Melatonin: Methods and Protocols

Preface
Contents
Contributors
Part I: Methods for the Detection of Melatonin and Its Biological Derivatives
Chapter 1: Two-Dimensional Thin Layer Chromatography of Melatonin and Related Compounds
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 2: Detection of Melatonin in Tissue Samples
1 Introduction
2 Materials
2.1 Tissue Dissection and Melatonin Extraction
2.2 [125I]-Iodine Melatonin Labeling
2.3 Radioimmunoassay (RIA)
3 Methods
3.1 Melatonin Extraction from Brain Tissue Dissections
3.2 Melatonin Extraction
3.3 Extraction Yield Measurement
3.4 [125I]-Iodine Melatonin Labeling
3.5 Quantification of Extracted Melatonin by Radioimmunoassay (RIA)
3.6 Data Analysis
4 Notes
References
Chapter 3: Measuring Dim Light Melatonin Onset in Humans
1 Introduction
2 Materials
2.1 General Requirements
2.2 Collection Tubes
2.3 Melatonin Assay Kits
3 Methods
3.1 General Requirements for Sample Collection
3.2 Plasma Sample Collection
3.3 Saliva Sample Collection
3.4 Melatonin Assay Results
3.5 Threshold-Based DLMO Analysis
4 Notes
References
Chapter 4: Measuring Urinary 6-Sulphatoxymelatonin in Humans
1 Introduction
2 Materials
3 Methods
3.1 Urine Sample Collection
3.2 aMT6s Assay Data Analysis
3.3 Cosinor Analysis
4 Notes
References
Chapter 5: MEL-Index: Estimation of Tissue Melatonin Levels Using Gene Expression Data
1 Introduction
2 Material
3 Methods
4 Notes
References
Chapter 6: Melatonin Synthesis Enzymes Activity: Radiometric Assays for AANAT, ASMT, and TPH
1 Introduction
2 Materials
2.1 Arylalkylamine-N-Acetyltransferase (AANAT) Activity
2.2 Acetylserotonin O-Methyltransferase Activity
2.3 Tryptophan Hydroxylase Activity
2.4 Materials and Equipment
3 Methods
3.1 Arylalkylamine-N-Acetyltransferase (AANAT) Activity Assay
3.2 Acetylserotonin O-Methyltransferase (ASMT) Activity Assay
3.3 Tryptophan Hydroxylase (TPH) Activity Assay
4 Notes
References
Chapter 7: Pinealectomy in Rats
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 8: Surgical Techniques and Nuances for Superior Cervical Ganglionectomy and Decentralization in Rats
1 Introduction
2 Materials
2.1 Animal Procedures
2.2 Surgery
3 Method
3.1 Preoperative Setup
3.2 Incision and Salivary Gland Dissection
3.3 Superior Cervical Ganglionectomy
3.4 Decentralization of the Sympathetic Trunk
3.5 Denervation of the Internal and/or External Carotid Nerves
3.6 Closure and Postoperative Care
3.7 Verification of Surgical Performance
4 Notes
References
Chapter 9: Pineal Microdialysis
1 Introduction
2 Materials
2.1 Probe Manufacturing
2.2 Probe Implantation
2.3 Sample Assembly and Collection
2.4 Verification of Probe Implantation
3 Methods
3.1 Probe Manufacturing (See Note 1)
3.2 Probe Implantation
3.3 System Assembly and Sample Collection
3.4 Verification of Probe Implantation Through Histology
4 Notes
References
Chapter 10: Radiochemical In Situ Hybridization in Developmental Studies of the Pineal Gland
1 Introduction
2 Materials
3 Methods
3.1 Preparing the Developing Brains for Histology
3.1.1 Removing, Fixing, and Freezing the Tissue
3.1.2 Cutting Cryostat Sections and Identifying the Pineal Gland
3.2 Preparing the DNA Probe
3.2.1 Design of the DNA Oligo Probe
3.2.2 Labeling of the DNA Oligo Probe
3.2.3 Purification of the DNA Oligo Probe
3.3 The Hybridization Procedure
3.3.1 Slide Prehybridization
3.3.2 Hybridization
3.3.3 Washing
3.4 Detection and Quantification of the Hybridization Signal
