Opioid Receptors: Methods and Protocols

Dedication
Preface
Contents
Contributors
Part I: Transcriptional and Post-Transcriptional Analysis and Cellular Detection of Opioid Receptors
	Chapter 1: Overview of Genetic Analysis of Human Opioid Receptors
		1 Introduction
		2 Genetic Polymorphisms of Opioid Receptors
		3 Analysis of Gene Variants of Opioid Receptors
		4 Pharmacogenetics of Opioid Addiction
		5 Opioid Receptor Genotyping and Personalized Medicine
		References
	Chapter 2: Renilla Luciferase Reporter Assay to Study 3′-UTR-Driven Posttranscriptional Regulation of OPRM1
		1 Introduction
		2 Materials
			2.1 Cell Plating
			2.2 Transfection
			2.3 Treatments and Readings
		3 Methods
			3.1 Cell Plating
			3.2 Transfection
			3.3 Treatments and Readings
			3.4 Data Analysis
		4 Notes
		References
	Chapter 3: Fluorescence Colocalization Analysis of Cellular Distribution of MOR-1
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Immunofluorescence Buffers
			2.3 Antibodies
			2.4 Other Materials
			2.5 Morphine Nociceptive Tolerance
		3 Methods
			3.1 Internalization of MOR-1 Assessed by Immunofluorescence Analysis
		4 Notes
		References
	Chapter 4: Monitoring Opioid Receptor Interaction in Living Cells by Bioluminescence Resonance Energy Transfer (BRET)
		1 Introduction
		2 Materials
			2.1 Generation of Fusion Proteins
			2.2 Cell Culture and Transfection
			2.3 Evaluation of Fusion Protein Expression and Detection of BRET Signal
		3 Methods
			3.1 Generation of Fusion Proteins for Monitoring KOR and DOR Heterodimerization
			3.2 Cell Culture and Transfection
			3.3 Expression of Fusion Proteins
			3.4 BRET Signal Measurement
		4 Notes
		References
	Chapter 5: Bioluminescence Resonance Energy Transfer (BRET) to Detect the Interactions Between Kappa Opioid Receptor and Nonvi...
		1 Introduction
		2 Materials
			2.1 Cell Transfection (Day 1)
			2.2 Plating Transfected Cells into 96-Well Plates (Day 2)
			2.3 Cell Stimulation with Opioid Ligands or Vehicle and BRET Signal Measurement (Day 3)
		3 Methods
			3.1 Cell Transfection (Day 1)
			3.2 Plating Transfected Cells into 96-Well Plates (Day 2)
			3.3 Cell Stimulation with Opioid Ligands or Vehicle and BRET Signal Measurement (Day 3)
			3.4 Data Analysis
		4 Notes
		References
	Chapter 6: Functionalization and Bioconjugation of Nanoruby for Long-Term, Ultrasensitive Imaging of u-Opioid Receptors
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents, Samples, and Consumables
		3 Methods
			3.1 Functionalization of Silica-Coated Nanoruby with Azide Groups, by Silanization
			3.2 Alkynation of NeutrAvidin with DBCO-NHS
			3.3 Bioconjugation of Azide-Functionalized Nanoruby to Alkynated NeutrAvidin by Click Chemistry
			3.4 Labeling μ-Opioid Receptor in Live Cells with NeutrAvidin-Conjugated Nanoruby
		4 Notes
		References
	Chapter 7: Immunohistochemical Analysis of Opioid Receptors in Peripheral Tissues
		1 Introduction
		2 Materials
			2.1 Buffers and Solutions
			2.2 Antibodies and Antigens
			2.3 Other Materials
		3 Methods
			3.1 Surgeries
			3.2 Animal Perfusion
			3.3 Tissue Isolation
			3.4 Single and Double Immunofluorescence
			3.5 DAB Immunohistochemistry
			3.6 Antibody Specificity Controls
		4 Notes
		References
	Chapter 8: Real-Time Quantitative Reverse Transcription PCR for Detection of Opioid Receptors in Immune Cells
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Enzymes
			2.3 Fluorescein Dyes
			2.4 Digestive Solution
			2.5 Commercial  Kits
			2.6 Surgical Dissection Tools for CCI
			2.7 Animals and Other Materials
		3 Methods
			3.1 CCI of the Sciatic Nerve
			3.2 Immune Cell Isolation from Blood
			3.3 Immune Cell Isolation from Injured Nerves
			3.4 Extraction of Total RNA by Trizol
			3.5 RNA Cleanup Protocol Using Qiagen RNeasy Plus Mini  Kit
			3.6 Measuring Total RNA Concentration and Quality by Using Spectrophotometer
			3.7 Reverse Transcription Protocol for Superscript IV VILO Master  Mix
			3.8 TaqMan Gene Expression Assays for MOR, DOR, and KOR
			3.9 Real-Time PCR
			3.10 DeltaDeltaCt Method
