Behavioral Neurogenetics (Neuromethods, 181)

Preface to the Series
Preface
Contents
Contributors
Chapter 1: Combined in Situ Hybridization Chain Reaction and Immunostaining to Visualize Gene Expression in Whole-Mount Drosop...
	1 Introduction
	2 Materials
		2.1 Split-Initiator Probes and Amplification Hairpins
		2.2 Reagents and Buffers for HCR and Immunostaining
		2.3 Reagents for Mounting
		2.4 Drosophila Strains
		2.5 Confocal Imaging
	3 Methods
		3.1 Multiplexable  HCR
		3.2 Combined HCR and  IHC
		3.3 Applications: Comparing Gene Expression in the Central Nervous System of Drosophila Species
		3.4 Applications: Validation of Single-Cell RNA-Seq Results
	4 Notes
		4.1 Sample Treatment: Proteinase K Digestion and Post-Hybridization Paraformaldehyde Fixation
		4.2 Detection Sensitivity: Probe Concentration, Probe-Set Size, and Amplification Conditions
		4.3 Cost
	References
Chapter 2: Mapping Brain-Wide Mesoscale Connectome from Single Animals with BRICseq
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
		2.3 Software
	3 Methods
		3.1 Viral Injection and Tissue Dissection
		3.2 BRICseq Library Preparation and Sequencing
		3.3 Reconstruction of Mesoscale Connectome
	4 Notes
	References
Chapter 3: Visualization of Mating-Dependent Activation of Neurons and Oogenesis in Drosophila melanogaster
	1 Introduction
	2 Materials
		2.1 Animal Preparations
		2.2 Other Reagents and Equipment
	3 Methods
		3.1 TRIC System Allows the Visualization of Mating-Dependent Activation of Neurons
			3.1.1 Preparation of Transgenic Flies
			3.1.2 Dissection and Immunostaining of Abdominal Ganglion of D. Melanogaster
			3.1.3 Acquisition of TRIC Signals with Microscope
			3.1.4 Data Analysis of TRIC Signal
			3.1.5 Note
		3.2 Ex Vivo Culture Experiment Reveals Direct Effect of Neurotransmitter and Hormones to the Peripheral Tissues
			3.2.1 Preparation of Flies
			3.2.2 Dissection and Ex Vivo Culture of the Ovary of D. melanogaster
			3.2.3 Immunostaining of the Ovary and Germline Stem Cells
			3.2.4 Imaging and Counting of Germline Stem Cells
			3.2.5 Notes
	References
Chapter 4: Measuring Sleep in Drosophila
	1 Introduction
		1.1 Methods to Quantify Sleep Amount in Drosophila
	2 Materials
		2.1 Overview of Drosophila Activity Monitoring (DAM) System
		2.2 A Laboratory and Incubators for Sleep Measurements
		2.3 Animals
		2.4 Apparatus, Accessories, and PCs for DAM System
	3 Methods
		3.1 Preparation of Locomotor Tubes
		3.2 Preparation of Flies
		3.3 Analysis of Sleep Using ``Insomniac3´´ Program
	4 Conclusion
	5 Notes
	References
Chapter 5: Gut Microbes and Drosophila Behavior
	1 Introduction
		1.1 Gut-Brain Interactions and Indigenous Microbes
		1.2 Gut-Associated and Gut-Invaded Microbes and Host Behaviors in Drosophila melanogaster
	2 Materials, Methods, and Notes
		2.1 Reduction of Indigenous Microbes Using Antibiotic Treatment
		2.2 Generation of Germ-Free Flies via Dechlorination
		2.3 Endosymbiont Wolbachia
		2.4 Confirmation of the Absence of Germs with the Colony-Forming Unit (CFU) Assay
		2.5 DNA Extraction from Gut Microbes
		2.6 Isolation of Gut Microbes and Identification of Bacterial Species Using PCR
		2.7 Oral Infection
	References
Chapter 6: Measurement of Thermoregulatory Behavior in Drosophila melanogaster
	1 Introduction
		1.1 Study of Thermoregulatory Behavior in Drosophila