3.4.1 Exposure and Development of the X-Ray Film
3.4.2 Exposure and Development of the X-Ray Film
4 Notes
References
Chapter 11: Pineal Cells Dissociation and Culture: Isolated Pinealocytes, Isolated Astrocytes, and Co-culture
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Dissociation
2.3 Cell Culture Characterization
3 Methods
3.1 Pinealocytes Dissociation and Culture
3.2 Astrocytes Separation and Culture
3.3 Pinealocytes and Astrocytes Co-culture
3.4 Cell Culture Characterization by Immunocytochemistry
4 Notes
References
Chapter 12: Pineal Gland Culture
1 Introduction
2 Materials
2.1 General Material
2.2 Culture Medium
3 Methods
4 Notes
References
Chapter 13: Preparation of Single Pineal Cells
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 14: RNA Sequencing of Single Pineal Cells
1 Introduction
2 Materials
3 Method
3.1 Generation and Barcoding of GEMs (See Note 1)
3.2 Clean GEM-RT and Amplification of cDNA
3.3 Construct 3′ Gene Expression Library
3.4 Biometric Analysis
4 Notes
References
Chapter 15: siRNA-Mediated Downregulation of Gene Expression in Cultured Rat Pineal Cells
1 Introduction
2 Materials
2.1 Pineal Gland Dissection
2.2 Pineal Medium
2.3 Dissociation and Isolation of Pineal Cells
2.4 siRNA Transfection
2.5 Stimulation of Pineal Cells
3 Methods
3.1 Pineal Medium
3.2 Pineal Cell Culture
3.2.1 Preparing the Papain Dissociation Reagents
3.2.2 Extracting the Pineal Gland
3.2.3 Dissociating the Pineal Cells (See Note 8)
3.3 siRNA Treatment of Pineal Cells
3.4 Pharmacological Stimulation of Pineal Cells
4 Notes
References
Chapter 16: Analysis of the Human Pineal Proteome by Mass Spectrometry
1 Introduction
2 Materials
2.1 Postmortem Samples
2.2 Protein Extraction
2.3 Protein Digestion
2.4 Filter-Aided Sample Preparation (FASP)
2.5 LC-MS/MS Analysis
3 Methods
3.1 Postmortem Sample Preparation
3.2 Protein Digestion
3.3 LC-MS/MS Analysis
3.4 Data Analysis
3.5 Imputation of Missing Values
3.6 Detection of Rhythmic Protein Abundance
3.7 Gene Ontology Analyses
3.8 Protein-Protein Interaction Visualization
3.9 Data Sharing Statement
4 Notes
References
Chapter 17: The Pineal Transcriptome Webpage
1 Introduction
2 The URL of the Website
3 Organization of Website Subpages
4 Notes
References
Part II: Methods to Measure Melatonin Effects at the Molecular and Cellular Level
Chapter 18: 2-[125I]iodomelatonin and [3H]melatonin Binding Assays for Melatonin Receptors
1 Introduction
2 Materials
2.1 For Membrane Preparation
2.2 For Binding Assay
3 Methods
3.1 Membrane Preparation
3.2 Saturation Binding Assay
3.3 Kinetic Binding Assay
3.4 Competition Binding Assay
3.5 Data Analysis
4 Notes
References
Chapter 19: Alternative Ligands at Melatonin Receptors
1 Background
2 New Ligands
3 Syntheses of New Radioligands
4 Pharmacology of the New Ligands
4.1 Binding of 2-[125I]Iodo- and [3H]-MLT at Human Receptors
4.2 Characteristics at Human Receptors as Radioligands
4.3 The Special Mention for [3H]-MLT
4.4 Binding in Physiological Conditions and Use in Autoradiography
5 Biasism: The Agonist/Antagonist Questions
6 Conclusions
References
Chapter 20: GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue
1 Introduction
2 Materials
2.1 Membrane Preparation
2.2 Binding
3 Methods
3.1 Membrane Preparation
3.2 [35S]GTPγS Binding Assay by Immunoprecipitation
4 Notes
References
Chapter 21: MT1 Melatonin Receptor Reconstitution in Nanodiscs
1 Introduction
2 Material
2.1 Membrane Preparation
2.2 MT1 Receptor Purification
