Muscarinic Receptor: From Structure to Animal Models (Neuromethods, 211)

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Radioligand Binding at Muscarinic Receptors
	1 Historical Background
	2 Principles of Ligand Binding
		2.1 Ligand Binding Definition
		2.2 Radioligands
		2.3 Ligand-Specific Binding
		2.4 Ligand Non-specific Binding
	3 Radioligand Binding Experiments at a Glance
	4 Available Radioligands
		4.1 Antagonist Radioligands
		4.2 Agonist Radioligands
		4.3 Irreversible Radioligands
	5 Source of Muscarinic Receptors
		5.1 Tissues
		5.2 Cell Lines
		5.3 Membranes
		5.4 Purified Receptors
	6 Incubation Conditions
		6.1 Buffers
		6.2 Sample  Size
		6.3 Incubation Volume
		6.4 Temperature
	7 Radioligand Separation
		7.1 Radioligand Binding in Cell Membranes
		7.2 Radioligand Binding in Intact Cells
	8 Experimental Arrangement
		8.1 Measurement of the Rate of Association
		8.2 Measurement of Dissociation
		8.3 Measurement of Binding Saturation
		8.4 Displacement (Competition) Binding
		8.5 Kinetics of Competitive Binding
		8.6 Determination of Radioligand-Specific Radioactivity
	9 Data Analysis
		9.1 Regression Analysis
		9.2 Software
	10 Conclusions
	References
Chapter 2: Tissue-Segment Binding Method for Detection of Muscarinic Acetylcholine Receptors in Receptor´s Natural Environment
	1 Introduction
	2 Materials
		2.1 Solution for Tissue Isolation
		2.2 Incubation Buffer
		2.3 Radioligands
	3 Methods
		3.1 Preparation of Tissue Segments
		3.2 Incubation Volume and Temperature
		3.3 Incubation Time
		3.4 Washing
		3.5 Tissue Solubilization and Measurement of Protein and Radioactivity
		3.6 Data Analysis
	4 Notes
		4.1 Binding Density and Affinity
		4.2 Identification of Surface and Intracellular mAChR
		4.3 Summary and Perspective
	References
Chapter 3: Use of Antibodies in the Research on Muscarinic Receptor Subtypes
	1 Introduction
	2 Selectivity Problems with Muscarinic Receptor Antibodies
	3 Methods for Immunohistochemical Detection of Muscarinic Receptors
		3.1 Tips for Immunoenzyme Staining
	4 Conclusions
	References
Chapter 4: Allosteric Modulation of Ligand Binding to Muscarinic Receptors
	1 Historic Overview
	2 Principles of Allosteric Modulation
		2.1 Allosteric Modulation of Ligand Binding
		2.2 Allosteric Modulation of Functional Response
	3 Promises of Allosteric Targeting of Muscarinic Receptors
		3.1 Subtype Selectivity by Targeting Less Conserved Domains
		3.2 Conservation of Space and Time Pattern of Signaling
		3.3 Absolute Selectivity
	4 Analyzing Allosteric Modulation of Ligand Binding
		4.1 Allosteric Modulation of Tracer Binding in Saturation Binding Experiments
		4.2 Displacement Binding Experiments
		4.3 Allosteric Modulation of Tracer Binding Kinetics
		4.4 Three-Ligand Systems
			4.4.1 Interaction of Two Orthosteric and One Allosteric Ligand
			4.4.2 Interaction of an Orthosteric, an Allosteric, and a Bitopic Ligand
			4.4.3 Interaction of an Orthosteric and Two Allosteric Ligands
	5 Conclusions
	References
Chapter 5: Allosteric Modulation of Functional Response of Muscarinic Receptors
	1 Introduction
	2 Analyzing Allosteric Modulation of Functional Responses
		2.1 Effects of Allosteric Modulators on Functional Response Under Equilibrium
			2.1.1 Agonists Effect on G-protein Binding and Receptor Activation
			2.1.2 Effects of an Allosteric Modulator on G-Protein Binding
			2.1.3 Conversions Between Various Receptor-G-protein Complexes
	3 Operational Model of Allosterically Modulated Agonism
		3.1 Principles of the Operational Model
		3.2 Allosteric Modulators
		3.3 Allosteric Antagonists
		3.4 Allosteric Agonists
	4 Effects of Allosteric Modulators on the Kinetics of Functional Responses
	5 Conclusions
	References
Chapter 6: Exploring Muscarinic Acetylcholine Receptor Binding Kinetics with Fluorescence Anisotropy
