Developmental, Physiological, and Functional Neurobiology of the Inner Ear

Preface to the Series
Preface
Contents
Contributors
Part I: Experimental Manipulation of the Inner Ear
Chapter 1: Pou4f3DTR Mice Enable Selective and Timed Ablation of Hair Cells in Postnatal Mice
1 Historical Background
2 Traditional Methods for Inducing Hair Cell Death in Mice
2.1 Cochlear Hair Cells
2.2 Vestibular Hair Cells
3 Selective Cell Ablation in Mice Expressing the Human Diphtheria Toxin Receptor
4 Auditory Hair Cell Ablation Using Pou4f3DTR Mice
5 Vestibular Hair Cell Ablation Using Pou4f3DTR Mice
6 Methods
6.1 Mouse Breeding
6.2 DT Administration
References
Chapter 2: Cochlear Explant Cultures: Creation and Application
1 Introduction
1.1 Cochlear Structure
1.2 Cochlear Development
1.3 History of Cochlear Explants
2 Materials and Reagents
2.1 Equipment
2.2 Reagents
3 Methods
3.1 Dissection of the Embryonic Cochlear Duct
3.2 Cochlear Explants
3.3 Perturbation of Cochlear Development
4 Notes/Troubleshooting
References
Chapter 3: Immunohistochemistry and In Situ mRNA Detection Using Inner Ear Vibratome Sections
1 Introduction
2 Materials and Methods
2.1 Preparation of the Chicken and Mouse Utricle
2.2 Preparation of the Chicken Cochlea
2.3 Preparation of the Adult Mouse Cochlea
2.4 Tissue Embedding
2.5 Vibratome Sections
2.6 Vibratome Leica VT1200/S and Compresstome VF-310-0Z
2.7 Immunolabeling of Vibratome Sections
2.8 In Situ mRNA Detection on Vibratome Sections
2.8.1 Probe Synthesis
2.8.2 In Situ Hybridization and Hybridization Chain Reaction
2.9 Confocal Microscopy
3 Results
3.1 Chicken Utricle
3.2 Chicken Basilar Papilla
3.3 Mature Mouse Cochlea
4 Conclusions
References
Chapter 4: Genetic Manipulation of the Embryonic Chicken Inner Ear
1 Introduction
1.1 Loss-of-Function in Avian Embryos: The CRISPR/Cas9 Revolution
1.2 Final Recommendations
2 Material
2.1 Electroporation Setup
2.2 Fertilized Eggs and Incubators
2.3 Other Material and Reagents
3 Methods
3.1 Windowing of the Eggs and Preparation of the Embryo
3.2 In Ovo Electroporation
3.3 Common Issues and Their Solution
4 Notes
References
Chapter 5: Molecular Tools to Study Regeneration of the Avian Cochlea and Utricle
1 Introduction: A Damage Model to Eliminate All Cochlear Hair Cells
2 Materials
2.1 Chicken Husbandry
2.2 Materials: Surgical
2.3 Materials: Antibodies, EdU and BrdU Staining, and syGlass Quantitation
2.4 Materials: Cold Peeling and Lysis
3 Methods
3.1 Chicken Husbandry
3.2 Surgical Method: Pre-operation
3.3 Surgical Method: Infusion and Post-operation
3.4 Dose Considerations and Partial Damage Phenotypes
3.5 EdU Bioavailability and Proliferative Window: 3D Quantification
3.6 Cold Peeling of Chicken Cochlear Sensory Epithelium and Downstream Applications
References
Part II: Molecular Analysis of the Inner Ear
Chapter 6: An Efficient Method to Detect Messenger RNA (mRNA) in the Inner Ear by RNAscope In Situ Hybridization
1 Introduction
2 Materials
2.1 Animal Care and Housing
2.2 Reagents
2.3 Dissection Tools
2.4 Instruments
2.5 RNAScope Reagents
3 Methods
3.1 Sample Collection and Fixation
3.1.1 Embryonic Samples
3.1.2 Neonatal/Postnatal Samples
3.1.3 Adult Samples
3.2 Decalcification
3.3 Sample Preparation
3.3.1 Whole-Mount Tissue
3.3.2 Cryosections
