Contemporary Approaches to the Study of Pain: From Molecules to Neural Networks

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Single-Cell RNA Sequencing of Somatosensory Neurons
1 Introduction
2 Preparations
2.1 Materials
2.1.1 Consumables
2.1.2 Surgery Tools
2.1.3 Chemicals
2.1.4 Equipment
2.2 Solutions
2.2.1 Stock Solution: 4x Concentrated Pre-cutting Solution (Pre-CS) in 1 l
2.2.2 Cutting Solution (CS) 100 ml (Prepare Shortly Before Use; see Chapter 5, Note 1)
2.2.3 Enzymes (Prepare Shortly Before Use to the Following Concentrations; see Chapter 5, Note 2)
2.3 Tissue Isolation
2.3.1 Animals
2.3.2 Surgery/Dissection Tools
2.3.3 Polish Pasteur Pipettes
3 Method
3.1 Dissection
3.1.1 Spinal Cord Removal
3.1.2 DRG Removal
3.1.3 Dorsal Horn Microdissection
3.1.4 Ganglia Microdissection
3.2 Dissociation
3.2.1 Dorsal Horn Dissociation
3.2.2 Ganglia Dissociation
3.3 Removal of Cell Debris
3.3.1 Dorsal Horn Gradient
3.3.2 Ganglia Gradient
4 Experimental Approach
4.1 Before the Start
4.1.1 What Is the Biological Question I Want to Answer?
4.1.2 How Many Cells Can I Collect from a Sample?
4.1.3 How Sensitive Are the Cells or Tissues with Respect to the Dissociation?
4.1.4 What Is my Budget?
4.2 scRNA-Seq Platforms
4.2.1 Quantification
Full-Length Transcript Methods
Tag-Based Methods
4.2.2 Cell Capturing
Low Throughput
High Throughput
4.3 Single Cell Vs. Single Nucleus Sequencing
4.4 scRNA-Seq Analysis
4.4.1 Data Processing
Quality Control
Software
Hardware
4.4.2 In Vivo Confirmation
5 Notes
References
Chapter 2: Preparation of Human and Rodent Spinal Cord Nuclei for Single-Nucleus Transcriptomic Analysis
1 Introduction
2 Materials
2.1 Buffer Preparation
2.1.1 General Comments
2.2 Workspace Decontamination
2.3 Personal Protective Equipment (PPE)
2.4 DEPC-Treated Water
2.5 DAPI Solution
2.5.1 Stock Solution
2.5.2 DAPI Work Solution
2.6 10 mM DTT Buffer
2.7 10% BSA
2.8 NRB with DAPI
2.9 Equipment Preparation
2.9.1 Surface Decontamination
2.10 Dounce Homogenizer Cleaning and Preparation
2.10.1 Decontamination
2.10.2 Washing
2.11 Centrifuge
3 Methods
3.1 Tissue Collection and Sample Factors
3.2 Nuclei Isolation
3.3 Tissue Preparation (Human)
3.4 Homogenization
3.5 Fluorescence Activated Nuclear Sorting (FANS)
3.5.1 Preparation of Collection Tubes and Media for Downstream Applications
3.6 Preparation of Collection Tubes for Bulk RNA-Seq
3.7 Preparation of Collection Tubes for 10x Chromium 3′ RNA-Seq v3
3.8 FANS Instrument Setup
3.9 Gating Strategy
3.10 10x Chromium 3′ RNA-Seq Assay
3.11 Bulk RNA-Seq
4 Notes
References
Chapter 3: Multiplex In Situ Hybridization of the Primate and Rodent DRG and Spinal Cord
1 Introduction
1.1 Technology
2 Materials
3 Method
3.1 Tissue Preparation
3.2 Before Starting the Assay
3.3 Pretreatment
3.4 Amplification
3.5 Preparation of the TSA Fluorophores
4 Immunohistochemistry
4.1 Materials
4.2 Method
5 Quantification
6 Notes
References
Chapter 4: Using Translating Ribosome Affinity Purification (TRAP) to Understand Cell-Specific Translatomes in Pain States
