Neurons: Methods and Applications for the Cell Biologist: Methods and Applications for the Cell Biologist
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Table of contents
- Contentsv
- Contributorsxi
- Prefacexv
- Chapter 1. Comparing the Properties of Neuronal Culture Systems: A Shopping Guide for the Cell Biolo1
- I. Introduction2
- II. Approaching Experimental Questions Using Neurons2
- III. Choosing a Neuronal Culture System8
- IV. Conclusions14
- References15
- Chapter 2. Growing and Working with Peripheral Neurons17
- I. Introduction18
- II. Methods19
- III. Cultures31
- References34
- Chapter 3. Dissection and Culturing of Chick Ciliary Ganglion Neurons: A System Well Suited to Synap37
- I. General Introduction38
- II. Materials42
- III. Methods43
- IV. Variations on Dissection, Dissociation, and Culturing Themes47
- V. Concluding Comments49
- References49
- Chapter 4. The Culture of Chick Forebrain Neurons51
- I. Introduction52
- II. Growth and Development Characteristics53
- III. Isolation of Single Chick Forebrain Neurons56
- IV. Culture Conditions for Chick Forebrain Neurons59
- References64
- Chapter 5. Growing and Working with Spinal Motor Neurons67
- I. Introduction68
- II. Incubation of Fertilized Chicken Eggs69
- III. Preparation of Chick Embryo71
- IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves73
- V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves77
- VI. Plating Motor Neurons Spinal Cord Neurons78
- VII. Motor Neuron Enrichment by Density Gradient Centrifugation79
- VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures80
- IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements82
- References85
- Chapter 6. Avian Purkinje Neuronal Cultures: Extrinsic Control of Morphology by Cell Type and Glutam89
- I. Introduction90
- II. Methods and Systems92
- III. Applications100
- References105
- Chapter 7. Culturing Hippocampal and Cortical Neurons111
- I. Introduction112
- II. Acquisition of Hippocampal and Cortical Neurons113
- III. Short-Term Culture Methods117
- IV. Long-Term Culture Methods121
- V. Summary126
- References127
- Chapter 8. Working with Xenopus Spinal Neurons in Live Cell Culture129
- I. Introduction130
- II. Neuronal Labeling131
- III. Culturing Xenopus Spinal Neurons139
- IV. Live Cell Imaging and Manipulations148
- V. Summary154
- References154
- Chapter 9. Culturing Neurons from the Snail Helisoma157
- I. Introduction158
- II. Maintaining Animals159
- III. Initial Dissection160
- IV. Culturing Nerve Cells163
- References170
- Chapter 10. The Tibial-1 Pioneer Pathway. An in Vivo Model for Neuronal Outgrowth and Guidance171
- I. Introduction172
- II. Development of the Tibial-1 (Ti1) Pathway173
- III. Guidance of Ti1 Axons176
- IV. Analysis of Axon Guidance Mechanisms in Grasshopper Procedures183
- V. Laboratory Protocols186
- References191
- Chapter 11. Techniques to Dissect Cellular and Subcellular Function in the Drosophila Nervous System195
- I. Introduction196
- II. Background Sources of Information196
- III. Drosophila as a Genetic Model System for Molecular Neurobiology199
- IV. Biochemistry of the Drosophila Nervous System203
- V. Cell Biology Techniques in the Drosophila Nervous System216
- VI. Functional Analysis Techniques in the Drosophila Nervous System224
- VII. Drosophila Electrophysiology228
- References238
- Chapter 12. PC12 Cells as a Model for Studies of Regulated Secretion in Neuronal and Endocrine Cells267
- I. Introduction268
- II. Propagation and Culture of PC12 Cells270
- III. Studying Secretion Using PC12 Cells271
- IV. Immunocytochemical Detection of Proteins in Intact and Permeable PC12 Cells283
- References285
- Chapter 13. B35 Neuroblastoma Cells: An Easily Transfected, Cultured Cell Model of Central Nervous S287
- I. Introduction288
- II. Applications Utilizing B35 and B50 Cells289
- III. Protocols295
- References302
- Chapter 14. Live-Cell Imaging of Slow Axonal Transport in Cultured Neurons305
- I. Introduction306
- II. Culturing Neurons from Superior Cervical Ganglia306
- III. Transfecting Neurons by Nuclear Injection310
- IV. Injecting Neurons with Fluorescent Proteins314
- V. Observing Movement314
- VI. Conclusion321
- References321
- Chapter 15. Making Proteins into Drugs: Assisted Delivery of Proteins and Peptides into Living Neuro325
- I. Introduction326
- II. Principles326
- III. Materials330
- IV. Methods331
- V. Conclusions and Perspectives336
- References337
- Chapter 16. Tranfection of Primary Central and Peripheral Nervous System Neurons by Electroporation339
- I. Introduction340
- II. Electroporation Theory and Principles341
- III. Electroporation Protocol for Chick Primary Neurons344
- IV. Rationale for Setting Electroporation Parameters345
- V. Discussion349
- References350
- Chapter 17. Biolistic Transfection353
- I. Introduction354
- II. Parameters that Affect Success of the Method355
- III. Factors that May Affect Performance but Have Not Been Tested365
- IV. Future Developments or Improvements365
- V. Conclusion367
- References367
- Chapter 18. Expression of Transgenes in Primary Neurons from Chick Peripheral and Central Nervous Sy369
- I. Introduction370
- II. Building Retroviral Vectors and Growing Functional Virus371
- III. Infecting Early Chick Embryos by Injection379
- IV. Dissecting Embryos and Culturing Neurons382
- V. Conclusion385
- References386
- Chapter 19. Production and Use of Replication-Deficient Adenovirus for Transgene Expression in Neuro387
- I. Introduction388
- II. Preparing Adenoviral Expression Vectors391
- III. Production of Adenovirus in HEK293 Cells397
- IV. Purification and Storage of Adenovirus Stocks400
- V. Titer Assays402
- VI. Safety Issues405
- VII. Infection of Primary Neurons406
- VIII. Conclusions and Perspectives413
- References414
- Index417
- Volume in Series435
This book lays out numerous simple techniques for growing and carrying out experiments with many varieties of neurons. Subjects include peripheral and central neurons from vertebrate and invertebrate sources, as well as neuron-like cell lines. It also explains recent advances in our ability to introduce exogenous proteins and genes to neurons in culture. Procedures for successful protein infiltration, biolistic transfection, electroporation, and viral transgenic methods in neurons are also presented.
* Contains culture methodology for more than a dozen types of CNS and PNS neurons
* Includes most recent and reliable techniques from expert practitioners for specific experimental applications
* Addresses the latest strategies for transfecting neurons
* Contains culture methodology for more than a dozen types of CNS and PNS neurons
* Includes most recent and reliable techniques from expert practitioners for specific experimental applications
* Addresses the latest strategies for transfecting neurons