3.3.3 Vibratome Sections
3.3.4 Zebrafish Embryos
3.4 RNAscope
3.4.1 Pretreatment
3.4.2 Hybridization and Signal Amplification
3.4.3 Color Reaction for Detection of a Single RNA of Interest by Brightfield or Fluorescence
3.4.4 Immunostaining and Mounting
3.5 Transcript Detection by Duplex Kit, Multiplex Fluorescent V2 Kit, and HiPlex Kit
4 Discussion
5 Conclusions
References
Chapter 7: A Manual Technique for Isolation and Single-Cell RNA Sequencing Analysis of Cochlear Hair Cells and Supporting Cells
1 Introduction
2 Materials
2.1 Equipment Required for the Described Surgical Procedure
3 Methods
3.1 Preparing the Micropipettes
3.2 Preparing Lysis Buffer
3.3 Performing the Dissection
3.4 Dissociating Cells of the Membranous Labyrinth
3.5 Harvesting Individual Cells
3.6 Record Keeping
3.7 Storage of Cells
3.8 Reverse Transcription
3.9 Library Preparation and Sequencing
4 Bioinformatics
4.1 Computing Environment
4.2 Expression Quantification
4.3 Expression Analysis
4.4 Quality Control
4.5 Normalization, Feature Selection, Scaling
4.6 Linear Dimensional Reduction
4.7 Clustering
4.8 Exploring Clusters
4.9 Quantification of Transcript Structure
5 Cares and Concerns
6 Conclusions
References
Headings0005225699
Chapter 8: Ribosomal Pulldown Assays and Their Use to Analyze Gene Expression in Multiple Inner Ear Cell Types
1 Introduction
2 Materials
2.1 The RiboTag Mouse Model
2.2 Selecting a Cre Recombinase Model
2.3 Materials and Tools for Collecting Mouse Inner Ear Tissues Followed by RiboTag RNA Extraction
2.3.1 Mouse Inner Ear Dissection
2.3.2 RiboTag RNA Extraction
2.4 Other Materials
3 Methods
3.1 Inner Ear Tissue Collection
3.1.1 Neonatal Mice
3.1.2 Adult Mice
3.2 Method for Ribosomal Immunoprecipitation and RNA Extraction from RiboTag Mouse Inner Ear Tissues (Fig. 4)
3.3 Assessing the Quality and Concentration of RiboTag IP and IN Samples
3.4 Testing the Efficiency of IP RNA Enrichment Using qPCR
3.4.1 Reverse Transcription
3.4.2 qPCR
3.5 Library Kit Selection and RNA-Seq
3.6 Data Analysis and Enrichment Factors
3.7 Validation
4 Conclusion
References
Part III: Hair Cell Function and Physiology
Chapter 9: In Vivo Analysis of Hair Cell Sensory Organs in Zebrafish: From Morphology to Function
1 Introduction
2 Materials and Methods
2.1 Propagation and Maintenance of Embryonic and Larval Zebrafish
2.2 Microinjection of Zebrafish Embryos to Investigate Gene Function
2.3 Viewing Gross Inner Ear Morphology and Auditory-Vestibular Behavior Under a Dissecting Scope
2.4 Mounting and Immobilizing Embryonic and Larval Zebrafish
2.5 Viewing and Imaging Zebrafish Hair Cell Epithelia
2.6 FM 1-43 Dye Labeling of Lateral Line Hair Cells
3 Results
3.1 Examination of Gross Development and Behavior
3.2 Closer Examination of the Zebrafish Inner Ear and Lateral Line
3.3 Subcellular Examination of Hair Cell Morphology
3.4 FM 1-43 Labeling of Hair Cells in the Lateral Line
4 Advanced In Vivo Methods: Moving Beyond the Methods Described Here
4.1 In Vivo Approaches to Study Hair Cell Activity
4.2 Advanced Behavioral and Optogenetic Approaches to Study Sensory System Function
4.3 Outlook and Future
5 Notes
References
Chapter 10: Electrophysiological Recordings of Voltage-Dependent and Mechanosensitive Currents in Sensory Hair Cells of the Au...
