Neural Development Methods and Protocols

Understanding the molecular and cellular mechanisms underlying the development of specific neural circuits is not just an intellectual curiosity but also central to our ability to develop therapeutic approaches to repair damaged pathways in the future. In Neural Development: Methods and Protocols, experts in the field contribute commonly used protocols to facilitate future research in developmental neuroscience. Split into four convenient sections, this detailed volume covers techniques of culturing neurons and glia as well as their growth and differentiation, methods of gene delivery and down regulation, protocols for analyzing axon growth and guidance plus synapse formation, and, finally, basic methods to analyze brain morphology and axon pathways in developing animals.  Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Comprehensive and accessible, Neural Development: Methods and Protocols provides key guidance for students and postdoctoral fellows new to developmental neurobiology.

Part I: Neuron and Glial Cell Culture

1. Isolation and Maintenance of Cortical Neural Progenitor Cells In Vitro

            Mi-Yeon Kim, Byoung-San Moon, and Kang-Yell Choi

2. Neural Induction with a Dopaminergic Phenotype from Human Pluripotent Stem Cells Through a Feeder-Free Floating Aggregation Culture

            Asuka Morizane, Daisuke Doi, and Jun Takahashi

3. Nucleoside Analog Labeling of Neural Stem Cells and Their Progeny

            Erik DeBoer and Mladen-Roko Rasin

4. Culture of Dissociated Hippocampal Neurons

            Yun Peng, Wen-Cheng Xiong, and Lin Mei

5. Culturing Mouse Cerebellar Granule Neurons

            Tharakeswari Selvakumar and Daniel L. Kilpatrick

6. Preparation of Primary Cultured Dopaminergic Neurons from Mouse Brain

            Won-Seok Choi, Hyung-Wook Kim, and Zhengui Xia

7. Culturing Astoryctes from Postnatal Rats

            Yangyang Huang and Cheryl Dreyfus

8. Culturing Oligodendrocyte Lineage Cells from Neonatal Rats

            Clifton G. Fulmer and Cheryl F. Dreyfus

9. Isolation and Culture of Schwann Cells

            Yanmei Tao

Part II: Gene Expression and Analysis

10. DNA Transfection: Calcium Phosphate Method

            Munjin Kwon and Bonnie L. Firestein

11. The Gene-Gun Approach for Transfection and Labeling of Cells in Brain Slices

            Anna Dunaevsky

12. Lentiviral Vector Production, Titration, and Transduction of Primary Neurons

            Baojin Ding and Daniel L. Kilpatrick

13. In-Ovo Electroporation in Embryonic Chick Spinal Cords

            Hui Wang and Michael P. Matise

14. Gene Transfer in Developing Chick Embryos: In Ovo Electroporation

            Emma K. Farley

15. In Utero Electroporation in Mice

            Chunlei Wang and Lin Mei

16. General Introduction to In Situ Hybridization Protocol Using Non-Radioactively Labeled Probes to Detect mRNAs on Tissue Sections

            Daehoon Lee, Shan Xiong, and Wen-Cheng Xiong

17. Immunofluorescence Staining with Frozen Mouse or Chick Embryonic Tissue Sections

            Hui Wang and Michael P. Matise

18. ß-Galactosidase Staining of LacZ Fusion Proteins in Whole Tissue Preparations

            Margaret A. Cooper and Renping Zhou

19. Chromatin Immunoprecipitation Assay of Brain Tissues Using Percoll Gradient-Purified Nuclei

            Baojin Ding and Daniel L. Kilpatrick

20. In Vivo Dual Luciferase Reporter Assay with Chick Neural Tube In Ovo Electroporation System

            Hui Wang and Michael P. Matise

Part III: Axon Pathways and Synapses

21. Growth Cone Collapse Assay

            Xin Yue, Alexander I. Son, and Renping Zhou

22. The Stripe Assay: Studying Growth Preference and Axon Guidance on Binary Choice Substrates In Vitro

            Markus Weschenfelder, Franco Weth, Bernd Knöll, and Martin Bastmeyer

23. Microcontact Printing of Substrate-Bound Protein Patterns for Cell- and Tissue Culture

            Martin Fritz and Martin Bastmeyer

24. Semi-Automated Analysis of Dendrite Morphology in Cell Culture

            Eric S. Sweet, Chris L. Langhammer, Melinda K. Kutzing, and Bonnie L. Firestein

25. Monitoring Synaptic Plasticity by Imaging AMPA Receptor Content and Dynamics on Dendritic Spines

            Hiroshi Makino and Bo Li

26. Morphological Analysis of Neuromuscular Junctions by Immunofluorescent Staining of Whole-Mount Mouse Diaphragms

            Haitao Wu and Lin Mei

Part IV: Morphological Analyses of the Developing Nervous System

27. Routine Histology Techniques for the Developing and Adult Central Nervous System

            W. Geoffrey McAuliffe

28. Cryosectioning

            Alexander I. Son, Katie Sokolowski, and Renping Zhou

29. The Golgi-Cox Method

            Gitanjali Das, Kenneth Reuhl, and Renping Zhou

30. Neuroanatomical Tract-Tracing Using Biotinylated Dextran Amine

            Nikolai E. Lazarov

31. Retrograde Tracing Technique for Neonatal Animals

            Kengo Funakoshi, Akira Yoshikawa, and Yoshitoshi Atobe