	Abbreviations
	1 Historical Background
	2 Principles of Fluorescence Polarization and Anisotropy
	3 Fluorescent Ligands for FA Assays
	4 Sources of Receptors
	5 Assay Protocol
		5.1 Materials
			5.1.1 For Cloning
			5.1.2 For Cell Culture and BBV Preparation
			5.1.3 For FA Experiment
			5.1.4 Software
		5.2 Generating BBV Particles
		5.3 FA Experiment
			5.3.1 Plate-Reader Calibration
			5.3.2 Saturation Experiment
			5.3.3 Competition Binding Experiment
	6 Data Analysis
		6.1 Simple (Simple GraphPad Prism, Excel, etc.)
		6.2 Medium (Advanced Graphpad Prism)
		6.3 Advance (IQMTools in Matlab)
	7 Notes
	8 Representative Results and Discussion
	9 Conclusion
	References
Chapter 7: MultiBacMam Technology for Studying the Downstream cAMP Signaling Pathway of M2 Muscarinic Acetylcholine Receptor
	1 Introduction
	2 Materials
		2.1 Plasmids and Generation of MultiBac Bacmid
		2.2 Production of MultiBacMam Virus
		2.3 cAMP Biosensor Protein Expression
		2.4 FRET-Based cAMP Assay
		2.5 Software
	3 Methods
		3.1 Cloning the MultiBac Bacmid
		3.2 Generation and Collection of MultiBacMam Virus
		3.3 Gsi-Epac-SH187 Expression and cAMP Assay
		3.4 Data Analysis
	4 Results
	5 Notes
	References
Chapter 8: Subcellular and Synaptic Distribution of Muscarinic Receptors in Neurons by Confocal and Electron Microscopy
	1 Introduction
	2 Different Models to Study the Localization of MRs
	3 Solutions and Material
		3.1 Preparation of Brain Sections
			3.1.1 Solutions
			3.1.2 Material
		3.2 Detection of MR by Immunohistochemistry
			3.2.1 Solutions
			3.2.2 Material
		3.3 Primary Neuronal Cell Cultures
			3.3.1 Solutions
			3.3.2 Material
		3.4 Organotypic Cultures
			3.4.1 Solutions
			3.4.2 Material
	4 Preparation of Samples
		4.1 Brain Sections
		4.2 Primary Neuronal Cultures
			4.2.1 Coverslip Coating
			4.2.2 Tissue Dissection and Neuronal Culture
			4.2.3 Transfection
		4.3 Organotypic Cultures
	5 Protocols for the Detection of MRs by Immunohistochemistry
		5.1 Fixed Brain and Organotypic Cultures and Sections
			5.1.1 Common Steps for Detection of MR at Light and Electron Microscopic Levels
			5.1.2 Immunofluorescence or Immunoperoxidase at Light Microscopic Levels
			5.1.3 Immunogold Detection at Electron Microscopic Level
				Single Detection of MRs
				Co-detection of MR and Another Protein of Interest
		5.2 Primary Neuronal Cultures
	6 Results
		6.1 Different Models to Study Localization and Dynamics of MRs
		6.2 Subcellular Localization of M2MR and M4MR in Normal Animals
		6.3 Subsynaptic Localization of M2R in Normal Animals
		6.4 Subcellular Redistribution of M2R and M4R After Acute and Chronic Activation
	7 Notes
		7.1 Perfusion
			7.1.1 Perfusion System
			7.1.2 Fixative
		7.2 Tissue Permeabilization
		7.3 Specificity of Primary Muscarinic Receptor Antibodies
		7.4 Adjustment of Primary Antibody Concentration
		7.5 Gold Coupled Secondary Antibodies or Streptavidin?
		7.6 Coating Grids with Pioloform
		7.7 Ultra-thin Sections Cutting Procedure
		7.8 Quantification
	8 Conclusions
	References
Chapter 9: Investigation of Muscarinic Receptors by Fluorescent Techniques
	1 Background
	2 Creating a Fluorescent Receptor
	3 Fluorescence Microscopy and  FRET
		3.1 Preparation of Cells for Microscopy
		3.2 The Microscope Setup
		3.3 Measuring  FRET
		3.4 FRET Experiments in the Plate Reader
	4 Conclusions
	References
Chapter 10: Autoradiography Assessment of Muscarinic Receptors in the Central Nervous System with a Special Focus on the Selec...