2.3 Nanodisc Assembly
2.4 Gi Protein Activation Assay (GTPγS) (See Note 4)
3 Methods
3.1 Membrane Preparation
3.2 MT1 Receptor Solubilization and Purification
3.3 Nanodisc Assembly and Purification
3.4 Gi Protein Activation Assay
4 Notes
References
Chapter 22: Functional Investigation of Melatonin Receptor Activation by Homogenous cAMP Assay
1 Introduction
2 Material
2.1 DNA Constructs
2.2 Cell Culture and Transfection
2.3 cAMP Assay
3 Methods
3.1 Cell Culture
3.2 Cell Transfection
3.3 Activation of Melatonin Receptors and cAMP Assay
3.4 Data Analysis
4 Notes
References
Chapter 23: Functionality of Melatonin Receptors: Internalization
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 24: Functionality of Melatonin Receptors: Recruitment of β-Arrestin at MT1
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 25: MT1 Receptor Signaling Pathways by Impedance Measurement
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 26: Measuring Protein-Protein Interactions of Melatonin Receptors by Bioluminescence Resonance Energy Transfer (BRET)
1 Introduction
2 Materials
2.1 Cells and Transfection
2.2 Luminescence, Fluorescence, and BRET-Ratio Measurements
3 Methods
3.1 Transfection and Preparation of Cells
3.2 BRET Measurement
3.3 Analysis
4 Notes
References
Chapter 27: Nonradioactive In Situ Hybridization of MT1 Melatonin Receptor for the Identification of Melatonin Target Cells
1 Introduction
2 Materials
2.1 Tissue Sections
2.2 RNAse-Free Technique
2.3 Reagents, Buffers, and Stock Solutions
2.4 Digoxigenin-Labeled Riboprobe
2.5 Equipment
3 Methods
3.1 Nonradioactive In Situ Hybridization: Standard Protocol
3.2 Nonradioactive In Situ Hybridization: Post-Hybridization RNAse Treatment (Optional)
3.3 Nonradioactive In Situ Hybridization: Adaptations for Frozen Sections
3.4 Nonradioactive In Situ Hybridization: Adaptations for Floating Sections
3.5 Adaptations for Radioactive In Situ Hybridization
3.6 Controls
4 Notes
References
Chapter 28: Beta-Galactosidase as a Transgenic Reporter for the Mapping and Phenotyping of MT1 and MT2 Melatonin Receptor-Expr...
1 Introduction
2 Materials
2.1 Animals
2.2 Reagents, Buffers, and Stock Solutions for LacZ Enzyme Histochemistry
2.3 Materials for LacZ Enzyme Histochemistry
2.4 Reagents, Buffers, and Stock Solutions for LacZ Immunodetection
2.5 Tissue Sections for LacZ Immunodetection
2.6 Materials for Floating Section Immunohistochemical LacZ Detection
3 Methods
3.1 Histochemical Detection of Beta-Galactosidase (LacZ) Enzyme Activity
3.2 Immunohistochemical Detection of Transgenic Beta-Galactosidase (LacZ) Protein
3.3 Controls
4 Notes
References
Chapter 29: Why Are We Still Cloning Melatonin Receptors? A Commentary
1 Introduction
2 Historical Background
3 Cloning History
4 Reported Sequences for Melatonin Receptors
4.1 MT1
4.2 MT2
4.3 Mel1c and GPR50
4.4 Mel1d
4.5 The Case of the Insects
5 Difficulties?
6 Why Still Trying to Clone (in the MLT Receptor Field)?
6.1 Pharmacology
6.2 Stability
6.3 Understanding Evolution, a Glimpse at the Case of GPR50/Mel1c in Mammals
7 Conclusion
References
Chapter 30: Measuring Binding at the Putative Melatonin Receptor MT3
1 Introduction
2 Materials
2.1 Brain Homogenate Preparation
2.2 Binding Experiment Setup
3 Methods
3.1 Preparation of Biological Source Homogenates
3.2 Binding Assay
4 Notes
References
Chapter 31: Cloning, Expression, Purification, Crystallization, and X-Ray Structural Determination of the Human NQO2 in Comple...