1 Introduction: Why Use TRAP in Pain Research?
1.1 Transcriptomes Do Not Equal Translatomes: mRNA Levels Do Not Always Equate to Protein Levels
1.2 History of Development of the TRAP Method, Advantages, and Limitations
1.3 Transgenic Mice Versus Viral Vector Approaches
1.4 TRAP Versus Whole Tissue Translation Profiling
1.5 TRAP Allows for the Study of Translation Regulation in Nociceptors in Vivo
2 Materials
2.1 Reagents
2.1.1 Magnetic Beads and Specific Antibodies
2.1.2 Ribosome Stalling Reagents/Protein Synthesis Inhibitors
2.1.3 Protein Solubilizing and Stabilizing Reagents
2.1.4 RNA-Related Reagents
2.1.5 Stock Solutions
2.2 Buffers
2.3 Equipment
2.4 Transgenic Animals: Nav1.8(het)-TRAP(EGFP-Homo) Mice
3 Methods
3.1 Protocol Optimization
3.2 Preparation of Beads and Antibodies (Affinity Matrix)
3.3 Tissue (DRG) Dissection
3.4 Tissue Homogenization
3.5 Incubation with Affinity Matrix
3.6 RNA Extraction
3.7 Direct-Zol RNA Extraction Kit Protocol
3.8 Downstream Processing
4 Notes
5 What Have We Learned from the TRAP Methodology?
References
Chapter 5: Ex Vivo Skin-Teased Fiber Recordings from Tibial Nerve
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 6: Single-Unit Electrophysiological Recordings of Primary Muscle Sensory Neurons Using a Novel Ex Vivo Preparation
1 Introduction
2 Materials
2.1 Recording Solutions
2.2 Dissection Dish and Surgical Equipment
2.3 Electrical Equipment
3 Methods
3.1 Intracardial Perfusion and Extraction of Target Tissues
3.2 Isolation of the Muscles, Nerves, DRGs, and Spinal Cord
3.2.1 Forepaw Dissection
3.2.2 Hind Limb
3.3 Transfer of the Preparation and Setup in the Recording Chamber
3.4 Recording Procedures, Stimulation Protocols, and Intracellular Staining
3.5 Limitations and Alternatives
4 Notes
References
Chapter 7: Electrophysiological Recording Techniques from Human Dorsal Root Ganglion
1 Introduction
2 Materials
3 Methods
3.1 Human DRG Recovery
3.2 Cleaning up the DRG
3.3 DRG Dissociation
3.4 Electrophysiological Recordings
3.4.1 Cleaning up the Central Nerve
3.4.2 Compound Action Potential Recordings
3.5 Calcium Imaging
3.6 Patch-Clamp Recording
4 Discussion
References
Chapter 8: Human Pluripotent Stem Cell-Derived Sensory Neurons: A New Translational Approach to Study Mechanisms of Sensitizat...
1 Introduction
2 The Generation of Human Sensory-like Neurons from Pluripotent Stem Cells
3 Chambers et al., 2012
3.1 Modifications and Amendments of Chambers et al.
4 Blanchard et al., 2015
5 Wainger et al., 2015
6 Schrenk-Siemens et al. 2015 and 2019
6.1 Generation of Mechanoreceptors
6.2 Generation of Nociceptor-like Neurons
6.3 Detailed Protocol for the Generation of Nociceptive-like Neurons
6.3.1 Formation of Neuroectodermal Spheres
Materials
6.3.2 Harvesting of Neural Crest-like Cells
6.3.3 Differentiation of NCLCs into Nociceptor-like Sensory Neurons
Materials
Preparations
6.3.4 Differentiation Procedure
Day -2
Day -1
Day 0
Day 1
Day 2
Day 3
Day 4: Splitting of the Progenitors
Day 5
Day 6-10
Day 11
Day 13
Day 14 till end
Day 26
7 In-Depth Characterization of hPSC-Derived Sensory-like Neurons
8 hiPSC-Derived Sensory-like Neurons in Research
8.1 hiPSC-Derived Neurons with Pain-Related Nav 1.7 Mutations
8.2 Patient-Derived iPSCs and their Role for Personalized Pain Therapies
9 Challenges and Limits Working with hPSC-Derived Sensory-like Neurons
10 Future Perspectives
References
Chapter 9: Intraspinal Transplantation of Precursors of Cortical GABAergic Interneurons to Treat Neuropathic Pain
1 Introduction
2 Materials
2.1 Mice
2.2 Euthanasia
2.3 Dissociation of MGE Cells
2.4 Laminectomy
2.5 MGE Cell Injection
3 Methods
3.1 Pretransplantation Steps
3.2 Transplantation Day
3.2.1 MGE Isolation and Dissection (~30-60 min)
3.2.2 MGE Cell Dissociation (~15 min)
3.2.3 Optional Step: Infection of MGE Cells with Lentivirus to Express cDNA (~1 h)
3.2.4 MGE Transplantation (~30-45 min per mouse)
Presurgical Steps
Surgery
3.3 Troubleshooting
3.3.1 No Cells Detected After Transplantation
3.3.2 MGE Cells Did Not Migrate After Transplantation
3.3.3 Behavioral Abnormalities Posttransplantation
4 Notes
References
Chapter 10: A Co-culture System for Studying Dorsal Spinal Cord Synaptogenesis
1 Introduction
2 Materials
2.1 Cell Culture Supplies
2.2 Immunofluorescence Staining, Imaging Acquisition, and Analysis
3 Method
3.1 Spinal Cord Neuron Isolation
3.2 DRG Neuron Isolation
3.3 Co-culture in Regular Culture Dishes (See Note 3)
3.4 Co-culture Using Campenot Chamber Cultures
3.5 Immunofluorescence Staining
3.5.1 For Regular Co-cultures
3.5.2 For Campenot Chamber Co-cultures
4 Notes
References
Chapter 11: Visualizing Synaptic Connectivity Using Confocal and Electron Microscopy: Neuroanatomical Approaches to Define Spi...