1 Introduction
2 Methods
2.1 Tissue Preparation (Acute and Culture)
2.1.1 Microdissection of the Neonatal Utricle for Electrophysiological Recordings
Organotypic Cultures
Materials
Solutions
2.1.2 Microdissection of the Organ of Corti for Electrophysiological Recordings
Dissection of the Neonatal Organ of Corti
Dissection of the Mature/Adult Organ of Corti (>P10)
Dissection of the Mature Apical Coil
Dissection of the Mature Basal/Middle Coil
2.2 Equipment
2.2.1 Electrophysiology
Vibration Isolation Table
Microscope
Recording Chamber
Camera
Patch-Clamp Amplifiers
Analog-to-Digital Signal Converter
Software
Filters, Power Supplies, and Oscilloscope
Micromanipulators
Perfusion Heating Devices and Heating and Cooling Platforms
2.2.2 Mechanical Stimulus: Stiff Glass Probes
Piezo Stack
Piezoelectric Bending Transducers
Calibration
Stiff Glass Probe
High Voltage Amplifiers
2.2.3 Mechanical Stimulus: Fluid Jet
Fluid Jet Design
Setting Up the Fluid Jet
Positioning the Fluid Jet
Delivery of the Stimulus
Calibration
Bundle Displacement
Rise Time
Commercial Pressure Clamp
2.2.4 Mechanical Stimulus: Kinocilium Coupled Probes
2.3 Electrophysiological Recording Procedures
2.3.1 General Approach
General Materials and Solutions Required
2.3.2 Voltage-Dependent Currents
Voltage Clamp
Current Clamp
2.3.3 Mechanosensitive Currents
Positioning the Stimulus Pipette
Resting Open Probability
Maximum Transduction Current and Operating Range
Kinetics of Activation and Adaptation
2.4 Additional Technical Tips
2.4.1 Dissection Tools
2.4.2 Perfusion
2.4.3 Tubing
2.4.4 Glass Fibers and Fire Polishing
2.4.5 Other Considerations
3 Conclusions
References
Chapter 11: Biophysical Recording from Adult Hair Cells
1 Introduction
2 Methods
2.1 Solutions and Recording Conditions
2.1.1 External Solution (in mM)
2.1.2 Internal Solution for the Patch Pipette Recording (in mM)
2.2 Further Notes
2.2.1 pH Buffering
2.2.2 Recording Temperature
2.2.3 Dye Loading with Patch Pipettes
2.2.4 Choice of Microscope
3 Dissection Protocols
3.1 Isolation of Hair Cells from the Organ of Corti of the Guinea Pig
4 In Situ Recording from the Mouse Cochlea
5 Subsidiary Recording Techniques
5.1 Measurement of Movements in Adult Cochlear Structures (OHCs, etc.)
5.2 Intracellular Calcium Measurements from IHCs
6 Conclusions
References
Chapter 12: Endocochlear Potential Measures, Local Drug Application, and Perilymph Sampling in the Mouse Inner Ear
Abbreviations
1 Introduction
2 Endocochlear Potential Recording
2.1 Rationale
2.2 Materials
2.2.1 Head Holder
2.2.2 Electrometer
2.2.3 Glass Microelectrodes
2.2.4 Audio Monitor
2.2.5 Micromanipulator/Microdrive
2.2.6 Surgical Instruments
2.2.7 Other Equipment
2.3 Detailed Methods
2.3.1 Initial Surgical Approach
2.3.2 Fenestration of the Cochlear Capsule
Repeated or Prolonged EP Measurements
Choices and Significance of EP Recording Location
2.4 EP Changes in Pathologic Conditions
2.5 Troubleshooting EP Recording
3 Local Drug Application Methods
3.1 Rationale
3.1.1 Limitations of Intratympanic Drug Application
3.2 Materials
3.2.1 Intratympanic Injections
3.2.2 Intralabyrinthine Injections
3.3 Detailed Methods
3.3.1 Intratympanic Injections
3.3.2 Intralabyrinthine Injections
3.4 Troubleshooting