		4 Notes
		References
Part II: Analysis of Signaling Events Modulated by Opioid Receptors
	Chapter 9: Quantitative Analysis of MOR-1 Internalization in Spinal Cord of Morphine-Tolerant Mice
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Western Blot Analysis
			2.3 Immunofluorescence
			2.4 Antibodies
			2.5 Morphine Nociceptive Tolerance
			2.6 Other Materials
		3 Methods
			3.1 Morphine Nociceptive Tolerance
			3.2 Preparation of Whole-Cell Lysate, Membrane, and Cytosolic Fraction
			3.3 Internalization of MOR-1 Assessed by Western Blotting Analysis
			3.4 Internalization of MOR-1 Assessed by Immunofluorescence
		4 Notes
		References
	Chapter 10: GTPγS-Autoradiography for Studies of Opioid Receptor Functionality
		1 Introduction
		2 Materials
			2.1 Coating of Slides
				2.1.1 Preparation of a Gelatin Buffer Containing 0.5% Gelatin and 0.05% KCr(SO4)2
				2.1.2 Cryostat/Sectioning
				2.1.3 Assay Buffer
				2.1.4 Washing Buffer
		3 Methods
			3.1 Preparation of Brain (Tissue)
			3.2 GTPγS Incubations
			3.3 Film or Phosphor Imaging
			3.4 Analysis of Data
		4 Notes
		References
	Chapter 11: Evaluating Opioid-Mediated Adenylyl Cyclase Inhibition in Live Cells Using a BRET-Based Assay
		1 Introduction
		2 Materials
			2.1 Cells, Culture Media, and Solutions
		3 Methods
			3.1 Cell Preparation
			3.2 Transfection of Biosensor (See Note 3)
			3.3 Replating Cells in Assay Plates
			3.4  Preparation of Drug and Substrate Solutions
			3.5  Set Up the Parameters in PHERAstar FS (see Note 10)
			3.6 Run the cAMP Assay
		4 Notes
		References
	Chapter 12: Patch Clamp Analysis of Opioid-Induced Kir3 Currents in Mouse Peripheral Sensory Neurons Following Nerve Injury
		1 Introduction
		2 Materials
			2.1 Surgical Dissection Tools for CCI
			2.2 Media and Reagents for Mouse DRG Neuron Cultures
			2.3 Buffers and Solutions for Patch Clamp
			2.4 Chemicals
			2.5 Expandable Materials and Equipment for DRG Dissection, DRG Neuron Culture, and Patch Clamp Experiments
			2.6 CCI of the Sciatic Nerve
			2.7 Preparation of the Cell Culture Media and Patch Clamp Solutions
			2.8 Preparation of Poly-l-Lysine-Coated Culture Plates
			2.9 Cultures of DRG Neurons
		3 Methods
			3.1 Performing the Patch Clamp Experiment
		4 Notes
		References
	Chapter 13: Opioid Modulation of Neuronal Iron and Potential Contributions to NeuroHIV
		1 Introduction
		2 μ-Opioid-Mediated Regulation of Neuronal Iron
			2.1 Opioid Modulation of Neuronal Endolysosomes
			2.2 Potential Mechanisms Driving Opioid-Mediated Iron Efflux from Endolysosomes
		3 Neuronal Responses to Increased Labile Iron Levels
			3.1 FHC Upregulation and Downstream Consequences
			3.2 Potential Mechanisms Leading to FHC Upregulation
			3.3 Iron-Mediated Regulation of Amyloid Precursor Protein Expression and Processing
		4 Novel Molecular Approaches to Study the Contributions of APP Processing in HAND with Opioid Abuse
		5 Conclusions
		References
	Chapter 14: An in Vitro Assay to Study the Role of Opioids in Modulating Immune Cell Adhesion
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Cell Adhesion Assay
			2.3 Instruments and Software
		3 Methods
			3.1 Cell Culture
			3.2 Coating of the Wells with a Specific Ligand
			3.3 Immune Cell Fluorescent Labeling
			3.4 Cell Adhesion Assay
			3.5 Data Analysis
		4 Notes
		References
	Chapter 15: Assessing Cell Viability Effects of Opioids in Primary Cortical Cells from Rat
		1 Introduction
		2 Materials
			2.1 Preparation of Primary Cortical Cell Culture from Rat
			2.2 Mitochondrial Function Assay
			2.3 Membrane Integrity Assay
			2.4 Other Materials
		3 Methods
			3.1 Preparation of Primary Cortical Cells from Rat
			3.2 Opioid Treatment
			3.3 Mitochondrial Function Assay (MTT Assay)
			3.4 Membrane Integrity Assay (LDH Assay)
			3.5 Procedure of an Assay Evaluating the Restorative Properties of a Compound of Interest on Opioid-Induced Toxicity
		4 Notes
		References
	Chapter 16: The Quest for More Effective Analgesics with Reduced Abuse Liability and Fewer Adverse Effects: Promises, Pitfalls...