1 Introduction
2 Materials
2.1 Mammalian Ringer´s Solution
2.2 Phosphate Buffer (PB)
2.3 Phosphate-Buffered Saline (PBS)
2.4 Phosphate-Buffered Saline with Triton (PBST)
2.5 Tris-Buffered Saline with Triton (TBST)
2.6 Fixative Solutions
2.7 Resins
2.8 Reynold´s Lead (II) Citrate
3 Methods
3.1 Perfusion Fixation
3.2 Post-fixation Times
3.3 Tissue Preparation
3.4 Immunohistochemistry for Confocal Microscopy
3.5 Pre-embedding Immunohistochemistry for Electron Microscopy (Peroxidase Labeling)
3.6 Immunohistochemistry for Correlated Confocal and Electron Microscopy
3.7 Tissue Preparation for Conventional Resin Embedding
3.8 Post-embedding Immunogold Labeling
3.9 Tissue Preparation for Freeze-Substitution Resin Embedding
4 Notes
Appendix 1: Immunofluorescence Labeling of Sections of Spinal Cord
Appendix 2: Protocol for Pre-embedding Immunohistochemistry (Peroxidase Labeling) of Sections Spinal Cord
Appendix 3: Preparing Tissues for Correlated Confocal and TEM Labeling
Appendix 4: Resin Embedding Spinal Cord Tissues for Electron Microscopy
Appendix 5: Post-embedding Immunogold Labeling for Electron Microscopy
Appendix 6: Freeze-Substitution Resin Embedding for Electron Microscopy
References
Chapter 12: Using Viral Vectors to Visualize Pain-Related Neural Circuits in Mice
1 Introduction
2 Materials and General Strategies
2.1 Mice
2.2 Viruses and Strategies
2.2.1 AAV Vectors for Tracing Axonal Projections and Synaptic Terminals and Their Limitations
2.2.2 Viral Vectors for Labeling Neurons that Project to a Target Region (Retrograde-Lentivirus, CAV-2, and rAAV2-Retro)
2.2.3 Viral Vectors for Transsynaptic Tracing of Presynaptic Inputs to Desired Neurons
2.2.4 Anterograde Transneuronal Tracing with High-Titer AAV1-Cre
2.2.5 Activity-Dependent Circuit Tracing Using a Viral-Genetic Combination Approach
3 Detailed Surgical Procedures for Viral Vector Delivery
3.1 Surgical Apparatus and Tools
3.2 Reagents and Drugs for Surgery
3.3 Preparations for Surgery
3.4 Surgical Procedures
4 Concluding Remarks
References
Chapter 13: Recording Pain-Related Brain Activity in Behaving Animals Using Calcium Imaging and Miniature Microscopes
1 Introduction
2 Materials
3 Methods
3.1 Animals
3.2 Viral Injection and Expression of Calcium Indicators in Specific Brain Regions
3.3 GRINjector: Fabrication of Lens Probe Injection Device
3.4 Microendoscope Implantation
3.5 Verification of GCaMP Expression in Fixed Tissue
3.6 Verification of Microendoscope Implantation and GCaMP Expression in Awake, Behaving Mice
3.7 Miniature Microscope Baseplate Mounting
3.8 Miniature Microscope Ca2+ Imaging of Pain
3.8.1 Miniature Microscope Behavior Recording Hardware
3.9 Behavioral Apparatus for Noxious Stimuli Delivery
3.10 Recording Neural Activity During Delivery of Noxious Stimuli
3.11 Miniature Microscope Recording Parameters
3.12 Noxious and Aversive Stimuli Experiments
3.13 Analysis of Locomotor Behavior in the Open-Field Assay
3.14 Preprocessing of Ca2+ Imaging Data
3.15 Extraction of Neuron Shapes, Locations, and Activity Traces
3.15.1 Manual Neuron Identification
3.16 Basic Analysis of Noxious-Stimuli Responsive Populations
3.16.1 Analysis of the Overlap in Neural Ensembles Responsive to Different Stimuli
3.16.2 Decoding of Stimuli Based on BLA Neuron Activity
3.16.3 Analysis of Accelerometer Traces