1 Introduction
2 Materials
2.1 Media and Antibiotics
2.2 Nucleic Acid Electrophoresis
2.3 Amplification of the Human NQO2 Gene Open Reading Frame (ORF) by PCR
2.4 Preparation of Linearized DNA Vector
2.5 Cloning of hNQO2 into pET23d Expression Vector
2.6 Colony PCR to Confirm Recombinant Clones
2.7 Recombinant hNQO2 Protein Expression in Escherichia coli
2.8 Purification of Recombinant hNQO2 Protein by Chromatographic Methods
2.9 Crystallization of Recombinant hNQO2 Protein in Complex with Melatonin
3 Methods
3.1 Amplification of the Human NQO2 Gene ORF by PCR
3.2 Preparation of the Linearized DNA Vector
3.3 Cloning of hNQO2 into pET23d Expression Vector
3.4 Colony PCR to Confirm Recombinant Clones
3.5 Recombinant hNQO2 Protein Expression in Escherichia coli
3.6 Purification of Recombinant hNQO2 Protein by Chromatographic Methods
3.7 Crystallization of Recombinant hNQO2 Protein in Complex with Melatonin
3.8 Data Collection and Structure Determination
4 Notes
References
Chapter 32: Melatonin Binding to Human NQO2 by Isothermal Titration Calorimetry
1 Introduction
2 Materials
2.1 Reagents and Apparatus
2.2 Buffers and Sample Preparation
3 Methods
4 Notes
References
Chapter 33: Measurement of NQO2 Catalytic Activity and of Its Inhibition by Melatonin
1 Introduction
2 Materials
3 Methods
3.1 Catalytic Activity
3.2 Measurement of the Inhibition by Melatonin
4 Notes
References
Chapter 34: Measuring the NQO2: Melatonin Complex by Native Nano-Electrospray Ionization Mass Spectrometry
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Mass Spectrometry Settings
3.3 Native MS Analysis of Deflavo-NQO2
3.4 Native MS Analysis of FAD-Saturated NQO2
3.5 Native MS Analysis of Melatonin Binding to Deflavo-NQO2
3.6 Native MS Analysis of Melatonin Binding to FAD-Saturated NQO2
4 Notes
References
Chapter 35: Human and Rodent Cell Lines as Models of Functional Melatonin-Responsive Pancreatic Islet Cells
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Cell Culture Medium
2.3 Chemicals for Incubation Experiments
2.4 Chemicals and Kits for Real-Time RT-PCR, Western Blot and Immunocytochemistry
2.4.1 Real-Time RT-PCR
2.4.2 Western Blot
2.4.3 Immunocytochemistry
2.5 Kits for the Analysis of Incubation Experiments
2.5.1 ELISA
2.5.2 Radioimmunoassay (RIA)
3 Methods
3.1 Cell Culture
3.2 Preparation of Cells for Batch Experiments
3.2.1 α-Cells
3.2.2 β-Cells
3.2.3 δ-Cells
3.3 Batch Experiment Protocols with Melatonin Alone or in Combination with Other Substances Described for Each Cell Line
3.3.1 α-Cells
3.3.2 β-Cells
3.3.3 δ-Cells
3.4 Analysis of Supernatants from Batch Experiments
3.5 Isolation of Cell Pellets from Batch Experiments
3.6 Analysis of Gene Expression with PCR
3.7 Protein Analysis with Western Blot
3.8 Use of Cells for Immunocytochemistry
3.9 Transient Transfection Experiments Employing δ-Cells
4 Notes
References
Chapter 36: Mesenchymal Stem Cell and Monocyte Co-cultures
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Reagents and Supplies
2.3 Equipment and Software
3 Methods
3.1 Culture Initiation
3.2 Transwell Co-cultures
3.3 Isolation of Mononuclear Cells from Blood
3.4 Isolation of Monocytes from Fresh Mononuclear Cells
3.5 Endpoint Analyses: Osteoblast and Osteoclast Differentiation and Activity
3.5.1 Osteoblast Assays: Qualitative and Quantitative Analyses
3.5.2 Osteoclast Assays: Qualitative and Quantitative Analyses
3.5.3 Measurement of Secreted Osteoprotegerin and RANKL
4 Notes
References
Part III: Methods to Measure Melatonin Effects In Vivo
Chapter 37: Real-Time Monitoring of Circadian Rhythms in the Eye
1 Introduction
2 Materials
3 Methods
3.1 Measurement of PER2::LUC Rhythm
3.2 Analysis of PER2::LUC Rhythm
4 Conclusion
5 Notes
References
Chapter 38: Assessing the Role of Melatonin in the Modulation of Visual Functions in the Mouse
1 Introduction
2 Materials
3 Methods