		1 Between Inadequate Pain Relief and Opioid Epidemics: The Urgent Need for More Effective, Safer, and Abuse-Liability-Free Ana...
		2 Identifying More Effective and Safer Analgesics: Holy Grail or Achievable Goal?
		3 Biased Agonism at MOR: Promises and Pitfalls
		4 Biased Agonism at KOR: Current State of the Art and Future Perspectives
		5 Developing Biased Agonists at KOR: Methodological Insights, Controversial Aspects, and Future Challenge
		6 Conclusions
		References
Part III: Model Systems to Studying Opioid Receptor-Mediated Functions
	Chapter 17: Mechanical Nociception in Mice and Rats: Measurement with Automated von Frey Equipment
		1 Introduction
		2 Material
			2.1 Apparatus
			2.2 Reagents
			2.3 Animals
		3 Methods
			3.1 Calibrate the Dynamic Plantar Aesthesiometer
			3.2 Carrying Out the Assay
		4 Notes
		References
	Chapter 18: Evaluation of Murine Macrophage Cytokine Production After In Vivo Morphine Treatment
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Drugs and Substances
			2.3 Macrophage Cultures
			2.4 ELISA for Cytokine Measurements
		3 Methods
			3.1 Treatment
			3.2 Harvest of Peritoneal Macrophages
			3.3 IL-1beta ELISA
		4 Notes
		References
	Chapter 19: Measurement of Macrophage Toll-Like Receptor 4 Expression After Morphine Treatment
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Drugs and Substances
			2.3 Setup of Macrophage Cultures
			2.4 RNA Expression Analysis
				2.4.1 RNA Extraction
				2.4.2 Genomic DNA Digestion
				2.4.3 RNA Quantification
		3 Methods
			3.1 RNA Extraction
			3.2 Genomic DNA Digestion and RNA Quantification
			3.3 Reverse Transcription
			3.4 Real-Time PCR
			3.5 Example of TLR4 mRNA Expression Analysis
		4 Notes
		References
Part IV: Behavioral Effects Mediated by Opioid Receptors
	Chapter 20: Conditioned Place Preference (CPP) in Rats: From Conditioning to Reinstatement Test
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Drugs
			2.3 Apparatus
		3 Methods
			3.1 Pretest Session
			3.2 Conditioning Sessions
			3.3 Test Session
			3.4 Extinction Sessions
			3.5 Reinstatement Test
		4 Notes
		References
	Chapter 21: Analysis of Opioid-Seeking Behavior Through the Intravenous Self-Administration Reinstatement Model in Rats
		1 Introduction
		2 Intravenous Heroin Self-Administration in  Rats
			2.1 Materials
			2.2 Surgery for Implantation of Venous Catheters
			2.3 Apparatus
		3 Methods
			3.1 Intravenous Self-Administration Training
			3.2 Acquisition
			3.3 Maintenance
			3.4 Extinction
			3.5 Heroin-Seeking Reinstatement
			3.6 Drug Primings
			3.7 Cue Priming
			3.8 Stress Primings
			3.9 Factors Modulating Heroin-Seeking Reinstatement
		4 Notes
		References
	Chapter 22: Induction of a High Alcohol Consumption in Rats and Mice: Role of Opioid Receptors in Rats and Mice
		1 Introduction
		2 Materials
			2.1 Housing Condition
			2.2 Equipment
		3 Methods
			3.1 Protocol for Animals with High Alcohol Consumption
				3.1.1 Forced alcohol drinking (4-7 days)
				3.1.2 Free-choice alcohol drinking
			3.2 Measurements from the Drinking Bottles Are Taken Daily at the Same Time
				3.2.1 Limited-access drinking
				3.2.2 Limited daily access
			3.3 Protocol for Unselected Heterogeneous Animals
				3.3.1 Two-Bottle Free Choices
				3.3.2 Limited Access
			3.4 Outcome Measure for Induction of High Alcohol Consumption
		4 Notes
		References
	Chapter 23: Early-Life Stress as a Probe to Study the Opioid System in Developing Rodents
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparatory Procedures
			3.2 Beginning of Postnatal Stress Procedures
			3.3 End of Postnatal Stress Procedures
			3.4 Tail-Flick Test
			3.5 Pathological Phenotype Obtainment
		4 Notes
		References
	Chapter 24: Evaluation of μ-Opioid System Functionality in Mouse Pups: Ultrasonic Vocalizations as an Index of Infant Attachme...
		1 Introduction
			1.1 Response to Separation from the Mother in the Mouse: Ultrasonic Vocalizations
			1.2 Experimental Conditions
			1.3 Behavioral Testing Procedure
			1.4 USV Analysis
		2 Materials
			2.1 Animals
			2.2 USV Recording System
			2.3 Soundproof-Attenuated Environment
			2.4 Pup´s Container
		3 Methods
		4 Notes
		References
Index