3.17 Cross-Day Analysis of Neuronal Activity
4 Outlook and Conclusions
References
Chapter 14: Optical Imaging of the Spinal Cord for the Study of Pain: From Molecules to Neural Networks
1 Introduction
1.1 Principles of In Vivo Optical Imaging of the Spinal Cord
1.2 Example Preparation for In Vivo Spinal Cord Imaging
2 Materials
3 Method
3.1 Advantages of Spinal Cord Imaging Techniques
3.1.1 The Scope of Information Collected
3.1.2 The Range of Cell Types Studied
3.1.3 The Range of Structures Studied Simultaneously
3.1.4 A Flexible Scale of the Recording
3.2 Disadvantages of Spinal Cord Imaging Techniques
3.2.1 The Penetration Depth of Optical Signals
3.2.2 Movement Artifacts
3.2.3 To Observe Is to Change
Box 1 The Early Choices
3.3 The Choice of Optical Labels
3.4 What Promises Does In Vivo Imaging of the Spinal Cord Hold
4 Conclusion
References
Chapter 15: In Vivo Calcium Imaging of Peripheral Ganglia
1 Introduction
2 Materials
3 Methods
4 Conclusions
References
Chapter 16: Optogenetic Modulation of the Visceromotor Response to Reveal Visceral Pain Mechanisms
1 Introduction
2 Materials
2.1 Laser/Balloon Device Materials
2.2 Experimental Setup
2.3 Electrode Implantation/Balloon Insertion
2.4 EMG Recording of VMR
3 Methods
3.1 Constructing Balloon/Laser Device
3.2 Equipment Setup
3.3 Electrode Implantation/Balloon Insertion
3.4 EMG Recording of VMR
4 Notes
References
Chapter 17: Use of Optogenetics for the Study of Skin-Nerve Communication
1 Introduction
2 Materials
2.1 Animals
2.1.1 Keratinocyte-Sensory Afferent Communication Studies
2.1.2 Sensory Afferent-Immune Communication
2.2 Preparation of Hairy Skin Prior to Laser Stimulation
2.3 Lasers
2.4 Construction of Place Preference Boxes for Behavioral Assessment
2.4.1 Components of LED Array-Can Be Ordered from Environmental Lights, San Diego, California, Unless Stated Otherwise
2.4.2 Assembly of LED Platform
2.4.3 Components and Assembly of Place Preference Behavior Box
Components
Box Assembly
2.5 Data Analysis
3 Methods
3.1 Keratinocytes and Sensory Afferent Communication-Ex Vivo Analysis
3.1.1 Photostimulation of ChR2 Expressed by Keratinocytes to Evoke Sensory Afferent Firing
3.2 Keratinocytes and Sensory Afferent Communication-In Vivo Behavioral Analysis
3.2.1 Paw Withdrawal Latency as a Measure of Keratinocyte-Induced Behavioral Response
3.2.2 Place Preference Assay to Measure Behavioral Response to Light Stimulation of Keratinocytes
3.3 Immune System and Sensory Afferent Communication
3.3.1 Mouse Model
3.3.2 Mouse Dorsal Skin-Optogenetic Stimulation
3.3.3 Mouse Ear Skin-Optogenetic Stimulation
3.3.4 Cultured Dorsal Root Sensory Neurons-Optogenetic Stimulation
4 Notes
References
Chapter 18: Channelrhodopsin-2 Assisted Circuit Mapping in the Spinal Cord Dorsal Horn
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment
2.3 Solutions
3 Methods
3.1 Characterization of ChR2 Recruitment
3.2 Excitatory Connections
3.3 Inhibitory Connections
3.4 Monosynaptic Versus Polysynaptic
4 Notes
References
Chapter 19: Production of AAVs and Injection into the Spinal Cord
1 Introduction
1.1 AAV Vector Production
2 Materials
2.1 HEK 293T Cells
2.2 Plasmid DNA
2.3 PEI-Mediated Split Transfection of HEK 293T Cells
2.4 Harvesting and Processing HEK 293T Cells and Supernatant Posttransfection