	1 Historical Overview
	2 Principles of Receptor Autoradiography
		2.1 Film Autoradiography
		2.2 Bioimaging/Phosphor Imaging
		2.3 Electronic Autoradiography
	3 Advantages and Disadvantages of the Method
	4 Equipment, Materials, and Setup
	5 Procedures
		5.1 Tissue Processing and Preparation of Glass Slide-Mounted Tissue Sections
		5.2 Specific Labeling of MRs in Brain Sections
			5.2.1 Labeling of MRs with [3H]-QNB
			5.2.2 Labeling of MRs with [3H]-NMS
			5.2.3 Labeling of M1 MRs with [3H]-pirenzepine
			5.2.4 Labeling of M2/M4 MRs with [3H]-AFDX384
		5.3 Generation of Autoradiograms
		5.4 Analysis of Autoradiograms
	6 Conclusions
	References
Chapter 11: Detection of Non-neuronal Acetylcholine
	1 Introduction
	2 Detection Methods for Acetylcholine
	3 Detection of Acetylcholine in So-Called Primitive Organisms Generated Very Early on the Evolutionary Time Scale
	4 Detection of Acetylcholine in the Plant Kingdom
	5 Detection of Acetylcholine or Positive Anti-ChAT Immunoreactivity in Mammalian Non-neuronal Cells
	6 Conclusion
	References
Chapter 12: Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor Autoregulation of Acetylcholine Release
	1 Background
	2 General Remarks
	3 Preparation and Superfusion of Cerebral Slices
		3.1 Stock Solutions
		3.2 Preparation of Krebs Buffer
		3.3 Labeling Medium
		3.4 Superfusion Medium
		3.5 Preparation of Cerebral Slices
		3.6 Labeling of Slices
		3.7 Assembling Superfusion Chambers
		3.8 Superfusion
		3.9 Scintillation Counting
		3.10 Processing of Data
	4 Typical Experiment
	5 Practical Tips
	6 Conclusions
	References
Chapter 13: Evaluation of Acetylcholine Synthesis and Release in Striatal Cholinergic Interneurons
	1 Introduction
	2 Protocols
		2.1 Measurement of [3H]Choline Uptake, CHT1 Activity, and [3H]ACh Synthesis
		2.2 Measurement of [3H]ACh Release from Cholinergic Interneurons
	3 Notes
	4 Summary
	References
Chapter 14: Regulation of Heart Contractility by M2 and M3 Muscarinic Receptors: Functional Studies Using Muscarinic Receptor ...
	1 Introduction
	2 General Approach for Analysis of Muscarinic Receptor Subtypes in the Heart
		2.1 Functional Approaches
		2.2 Immunological Study
		2.3 Molecular Biology Study
	3 Contraction Study Using Muscarinic Receptor Knockout (KO)  Mice
		3.1 Chronotropic Actions
		3.2 Inotropic Actions
		3.3 Distribution of M2 and M3 Muscarinic Receptors in the Mouse Atrium
		3.4 Regulation of COX-2 Expression in the Isolated Mouse Atrium
		3.5 Cardiac Intrinsic Neurons Regulating Mouse Atrial Contractility
	4 Protocols
	5 Conclusions
	References
Chapter 15: Muscarinic Regulation of Gastrointestinal Motility
	1 Introduction
	2 Muscarinic Regulation of Stomach Motility
		2.1 Carbachol-Induced Gastric Contraction
		2.2 Electrical Field Stimulation (EFS)-Induced Cholinergic Contraction
		2.3 Gastric Emptying
	3 Muscarinic Regulation of Contractile Activity in the Ileum and Colon
		3.1 Contractile Response and Peristaltic Reflex in the Ileum of Muscarinic Receptor KO Mice
			3.1.1 Muscarinic and Cholinergic Contractions
			3.1.2 Peristaltic Reflex
		3.2 Contractile Response and Peristaltic Reflex in the Colon of Muscarinic Receptor Knockout Mice
			3.2.1 Spontaneous Contraction Pattern of Colonic Strips
			3.2.2 Carbachol-Induced Colonic Longitudinal Smooth Muscle Contractions
			3.2.3 Carbachol- and EFS-Induced Colonic Circular Smooth Muscle Contraction
			3.2.4 Peristaltic Reflex
			3.2.5 Defecation
			3.2.6 Colonic Propulsion
	4 Role of M2 and M3 Subtypes in the Functions of the Gastrointestinal Tract
	5 Concluding Remarks
	6 Protocols
		6.1 Recording of Peristalsis in the Mouse Small Intestine
		6.2 Recording of Peristalsis in the Mouse Colon
		6.3 Measurement of Gastric Emptying and Defecation
			6.3.1 Gastric Emptying
			6.3.2 Fecal Excretion
			6.3.3 Colonic Propulsion
	References
Chapter 16: Systems for a Long-Term Record of Animal Activity, Temperature, and Heart Rate Affected by Muscarinic Receptors
	1 Introduction
	2 Telemetry Measurement of Parameters Affected by Neurotransmitters
	3 Advantages and Disadvantages of the Method
	4 Equipment, Materials, and Setup for Muscarinic Pharmacology Investigation
		4.1 Procedures
		4.2 Device Preparation
		4.3 Animal Preparation
		4.4 Implantation
		4.5 Post-operative  Care
		4.6 Notes for the Specific Experiment
	5 Conclusions
	References
Index