.6.2 Tissue Process for LM with Preserved Section Thickness (Fig. 7)
				3.6.3 Tissue Process for EM (Fig. 7)
				3.6.4 Dehydration Using Temperature-Controlled Microwave (Fig. 8)
				3.6.5 Tissue Processed for Serial Block-Face Scanning Electron Microscopy (SBEM)
				3.6.6 Perfusion for In Vivo Recording
		4 Notes
			4.1 Tips for Good Immunohistochemistry
			4.2 Comparison of Nickel-DAB and DAB Staining Methods
			4.3 Rapid Microwave-Enhanced Fixation and Dehydration
			4.4 Embedding Sections with the Resin
			4.5 Heavy Metal Staining Procedure for SBEM
			4.6 Tips for Good Perfusion
		References
Part III: Functional Techniques
	Chapter 25: Using Electrophysiology to Study Synaptic and Extrasynaptic Ionotropic Receptors in Hippocampal Neurons
		1 Introduction
		2 Material
			2.1 Hippocampal Pyramidal Neurons Culture Preparation
				2.1.1 Buffers and Solutions
				2.1.2 Poly-d-Lysine Preparation
				2.1.3 Dissection Material
			2.2 Electrophysiological Recordings
				2.2.1 Electrophysiological Solutions and Blockers
				2.2.2 Building a Fast Application Tool for Rapid Solution Exchange
				2.2.3 Electrophysiology Equipment
		3 Methods
			3.1 Hippocampal Pyramidal Neuron Culture (Modified from)
			3.2 Patch Clamp Electrophysiology
				3.2.1 Setting Up the Experimental Rig: The Perfusion System
				3.2.2 Pipette Preparation
				3.2.3 Sealing onto the Neurons
				3.2.4 Whole-Cell Configuration
			3.3 Miniature EPSC Recordings
				3.3.1 mEPSCs Recordings
				3.3.2 mEPSCs Analysis
			3.4 Outside-out Recordings
				3.4.1 Fast Agonist Application Preparation
				3.4.2 Patch Excision
				3.4.3 Study of Channel Properties by Means of NSFA
		4 Notes
		Appendix 1: Igor Macro for Peak-Scaled Nonstationary Fluctuation Analysis
		Appendix 2: SigworthNSNA Function
		Appendix 3: Igor Macro for Nonstationary Fluctuation Analysis
		References
	Chapter 26: Biophysical Methods to Analyze Direct G-Protein Regulation of Neuronal Voltage-Gated Calcium Channels
		1 Introduction
		2 Methods
			2.1 Biophysical Analysis of G-Protein Regulation by the ``Double Pulse´´ Method
			2.2 Biophysical Analysis of G-Protein Regulation by the ``Subtraction´´ Method
		3 Concluding Remarks
		4 Notes
		References
	Chapter 27: Electrophysiological Recordings in Behaving Animals
		Abbreviations
		1 Introduction
		2 Materials
			2.1 Electrode Preparation
				2.1.1 Stimulating Electrodes
				2.1.2 Recording Electrodes
				2.1.3 General Material
			2.2 Electrode Implantation During Surgery
			2.3 Electrophysiological Recordings
		3 Methods
			3.1 Electrode Preparation
				3.1.1 Stimulating Electrodes
				3.1.2 Recording Electrodes
			3.2 Electrode Implantation During Surgery
			3.3 Electrophysiological Recordings
				3.3.1 Stimulation
				3.3.2 Recording
				3.3.3 Evoked Field Potentials
				3.3.4 Neuronal Identification
				3.3.5 Synaptic Effects
				3.3.6 Relationships Between Neural Activities and Behavior
		4 Notes
		References
	Chapter 28: Voltammetry in Behaving Animals
		1 Introduction
		2 Materials
			2.1 Electrochemistry
			2.2 Instrumentation and Software
			2.3 Electrical Stimulation
			2.4 Electrode Construction
			2.5 Surgery
			2.6 Experiment
			2.7 Electrode Calibration
			2.8 Data Analysis
			2.9 Histology/Electrode Placement Verification
		3 Methods
			3.1 Electrochemistry
			3.2 Instrumentation and Software
			3.3 Selecting a Waveform
			3.4 Electrical Stimulation
			3.5 Electrode Construction
				3.5.1 Acute Carbon Fiber Microelectrodes
				3.5.2 Chronic Carbon Fiber Microelectrodes
				3.5.3 Ag/AgCl Reference Electrode
			3.6 Stereotaxic Surgery
				3.6.1 Acute Electrode Implantation in Rats
				3.6.2 Chronic Electrode Implantation in Rats
				3.6.3 Acute Electrode Implantation in Mice
				3.6.4 Chronic Electrode Implantation in Mice
			3.7 Experiment Design
				3.7.1 Basic Procedure
			3.8 Electrode Calibration
			3.9 Data Analysis
			3.10 Histology/Electrode Placement Verification
		4 Notes
		References
	Chapter 29: In Vivo Brain Microdialysis of Monoamines
		1 Introduction
		2 Materials
			2.1 Materials for Microdialysis Probe Manufacturing
			2.2 Material for Microdialysis Probe Calibration
			2.3 Small Animal Stereotaxic Surgery
				2.3.1 Stereotaxic Equipment
				2.3.2 Surgical Instruments
				2.3.3 Microdialysis Equipment for Freely Moving Animals
				2.3.4 Chromatographic Equipment
		3 Methods
			3.1 Manufacturing of Microdialysis Probes
			3.2 Stereotaxic Surgery
			3.3 Microdialysis Procedure
				3.3.1 In Vitro Microdialysis
				3.3.2 In Vivo Microdialysis
			3.4 Chromatographic Analysis
				3.4.1 Chromatographic Conditions for the Separation of DA and 5-HT with a HPLC Equipment
					Chromatographic Column
					Analysis
				3.4.2 Chromatographic Conditions for the Separation of NA with a HPLC Equipment
					Analysis
				3.4.3 Chromatographic Conditions for the Detection of NA, DA, and 5-HT with a UHPLC Equipment
					Chromatographic Column
					Analysis
		4 Notes
		References
	Chapter 30: Optical Control of Brain Receptors Using Photoactive Drugs in Behaving Animals
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Formalin  Test
		4 Notes
		References
	Chapter 31: Dynamic Recording of Membrane Potential from Hippocampal Neurons by Using a Fluorescence Resonance Energy Transfer...
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Preparation of Primary Hippocampal Neuronal Cultures
			3.2 Transfection of the Voltage Biosensor in Primary Hippocampal Neuronal Cultures
			3.3 FRET Assay in Hippocampal Neurons Expressing the Voltage Biosensor
		4 Notes
		References
	Chapter 32: Monitoring GPCR-Mediated cAMP Accumulation in Rat Striatal Synaptosomes
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment and Software
		3 Methods
			3.1 Preparation of Striatal Synaptosomes
			3.2 Time-Resolved Fluorescence Energy Transfer (TR-FRET)-Based cAMP Determination
			3.3 Data Analysis
		4 Notes
		References
	Chapter 33: GPCR-Mediated MAPK/ERK Cascade Activation in Mouse Striatal Slices
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Buffers and Reagents
			2.3 Instrumentation, Equipment, and Software
		3 Methods
			3.1 Brain Slice Preparation and Receptor-Ligand Incubation
			3.2 Western-Blot Determination of ERK Phosphorylation
		4 Notes
		References
Index