Measuring Cerebellar Function

Preface
Preface to the Series
Contents
Contributors
Chapter 1: Cerebellar Modelling Using Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Human Induced Pluripotent Cell Culture
2.2 Cerebellar Lineage Induction and Differentiation
2.3 Dissociated Culture
2.4 Three-Dimensional Organoid Culture
2.5 Sample Harvest
3 Methods
3.1 Human iPSC Culture and Maintenance
3.1.1 Matrigel Coating
3.1.2 Thawing hiPSCs
3.1.3 hiPSC Maintenance
3.2 Base Protocol for the First 35 Days of Differentiation
3.2.1 Embryoid Body Formation
3.2.2 Cerebellar Lineage Induction
3.2.3 Cerebellar Progenitor Maturation up to Day 35
Method 1: Matrigel Embedding Using Parafilm Sheet
Method 2: Matrigel Embedding in a V-Bottom 96-Well Plate
3.2.4 Neural Aggregate Culture Between Day 21 and 35
3.3 Dissociated Culture of hiPSC-Derived Cerebellar Neurons
3.4 Cerebellar Organoid Culture
3.4.1 Organoid Culture in Suspension
3.4.2 Organoid Culture at Air-Liquid Interface (ALI)
3.5 Analysis
3.5.1 Sample Preparation for Pooled Analysis
Preparation for Reverse Transcriptase-Polymerase Chain Reaction
Preparation for Immunostaining
Preparation for Flow Cytometry Analysis
3.5.2 Sample Preparation for Single-Cell Transcriptomics
4 Notes
5 Conclusions
References
Chapter 2: From Cerebellar Genes to Behaviors in Zebrafish
1 Introduction
1.1 Anatomy of Zebrafish Cerebellar Circuits
1.2 Development of Zebrafish Cerebellar Circuits
1.2.1 Establishment of the Cerebellum Domain
1.2.2 Differentiation of Neurons
1.3 Function of Zebrafish Cerebellar Circuits
1.3.1 Eyeblink Conditioning, Vestibular-Ocular Reflex, and Optokinetic Response
1.3.2 Motor Adaptation
1.3.3 Fear Conditioning
2 Materials
2.1 Molecular Markers
2.2 Transgenic Fish
2.3 Zebrafish Mutants
3 Methods
3.1 Histology, Mutagenesis, Transgenesis, and Electrophysiology
3.2 Classical Fear Conditioning
4 Discussion and Prospective
References
Chapter 3: Silencing the Output of Cerebellar Neurons Using Cell Type-Specific Genetic Deletion of Vesicular GABA and Glutamat...
1 Introduction
2 Materials
2.1 Mouse Lines
2.2 Visualization of Molecular Expression
2.3 In Vivo Electrophysiology
3 Methods
3.1 Generation of Conditional Vgat and Vglut Knockout Mice Using Existing Alleles
3.2 Visualization of Gene and Protein Expression in Tissue Slices
3.3 Electrophysiology-Genetic Crosses and Required Recording Components
3.4 Phenotyping the Genetically Manipulated Mice
4 Notes
References
Chapter 4: Mapping Structure-Function Relationships within Cerebellar Circuits
1 Introduction
2 Materials
2.1 Anesthetics
2.2 Glass Pipettes
2.3 Tracers
2.4 Tracer Delivery
2.5 Surgical Procedures
2.6 Electrophysiological Mapping
2.7 Chronic Implants
2.8 Tissue Processing
2.9 Microscopy and Imaging
3 Methods
3.1 Preparation for Anatomical Tracing
3.2 General Surgical Procedures
3.3 Electrophysiology Mapping
3.4 Tracer Delivery (if Carrying out Anatomical Investigations)
3.5 Chronic Implants
3.6 Survival Time, Perfusion, and Histology
3.7 Microscopy and Anatomical Mapping
3.8 Chronic Stimulation
4 Notes
References
Chapter 5: Designing Behavioral Tasks for Cerebellar Functional Analysis in the Mouse
1 Introduction
1.1 Established Behaviors for Cerebellar Functional Analysis
2 Innate Behaviors
2.1 Modification of Innate Behaviors
2.2 Licking Tasks