4 Perilymph Sampling
4.1 Rationale
4.2 Materials
4.3 Detailed Methods
4.3.1 Sample Handling
4.4 Troubleshooting
5 Conclusions
References
Part IV: The Central Auditory Pathway
Chapter 13: In Vivo Whole-Cell Recording in the Gerbil Cochlear Nucleus
1 Introduction
2 Materials
2.1 Animal
2.2 Anesthetics
2.3 Surgical Tools
2.4 Electrophysiology
2.5 Acoustic System
3 Methods
3.1 Surgery
3.1.1 Anesthesia: Induction and Maintenance
3.1.2 Mounting the Head Bar
3.1.3 Craniotomy and Exposure of the Cochlear Nucleus
3.1.4 Electrophysiology
3.1.5 Perfusion
4 Notes
5 Conclusion
References
Chapter 14: Measurement of Human Cochlear and Auditory Nerve Potentials
1 Introduction
2 Material and Methods
2.1 Subject Screening and Preparation
2.2 Custom Earmold
2.3 In Situ Acoustic Calibration
2.4 Local Anesthetics
2.5 Transtympanic Electrode Placement
2.6 Adjustable Frame
2.7 Experimental Apparatus: Measurement Booth
2.8 Experimental Apparatus: Acoustical Stimulation
2.9 Experimental Apparatus: Electrophysiological Recordings
2.10 Stimulus Paradigms and Data Processing
3 Conclusion
References
Chapter 15: Strategies for Identification of Medial Olivocochlear Neurons for Patch-Clamp Studies of Synaptic Function Using E...
1 Introduction
2 Materials
2.1 Materials for Retrograde Label of Axons and Brain Slice Preparation
2.1.1 Materials for Retrograde Fluorescent Tracer Application
2.1.2 Equipment for Brain Slice Preparation
2.2 Materials for Patch-Clamp Recordings
2.3 Materials for Optogenetic Stimulation of Presynaptic MNTB Neurons
3 Methods
3.1 Acute Application of Dextran Fluorescein Crystals to the Cochlea and Brainstem Slice Preparation
3.2 Patch-Clamp Recordings from Fluorescent MOC Neurons in Brainstem Slices with Electrical Stimulation of Presynaptic Axons
3.3 Optimizing Optogenetic Stimulation of MNTB Neurons
4 Notes
5 Conclusions
References
Chapter 16: Auditory Brainstem Response (ABR) Measurements in Small Mammals
1 Introduction/Overview
2 Materials
2.1 Equipment
3 Methods
3.1 General Setup
3.2 ABR Protocol
3.3 Animal Preparation/Anesthesia
3.4 ABR Testing
3.5 Recovery
3.6 After ABR Testing
3.7 ABR Threshold Analysis
3.8 ABR Waveform Analysis
3.9 Best Practices
4 Information We Can Obtain from ABRs
5 Considerations
5.1 Protocol Factors that Can Affect ABRs
5.2 Stimulus/Recording Parameters that Can Affect ABRs
5.3 Subject and Environmental Factors that Can Affect ABRs
References
Chapter 17: Behavioral Models Loudness, Hyperacusis, and Sound Avoidance
Abbreviations
1 Introduction
1.1 Loudness Dynamic Range
1.2 Loudness Recruitment
1.3 Loudness Hyperacusis
1.4 Sound Avoidance Hyperacusis
2 Measuring Loudness: Recruitment and Hyperacusis
2.1 Reaction Time-Intensity Functions
2.2 Subjects
2.3 Equipment
2.4 Procedures
2.5 Effects of Stimulus Duration, Bandwidth, and Frequency on Loudness Growth
3 Animal Models of Recruitment and Hyperacusis
3.1 Noise-Induced RT-I Functions with Recruitment-Like Features
3.2 Genetic and Noise-Induced RT-I Functions with Hyperacusis-like Features
3.3 Transient Ototoxic Drug-Induced Hyperacusis
4 Active Sound Avoidance Paradigm (ASAP) to Assess Fear or Avoidance Hyperacusis
4.1 Subjects
4.2 Equipment
4.3 Procedures
4.4 Analysis of Results
5 Summary
References
Index