		1.2 Thermosensory Proteins Involved in Thermoregulatory Behavior of Drosophila
		1.3 Devices for Analyzing Thermoregulatory Behavior of Drosophila
	2 Materials
		2.1 Fly Larvae
		2.2 Temperature Gradient Assay
			2.2.1 Agarose-Covered Glass Plate
			2.2.2 Temperature Gradient Assay Instrument
			2.2.3 Image Capture and Analysis
		2.3 Video Recording Setup for Tracking Drosophila Behavior on the Temperature Gradient
	3 Methods
		3.1 Preparation of Larvae
		3.2 Temperature Gradient Assay
			3.2.1 Preparation of Agarose-Covered Glass Plates
			3.2.2 Preparation of the Peltier Device
			3.2.3 Recoding and Analysis of the Distribution of Larvae
			3.2.4 Representative Results of the Temperature Gradient Assay
		3.3 Behavioral Tracking on a Temperature Gradient
	4 Conclusions
	References
Chapter 7: Quantifying Social Interactions in Medaka Fish
	1 Introduction
		1.1 Female Mate Preference for Visually-Familiarized  Male
		1.2 Male Mate-Guarding Behaviors in Medaka  Fish
		1.3 Visually Mediated Social Attraction
	2 Materials
		2.1 Preparation for Medaka  Fish
	3 Methods
		3.1 Mating Test to Confirm Sexual Maturation
		3.2 Female Preference for Visual Familiarization
		3.3 Paternity Test Using Transgenic  Fish
		3.4 Mate-Guarding Behaviors
		3.5 Mate-Guarding Behaviors in a Three-Chamber  Tank
		3.6 Social Attraction
	4 Notes
		4.1 Breeding Condition Strongly Influence Ovarian Cycle and Female Behaviors
		4.2 Recording Conditions for the Automatic Tracking
	References
Chapter 8: A Method for Selective Breeding to Domesticate Mice
	1 Introduction
		1.1 Domestication
		1.2 Tameness
		1.3 Development of Wild-Derived Heterogeneous Stock
	2 Materials
		2.1 Mice
		2.2 Breeding Equipment and Environment
		2.3 Preparation for the Tameness  Test
		2.4 Preparation for Video Analysis
		2.5 Breeding Records
	3 Methods
		3.1 Handling of Mice
		3.2 Mating Mice to Establish Heterogeneous Stock
		3.3 Animal Preparation
		3.4 Tameness  Test
		3.5 Scoring Tameness Using Video  Data
		3.6 Selective Breeding
	4 Notes
	References
Chapter 9: Bioassaying the Function of Pheromones in Drosophila melanogaster´s Social Behavior
	1 Introduction
	2 How to Rear Drosophila melanogaster for Behavioral Experiments with Pheromones
		2.1 Rearing Flies for Behavioral Experiments
		2.2 Controlling the Mating Status of Flies
		2.3 Generating Oenocyte Ablated Flies
	3 Bioassaying the Role of Single or Combinations of Pheromones in Courtship Behavior and Mate Choice
		3.1 Perfuming Drosophila Melanogaster Using the Tumbling Method
		3.2 Assaying Courtship Behavior
		3.3 Assaying Mate Choice Behavior
	4 Assaying Volatile and Contact Pheromones for Oviposition Site Selection
		4.1 Preparing the Oviposition Assays
			4.1.1 Preparing the Olfactory Oviposition Assay
			4.1.2 Preparing the Contact Oviposition Assay
		4.2 Isolating Females for Oviposition Assays
		4.3 Extracting Cuticular Pheromones for the Oviposition Assays
		4.4 Preparing Pheromone Dilutions for the Oviposition Assay
		4.5 Running the Olfactory Oviposition Assays
		4.6 Running the Contact Oviposition Assays
	5 Laboratory Practices When Working with Pheromones
		5.1 General Laboratory Practices
		5.2 Cleaning Work Surfaces
		5.3 Cleaning Glassware
		5.4 Cleaning Laboratory Utensils
	References
Chapter 10: Miniature Microscopy of Hippocampal CA1 to Identify Engram Cells and Record Calcium Transients for Analyses of Ens...