2.3 Locomotion
3 Freely Moving Locomotion
3.1 Head-Fixed Locomotion
3.2 Operant Behaviors
3.3 Forelimb Tasks
3.3.1 Skilled Reaching
3.3.2 Lever-Based Tasks
3.4 Specific Considerations for Establishing Head-Fixed, Forelimb-Based Operant Behaviors
3.4.1 Methods for Head Fixation
3.5 Optimizing Experimental Ergonomics
3.6 Behavioral Design
3.6.1 Water Restriction
3.6.2 Habituation/Training
3.6.3 Shaping Behavior
3.6.4 Performance Standards/Metrics
3.6.5 Detecting Alternate Strategies
References
Chapter 6: Learning Paradigms and Genetic Tools for the Study of Cerebellum-Dependent Learning and Memory
1 Introduction
1.1 Reflexive Eye Movements
1.2 Gait Activity
1.3 Adaptive Motor Learning and Relevant Region in the Brain
1.4 Potential Synaptic Plasticity Mechanisms for Cerebellum-Dependent Motor Learning
2 Methods
2.1 How to Measure Reflexive Eye Movements in Mice
2.2 New Approach to Synaptic Plasticity Mechanisms for Cerebellum-Dependent Motor Learning
2.3 New Training Paradigms for Adaptive Motor Learning in VOR and OKR
2.4 New Methods to Approach Cerebellum-Dependent Control in Gait Activity
3 Results and Discussion
3.1 Synaptic Plasticity Mechanisms for Adaptive Motor Leaning
3.2 New Aspects of Adaptive Motor Learning in the VOR and OKR
3.3 Cerebellum-Dependent Control on Gait
4 Conclusions
References
Chapter 7: Activity-Dependent Chromatin Mechanisms in Cerebellar Motor Learning
1 Introduction
2 Materials
2.1 Equipment and Tools
2.1.1 Surgery
2.1.2 Optogenetic Stimulation
2.1.3 Viral Delivery
2.1.4 Delay Tactile Startle Conditioning Apparatus
2.1.5 Tissue Collection and Biochemical Procedures
2.1.6 Hi-C or PLAC-Seq, Additional Reagents Described in
2.1.7 RNA-Seq
2.2 Mouse Genetic Lines
2.3 General Surgical Procedures
2.4 Fiber-Optic Cannula Implantation
2.5 Viral Injection
3 Methods
3.1 Optogenetic Stimulation of Granule Neurons in ADCV
3.2 Conditional CRISPR-Mediated Gene Knockout Using Viral Infection in ADCV
3.3 Delay Tactile Startle Conditioning
3.4 Biochemical Procedures to Examine the Chromatin Landscape
4 Conclusions
References
Chapter 8: In Vitro Voltage Imaging of Subthreshold Activity in Inferior Olive Neurons with ANNINE-6plus
1 Introduction
2 Materials
2.1 Preparation of ANNINE-6plus (A6+) Stock Solution
2.2 In-Vivo Stereotactic Injection of A6+ into Inferior Olive of Adult Anesthetized Mouse
2.2.1 Injection Setup
2.2.2 Pipette Preparation
2.2.3 Surgery
2.3 Acute Brainstem Slice Preparation at Physiological Temperature
2.3.1 Slicing Device
2.3.2 Surgical Tools
2.3.3 Chemicals
2.4 In Vitro Voltage Imaging
2.4.1 Illumination
2.4.2 Camera
3 Methods
3.1 Preparation of ANNINE-6plus (A6+) Solutions
3.2 In-Vivo Stereotactic Injection of A6+ into Inferior Olive of an Adult Anesthetized Mouse
3.2.1 Pipette Fabrication
3.2.2 Anesthesia and Mouse Well-Being
3.2.3 Mouse Body and Skull Alignment
3.2.4 Craniotomy and Dye Delivery into the Inferior Olive
3.2.5 Post-operative Care
3.2.6 Acute Brainstem Slice Preparation at Physiological Temperature
3.2.7 Preparation of Standard Physiological Solution (SPS)
3.2.8 Workspace Preparation
3.2.9 Brainstem Dissection
Brainstem Slicing
3.2.10 Assessing Slice Quality
3.2.11 In Vitro Voltage Imaging
Experimental Setup
Acquisition Steps
Basic Illumination and Acquisition Parameters
Adjusting Signal Acquisition