3.1 Melatonin Administration and Dark Adaptation 1 h Prior to ERG Measurement
3.2 Pupillary Dilation 30 min Prior to ERG Measurement (30 min After Dark Adaptation)
3.3 Anesthesia < 3 min Prior to ERG Measurement
3.4 Recording Preparation
3.5 ERG Recording
3.6 ERG Wave Form Analysis
4 Conclusions
5 Notes
References
Chapter 39: Methods to Assess Melatonin Receptor-Mediated Phase-Shift and Re-entrainment of Rhythmic Behaviors in Mouse Models
Abbreviations
1 Introduction
1.1 Melatonin and Its Receptors
1.2 Modulation of Rhythmic Behaviors by Melatonin Receptor Agonists
1.3 Phase-shift of Circadian Activity Rhythms by Melatonin Receptor Ligands
1.4 Modulation of Re-entrainment by Melatonin Receptor Ligands
2 Materials
2.1 Facilities
2.2 Equipment
2.3 Data Acquisition
2.4 Measurement of Environmental Conditions
2.5 Circadian and Zeitgeber Time
2.6 Animal Information
2.7 Drug Preparation
3 Methods
3.1 Phase-Shift of Circadian Activity Rhythms Protocol
3.2 Eastbound and Westbound Jet Lag Re-entrainment Paradigm Protocols
4 Notes
References
Chapter 40: Neurotransmission Recovery by Melatonin Measured by CMAP
1 Introduction
2 Materials
3 Methods
3.1 Compression of the Sciatic Nerve
3.2 CMAP Recordings
3.3 Data Analysis
4 Notes
References
Chapter 41: Olfaction and Melatonin: The Use of the Olfactory Discrimination Test
1 Introduction
2 Materials
2.1 Animals
2.2 Zinc Gluconate Infusion
2.3 Olfactory Discrimination Test
3 Methods
3.1 Zinc Gluconate Infusion (for a Positive Control of Hyposmia)
3.2 Isolation Period
3.3 Habituation Period
3.4 Olfactory Discrimination Test
3.5 Analyzing the Test
4 Notes
References
Chapter 42: Photothrombotic Mouse Models for the Study of Melatonin as a Therapeutic Tool After Ischemic Stroke
1 Introduction
2 Materials
3 Methods
3.1 Photothrombotic Stroke Model
3.2 Anesthesia and Surgical Preparation
3.3 Surgical Operation for Illumination of Target Area
3.4 Postsurgical Preparation
4 Notes
References
Chapter 43: Hypnotic Effects of Melatonergic Compounds Measured in Mice or Rats
1 Introduction
2 Materials
3 Methods
3.1 Anesthetize the Animal
3.2 EEG and EMG Electrode Implant
3.3 EEG/EMG Recordings
3.4 Analysis of the Sleep Stages
4 Notes
References
Chapter 44: Acute and Chronic Pain Preclinical Models to Study the Analgesic Properties of Melatonergic Compounds
1 Introduction
2 Materials
3 Methods
3.1 Spared Nerve Injury (SNI) Model
3.2 Anesthesia and Surgical Preparation
3.3 Surgical Procedure
3.4 Postsurgical Recovery
3.5 Experimental Paw Withdrawal Threshold Measurement
3.6 Measure of Chronic Pain Using Von Frey Test
3.7 Measure of Acute Pain Using the Hot Plate Test
4 Notes
References
Chapter 45: Behavioral and Fluorescent-Based Immunohistochemistry Protocols for Examining Antidepressant-Like Effects of Melat...
1 Introduction
2 Materials
2.1 Solutions
2.2 Intracardiac Perfusion
2.3 Immunofluorescence of Free-Floating Brain Sections
2.4 Experimental Animals
2.5 Devices for Behavioral Tests
3 Methods
3.1 Doses and Administration Schedules
3.2 Forced Swimming Test
3.3 Open Field Test
3.4 Videotaping of Antidepressant- Like Behavior and Locomotor Activity
3.5 Brain Obtention and Fixation for Immunohistochemistry Assay
3.6 Tissue Processing for Immunofluorescent Labeling
3.7 Data Analysis
4 Notes
References
Chapter 46: A Method for Perfusion of Tissue-Isolated Human Tumor Xenografts in Nude Rats to Investigate the Oncostatic Role o...
1 Introduction
2 Materials
2.1 Artificial Lung and Perfusion Apparatus
2.2 Surgical Equipment
3 Methods
3.1 Collection of Donor Blood
3.2 Tissue-Isolated Human Tumor Perfusions
4 Notes
References
Chapter 47: A Method for Growing Tissue-Isolated Human Tumor Xenografts in Nude Rats for Melatonin/Cancer Studies
1 Introduction
2 Materials
2.1 Animals
2.2 Surgical Equipment
3 Methods
3.1 Tissue-Isolated Tumor Implantation
4 Notes
References
Index