	1 Introduction
		1.1 Engram Cells: Memory-Encoding Cells
		1.2 Visualization of Neuronal Activity with a Miniature Microscope
	2 Materials
		2.1 LV for Gene Transfer
		2.2 Miniature Microscope
		2.3 Animals
		2.4 LED for Photoconversion
	3 Methods
		3.1 Animal Preparations
		3.2 Identification and Photoconversion of KikGR Signals from Engram Cells
		3.3 Ca2+ Imaging During Memory Processing
		3.4 Imaging Data Processing
		3.5 Identification of Cells and Quantification of Their Activities by the Automatic Cell Extraction Program
		3.6 Merging of Ca2+ Transients and Engram Cell  ROIs
	4 Conclusions
	References
Chapter 11: Optical Recording of Brain Neuron Activities from a Male Drosophila Behaving on a Treadmill
	1 Introduction
	2 Materials
		2.1 Flies
		2.2 Fly Treadmill
		2.3 Preparation Making
			2.3.1 Sample Holder
			2.3.2 Preparation Block
			2.3.3 Tools for Surgery
		2.4 Recording Setup
		2.5 Visual Stimulus
		2.6 Software
	3 Methods
		3.1 Preparation
		3.2 Imaging
		3.3 Data Analysis
	4 Notes
		4.1 Fly Treadmill
		4.2 Preparation
		4.3 Imaging
		4.4 Data Analysis
	References
Chapter 12: In Vivo Brain Imaging in Freely Moving and Socially Interacting Drosophila
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sample Preparation
		3.2 System Hardware Assembly
		3.3 System Operation
		3.4 Analyzing the  Data
	4 Notes
		4.1 Fly Brain Window Surgery
		4.2 Customizing Flyception Light Paths for Fluorescence Sensors of Other Wavelengths
	5 Conclusion
	References
Chapter 13: Drosophila as a Model to Explore Individuality
	1 Introduction: The Individuality of Flies
		1.1 The Possible Neural Basis of Individuality
		1.2 Behavior Paradigms Used to Study Drosophila Individuality
		1.3 The Drosophila Olfactory System
	2 Materials
		2.1 Choices of Fly Genetic Backgrounds
		2.2 Culture Conditions for Flies
			2.2.1 The Environment
			2.2.2 Fly Food Ingredients
			2.2.3 Preparation of Flies: Single versus Group Housing
		2.3 Current Treadmill Systems for Drosophila
		2.4 Design of the Camera-Mode Fly Treadmill System
		2.5 Design of the Odor Delivery System
		2.6 Data Acquisition and Feature Extraction
		2.7 Individual Fly Walking Trajectories
	3 Methods
		3.1 Building the Camera-Mode Treadmill System
		3.2 Building the Odor Delivery System
		3.3 Monitoring the Walking Behavior of a Single  Fly
			3.3.1 Fly Preparation (Starvation/Agar)
			3.3.2 Preparation of the Odor Delivery System
			3.3.3 Preparation of the Treadmill System
			3.3.4 Tethering Flies
			3.3.5 Place the Tethered Fly on the Treadmill
			3.3.6 Computer Interface and Program Initiation/Termination
			3.3.7 Locomotor Activity  Test
		3.4 Photoionization Detection of Odor Delay Time and Concentration Dynamics
	4 Conclusion
	References
Chapter 14: Automated Behavior Analysis Using a YOLO-Based Object Detection System
	1 Introduction
		1.1 Automated Behavior Analysis in Neurogenetics
		1.2 Conventional Methods
		1.3 Integration of  AI
		1.4 YOLO in Behavioral Analysis
	2 Materials
		2.1 Target Species
		2.2 Insect Preparation
		2.3 Video Recording
	3 Methods
		3.1 Pre-processing
		3.2 Training Datasets
		3.3 Training of the YOLO v2 Network and Detection
		3.4 Reliability: Accuracy of Detection and Correction of Errors
		3.5 Analysis of the Relationship Between Aggression and Courtship
		3.6 Weaknesses
	4 Future Directions
		4.1 Use of Free-Moving Females
		4.2 Multiple Class Detection
		4.3 Real-Time Analysis
	5 Conclusions
	References
Chapter 15: Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling
	1 Introduction
	2 Materials and Methods
		2.1 TurboID-Based Enzymatic Protein Labeling and Extraction of Biotinylated Proteins from C. Elegans
			2.1.1 Materials
		2.2 Methods
			2.2.1 Worm Husbandry
			2.2.2 Protein Extraction
			2.2.3 Desalting to Remove Free Biotin
	3 Affinity Purification Using Streptavidin Beads
		3.1 Streptavidin-Based Affinity Purification of Biotinylated Proteins and Streptavidin Magnetic Bead Washing
			3.1.1 Materials
			3.1.2 Method
		3.2 ``Elution-Digestion´´ Protocol
			3.2.1 Materials
			3.2.2 Method
		3.3 Digestion and Mass Spectrometry (Performed by Mass Spectrometry Facility)
		3.4 ``On-Bead Digestion´´ Protocol
			3.4.1 Streptavidin Magnetic Bead Acetylation
				Materials
				Method
			3.4.2 Digestion and Mass Spectrometry (Performed by Mass Spectrometry Facility)
			3.4.3 Data Analysis Using R Scripts
			3.4.4 Proteomics Data Deposition
	4 Analytical SDS-PAGE and Immunoblotting
		4.1 Materials
		4.2 Method
	5 Experimental Design and Analysis of Mass Spectrometry  Data
		5.1 Ensuring Tagged Proteins Are Functional and Appropriately Localized
			5.1.1 Background Noise
			5.1.2 Controls
	6 Conclusion
	References
Index