Special Care for High Magnification Objectives
3.2.12 Tips for Improving the Quality of Recordings
Dust in Perfusion
Bath Liquid Level
Limiting Photobleaching
Slice Immobilization
4 Notes
References
Chapter 9: Mapping Synaptic Connectivity in the Cerebellar Cortex Using RuBi-Glutamate Uncaging
1 Introduction
2 Materials
2.1 Mice
2.2 Setups for Photostimulation
2.3 Electrophysiological Setup
2.4 Pipettes
2.5 Epifluorescence System
3 Methods
3.1 Slice Preparation and Solutions
3.2 Calibration of Photostimulation
3.3 Purkinje Cell Recording and Synaptic Mappings
3.4 Slice Reconstruction
3.5 Connectivity Map Establishment
4 Additional Recommendations
4.1 A Simplified Setup
4.2 Controls for RuBi-Glutamate
References
Chapter 10: Multi-site Extracellular Electrode Neuronal Recordings in the Rodent Cerebellar Cortex and Nuclei
1 Introduction
2 General Considerations
2.1 The Extracellular Signal
2.2 Choosing (or Designing) an Electrode
2.2.1 Size/Impedance/Surface Treatment
2.2.2 Mechanical Stress and Trauma
2.2.3 Stability, Head-Fixed vs. Freely Moving Animals
2.3 Multi-site Electrode Typology
2.3.1 Wire Bundles
2.3.2 Glass-Insulated Multi-site Electrodes
2.3.3 Silicon Probes
2.4 The Future of Multi-site Electrodes
2.4.1 Acquisition Hardware and Software
3 Experimental Procedures
3.1 Electrodes and Implants
3.1.1 Protocol for Bundles
3.1.2 Bundle Creation
3.1.3 Preparation of the Guide Cannulas
3.1.4 Assembly of the Implant
3.1.5 Impedance Adjustment
3.2 Microdrive
3.3 Surgery
3.3.1 Before the Surgery
3.3.2 Pain Management
3.3.3 Preparing the Animal
3.3.4 Preparing the Skull
3.3.5 Craniotomy
3.3.6 Electrode Insertion and Sealing
3.3.7 Final Steps and Postoperative Management
3.4 Spike Sorting
3.4.1 Spike Sorting in the Cerebellum
3.4.2 Sorting Complex Spikes
3.4.3 Cell Identification
References
Chapter 11: Investigating Cerebrocerebellar Neuronal Interactions in Freely Moving Mice Using Multi-electrode, Multi-site Reco...
1 Introduction
2 Materials
2.1 Surgical Procedures
2.2 Electrophysiological Recordings
2.3 Behavioral Tasks
3 Methods
3.1 Triple Site Recordings in Head-Fixed Mice
3.1.1 Surgical Procedures
3.1.2 Electrophysiological Recordings
3.2 Triple Site Recordings in Freely Moving Mice
3.2.1 Micro-Drive Assembly
3.2.2 Surgical Procedures
3.2.3 Electrophysiological Recordings
3.2.4 Behavioral Task
3.2.5 Optical Stimulation of LS PCs
3.3 Data Analysis
3.3.1 Histology
3.3.2 Data Preprocessing
3.3.3 Analysis of PC Activity
3.3.4 Estimation of LFP Phase and Phase Relationships
3.3.5 Time-Resolved Coherence Analysis of LFP Data
3.3.6 Time-Frequency Analysis of LFP Data
4 Notes
References
Chapter 12: In Vivo Optical Detection of Membrane Potentials in the Cerebellum: Voltage Imaging of Zebrafish
1 Introduction
2 Materials
2.1 Staining of the Zebrafish Cerebellum by Voltage-Sensitive Dye
2.2 Establishment of Transgenic Zebrafish Expressing GEVIs
2.3 Voltage Imaging of the Zebrafish Cerebellum and Spinal Cord
2.4 Drug Treatments
2.5 Electrical Stimulation, Electrophysiology
2.6 Analysis
3 Methods
3.1 Zebrafish Lines
3.2 Staining of the Zebrafish Cerebellum by Voltage-Sensitive Dye
3.3 Establishment of Transgenic Zebrafish Expressing GEVIs
3.4 Voltage Imaging of Zebrafish Cerebellum and Spinal Cord
3.5 Drug Treatments
3.6 Electrical Stimulation
