Neurons: Methods and Applications for the Cell Biologist (eBook)

Cover 1
Contents 6
Contributors 12
Preface 16
Chapter 1. Comparing the Properties of Neuronal Culture Systems: A Shopping Guide for the Cell Biologist 18
I. Introduction 19
II. Approaching Experimental Questions Using Neurons 19
III. Choosing a Neuronal Culture System 25
IV. Conclusions 31
References 32
Chapter 2. Growing and Working with Peripheral Neurons 34
I. Introduction 35
II. Methods 36
III. Cultures 48
References 51
Chapter 3. Dissection and Culturing of Chick Ciliary Ganglion Neurons: A System Well Suited to Synaptic Study 54
I. General Introduction 55
II. Materials 59
III. Methods 60
IV. Variations on Dissection, Dissociation, and Culturing Themes 64
V. Concluding Comments 66
References 66
Chapter 4. The Culture of Chick Forebrain Neurons 68
I. Introduction 69
II. Growth and Development Characteristics 70
III. Isolation of Single Chick Forebrain Neurons 73
IV. Culture Conditions for Chick Forebrain Neurons 76
References 81
Chapter 5. Growing and Working with Spinal Motor Neurons 84
I. Introduction 85
II. Incubation of Fertilized Chicken Eggs 86
III. Preparation of Chick Embryo 88
IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves 90
V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves 94
VI. Plating Motor Neurons Spinal Cord Neurons 95
VII. Motor Neuron Enrichment by Density Gradient Centrifugation 96
VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures 97
IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements 99
References 102
Chapter 6. Avian Purkinje Neuronal Cultures: Extrinsic Control of Morphology by Cell Type and Glutamate 106
I. Introduction 107
II. Methods and Systems 109
III. Applications 117
References 122
Chapter 7. Culturing Hippocampal and Cortical Neurons 128
I. Introduction 129
II. Acquisition of Hippocampal and Cortical Neurons 130
III. Short-Term Culture Methods 134
IV. Long-Term Culture Methods 138
V. Summary 143
References 144
Chapter 8. Working with Xenopus Spinal Neurons in Live Cell Culture 146
I. Introduction 147
II. Neuronal Labeling 148
III. Culturing Xenopus Spinal Neurons 156
IV. Live Cell Imaging and Manipulations 165
V. Summary 171
References 171
Chapter 9. Culturing Neurons from the Snail Helisoma 174
I. Introduction 175
II. Maintaining Animals 176
III. Initial Dissection 177
IV. Culturing Nerve Cells 180
References 187
Chapter 10. The Tibial-1 Pioneer Pathway. An in Vivo Model for Neuronal Outgrowth and Guidance 188
I. Introduction 189
II. Development of the Tibial-1 (Ti1) Pathway 190
III. Guidance of Ti1 Axons 193
IV. Analysis of Axon Guidance Mechanisms in Grasshopper Procedures 200
V. Laboratory Protocols 203
References 208
Chapter 11. Techniques to Dissect Cellular and Subcellular Function in the Drosophila Nervous System 212
I. Introduction 213
II. Background Sources of Information 213
III. Drosophila as a Genetic Model System for Molecular Neurobiology 216
IV. Biochemistry of the Drosophila Nervous System 220
V. Cell Biology Techniques in the Drosophila Nervous System 233
VI. Functional Analysis Techniques in the Drosophila Nervous System 241
VII. Drosophila Electrophysiology 245
References 255
Chapter 12. PC12 Cells as a Model for Studies of Regulated Secretion in Neuronal and Endocrine Cells 284
I. Introduction 285
II. Propagation and Culture of PC12 Cells 287
III. Studying Secretion Using PC12 Cells 288
IV. Immunocytochemical Detection of Proteins in Intact and Permeable PC12 Cells 300
References 302
Chapter 13. B35 Neuroblastoma Cells: An Easily Transfected, Cultured Cell Model of Central Nervous System Neurons 304
I. Introduction 305
II. Applications Utilizing B35 and B50 Cells 306
III. Protocols 312
References 319
Chapter 14. Live-Cell Imaging of Slow Axonal Transport in Cultured Neurons 322
I. Introduction 323
II. Culturing Neurons from Superior Cervical Ganglia 323
III. Transfecting Neurons by Nuclear Injection 327
IV. Injecting Neurons with Fluorescent Proteins 331
V. Observing Movement 331
VI. Conclusion 338
References 338
Chapter 15. Making Proteins into Drugs: Assisted Delivery of Proteins and Peptides into Living Neurons 342
I. Introduction 343
II. Principles 343
III. Materials 347
IV. Methods 348
V. Conclusions and Perspectives 353
References 354
Chapter 16. Tranfection of Primary Central and Peripheral Nervous System Neurons by Electroporation 356
I. Introduction 357
II. Electroporation Theory and Principles 358
III. Electroporation Protocol for Chick Primary Neurons 361
IV. Rationale for Setting Electroporation Parameters 362
V. Discussion 366
References 367
Chapter 17. Biolistic Transfection 370
I. Introduction 371
II. Parameters that Affect Success of the Method 372
III. Factors that May Affect Performance but Have Not Been Tested 382
IV. Future Developments or Improvements 382
V. Conclusion 384
References 384
Chapter 18. Expression of Transgenes in Primary Neurons from Chick Peripheral and Central Nervous Systems by Retroviral Infection of Early Embryos 386
I. Introduction 387
II. Building Retroviral Vectors and Growing Functional Virus 388
III. Infecting Early Chick Embryos by Injection 396
IV. Dissecting Embryos and Culturing Neurons 399
V. Conclusion 402
References 403
Chapter 19. Production and Use of Replication-Deficient Adenovirus for Transgene Expression in Neurons 404
I. Introduction 405
II. Preparing Adenoviral Expression Vectors 408
III. Production of Adenovirus in HEK293 Cells 414
IV. Purification and Storage of Adenovirus Stocks 417
V. Titer Assays 419
VI. Safety Issues 422
VII. Infection of Primary Neurons 423
VIII. Conclusions and Perspectives 430
References 431
Index 434
Volume in Series 452