2.5 Discontinuous Iodixanol Density Gradient Ultracentrifugation
2.6 Diafilstration
2.7 Physical Titer Quantification
2.8 Identity Check
3 Methods
3.1 HEK 293T Cells
3.1.1 Starting Adherent HEK 293T Cells from Frozen Stocks
3.1.2 Passaging HEK 293T Cells
3.2 Plasmid DNA
3.3 PEI-Mediated Split Transfection of HEK 293T Cells
3.3.1 Plasmid DNA and PEI Solutions
3.3.2 Split Transfection of HEK 293T Cells
3.4 Harvesting and Processing of HEK 293T Cells and Their Supernatants Posttransfection
3.5 Discontinuous Iodixanol Density Gradient Ultracentrifugation
3.5.1 Preparing the Crude and Cleared Cell Lysates
3.5.2 Preparing the Resuspended Pellet of the PEG 8000 Precipitated Supernatant for Ultracentrifugation
3.5.3 Preparing the Discontinuous Iodixanol Density Gradients
3.5.4 Ultracentrifugation
3.5.5 Collecting the AAV Vectors
3.6 Diafiltration
3.7 Physical Titer Quantification
3.7.1 Measuring Genomic AAV Vector DNA Concentration
3.7.2 Calculations
3.8 Quality Controls
3.8.1 Identity Check
4 Notes
5 Intraspinal Injections
5.1 Materials
5.1.1 Equipment
5.1.2 Surgical Tools
5.1.3 Consumables
6 Methods
6.1 Intraspinal Injections
6.1.1 Preparation for the Surgery
6.1.2 Surgical Exposure of the Spinal Cord
6.1.3 Intraspinal Injection
6.1.4 Completion of the Surgery
7 Notes
References
Chapter 20: Use of Intraspinally Delivered Chemogenetic Receptor, PSAM-GlyR, to Probe the Behavioral Role of Spinal Dorsal Hor...
1 Introduction
1.1 ``Minimally Invasive´´ Intraspinal Injection
2 Materials
2.1 Reagents
2.2 Surgical Tools
2.3 Equipment
3 Methods
3.1 Custom Stereotaxic Injection Setup (Optional)
3.2 Preparation for the Surgery
3.3 Preparation for the Stereotaxic Injector
3.4 Surgical Procedures to Expose the Spinal Cord Segment
3.5 Intraspinal Injection
3.6 Post-operative Care of Animals
4 Notes
5 Chemogenetics to Assess the Behavioral Role of Dorsal Horn Neurons
5.1 Materials
5.2 Methods
5.2.1 Acclimation Prior to Behavior
5.2.2 Acclimation Period for Cotton Swab and von Frey
5.2.3 Acclimation Period for Hargreaves
5.3 Control Experiments for Off-target Behavioral Effects of the Ligand
5.3.1 Cotton Swab and von Frey Assay
5.3.2 Hargreaves Thermal Assay
5.4 Baseline Measurements
5.4.1 Cotton Swab and von Frey Assay
5.4.2 Hargreaves Thermal Assay
5.4.3 Acetone Evaporation Test
5.5 Notes
6 Persistent Pain Models
6.1 Materials
6.1.1 Spared Nerve Injury Neuropathic Pain Model
6.1.2 Complete Freund´s Adjuvant Inflammatory Pain Model
6.2 Methods
6.2.1 Spared Nerve Injury Neuropathic Pain Model
6.2.2 Complete Freund´s Adjuvant Inflammatory Pain Model
7 Validation of Viral Injection with Immunohistochemistry
7.1 Materials
7.2 Methods
References
Chapter 21: Measuring Mouse Somatosensory Reflexive Behaviors with High-Speed Videography, Statistical Modeling, and Machine L...
1 Introduction
2 Materials
2.1 Animals
2.2 Somatosensory Stimuli
2.3 Testing Platform and Holding Chambers
2.4 High-Speed Camera
3 Methods
3.1 Performing Somatosensory Behaviors
3.2 Scoring Subsecond Withdrawal and Escape Behaviors
3.3 Transforming Data into a Single Dimension with Principal Component Analysis
3.4 Supervised Machine Learning to Make Predictions about Pain-like Probabilities
3.5 Application of our Method to Study VFH and Peripheral Optical Stimuli Triggered Paw Withdrawal Reflexes
4 Discussion
References
Index