3.7 Electrophysiological Recordings
3.8 Analysis
4 Notes
References
Chapter 13: Imaging Neuronal Activity in Cerebellar Cortex of Behaving Mice
1 Introduction
2 Materials and Methods
2.1 Preparing Animals for In Vivo Imaging
2.1.1 Surgery: Implantation and Craniotomy
2.1.2 Targeted Genetically Encodable Activity Indicator Expression in Cerebellum
2.2 Imaging and Data Analysis
2.2.1 Imaging Cerebellar Granule Cells and Molecular Layer Interneurons In Vivo
2.2.2 Purkinje Cell-Specific Two-Photon Imaging for Elucidation of Olivocortical Modules
2.2.3 Targeting Approaches to Overcome Constraints of Wide-Field Imaging of Purkinje Cells
3 Future Directions
References
Chapter 14: Measuring Cerebellar Processing and Sensorimotor Functions in Non-Human Primates
1 Introduction
2 Materials: Implants Designs and Manufacturing Process
2.1 CT/MRI Image Processing
2.1.1 Import and Re-alignment
2.1.2 Segmentation
2.1.3 Export of the 3D structures (File format: .stl or .obj)
2.2 Adapting Implant Design to CT/MRI Images
2.3 Headpost
2.4 Recording chamber
2.5 Coatings
3 Methods: CT and MRI Scans
4 Methods: Surgical Procedures
4.1 Anesthesia
4.1.1 Anesthesia for Minor Procedures
4.1.2 Anesthesia for Major Procedures
4.2 Headpost Implantation
4.3 Chamber Placement and Craniotomy
4.4 Postoperative Care
5 Methods: Behavior
5.1 Eye Tracking
5.2 Orofacial Behavior
5.3 Hand and Whole-Body Tracking
5.4 Training of the Behavioral Tasks
6 Methods: Cerebellar Electrophysiology
7 Methods: Pharmacological Interventions
8 Notes
9 Conclusions
References
Chapter 15: Optogenetics in Complex Model Systems (Non-Human Primate)
1 Introduction
2 Material and Methods
2.1 Animal Preparations
2.2 Single Unit Recording
2.2.1 Materials
2.3 Injectrode
2.3.1 Materials
2.4 Data Acquisition and Behavioral Control System
2.4.1 Materials
2.5 Light Delivery to Target Area
2.5.1 Materials
2.6 Laser Light Source
2.6.1 Materials
2.7 Experiment Specific Methods
2.7.1 Experiment to Examine the Role of OMV Purkinje Cells Simple Spike Activity on Saccade Dynamics
Identifying OMV Purkinje Cells
Injecting Vector Solution
Technical Requirements
The Effects of Perturbing SS Activity on Saccades
2.7.2 Experiment to Examine the Role of Mossy Fiber Inputs to OMV P-Cell Simple Spike Activity
Identifying the OMV and NRTP and Injecting AAV-ArchT
Histological Verification of ArchT Expression in OMV
Identifying the Affected OMV Sites Electrophysiologically
The Effects of Partial NRTP Mossy Fiber Inhibition on Purkinje Cell Activity
3 Conclusion
References
Chapter 16: Clinical Assessment of the Cerebellum
1 Introduction: Clinical Investigation of Cerebellar Dysfunction
2 Materials
2.1 Assessment of Limb, Balance, and Gait Function
2.2 Evaluation of Oculomotor Dysfunction
2.3 Evaluation of Non-motor Cerebellar Dysfunction
2.4 Computed Tomography Scanning
2.5 Magnetic Resonance Imaging
3 Methods: Cerebellar Clinical Assessment
3.1 Clinical Examination
3.1.1 Motor Assessment
Limb Findings
Balance and Gait Findings
Oculomotor Findings
3.1.2 Non-motor Complications and Cerebellar Cognitive Affective Syndrome
3.2 Neuroimaging
3.2.1 Conventional Angiography
3.2.2 Computed Tomography (CT) Scan
3.2.3 Magnetic Resonance Imaging (MRI)
3.2.4 Application of Neuroimaging Modalities in Cerebellar Disease
4 Conclusions
References
Index