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Dorn Spinal Therapy

Spine Healing Therapy

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Contributors xi

Preface xv

1. Comparing the Properties of Neuronal Culture Systems: A Shopping Guide for the Cell Biologist

Peter J. Hollenbeck and James R. Bamburg

I. Introduction 2

II. Approaching Experimental Questions Using Neurons 2

III. Choosing a Neuronal Culture System 8

IV. Conclusions 14 References 15

2. Growing and Working with Peripheral Neurons Yan He and Peter W. Baas

I. Introduction 18

II. Methods 19

III. Cultures 31

References 34

3. Dissection and Culturing of Chick Ciliary Ganglion Neurons: A System Well Suited to Synaptic Study

Barbara W. Bernstein

I. General Introduction 38

II. Materials 42

III. Methods 43

IV. Variations on Dissection, Dissociation, and Culturing Themes 47

V. Concluding Comments 49 References 49

4. The Culture of Chick Forebrain Neurons

Steven R. Heidemann, Matthew Reynolds, Kha Ngo, and Phillip Lamoureux

I. Introduction 52

II. Growth and Development Characteristics 53

III. Isolation of Single Chick Forebrain Neurons 56

IV. Culture Conditions for Chick Forebrain Neurons 59 References 64

5. Growing and Working with Spinal Motor Neurons Thomas B. Kuhn

I. Introduction 68

II. Incubation of Fertilized Chicken Eggs 69

III. Preparation of Chick Embryo 71

IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves 73

V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or

Ventral Halves 77

VI. Plating Motor Neurons Spinal Cord Neurons 78

VII. Motor Neuron Enrichment by Density Gradient Centrifugation 79

VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures 80

IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements 82

References 85

6. Avian Purkinje Neuronal Cultures: Extrinsic Control of Morphology by Cell Type and Glutamate

Peter L. Jeffrey, Vladimir J. Balcar, Ornella Tolhurst, Ron P. Weinberger, and Jenny A. Meany

I. Introduction 90

II. Methods and Systems 92

III. Applications 100

References 105

7. Culturing Hippocampal and Cortical Neurons Peter J. Meberg and Matthew W. Miller

I. Introduction 112

II. Acquisition of Hippocampal and Cortical Neurons 113

III. Short-Term Culture Methods 117

IV. Long-Term Culture Methods 121

V. Summary 126 References 127

8. Working with Xenopus Spinal Neurons in Live Cell Culture

Timothy M. Gomez, Dan Harrigan, John Henley, and Estuardo Robles

I. Introduction 130

II. Neuronal Labeling 131

III. Culturing Xenopus Spinal Neurons 139

IV. Live Cell Imaging and Manipulations 148

V. Summary 154 References 154

9. Culturing Neurons from the Snail Helisoma

Christohper S. Cohan, James L. Karnes, and Feng-Quan Zhou

I. Introduction 158

II. Maintaining Animals 159

III. Initial Dissection 160

IV. Culturing Nerve Cells 163 References 170

10. The Tibial-1 Pioneer Pathway: An in Vivo Model for Neuronal Outgrowth and Guidance

Jennifer Bonner, Kimberly A. Gerrow, and Timothy P. O'Connor

I. Introduction 172

II. Development of the Tibial-1 (Ti1) Pathway 173

III. Guidance ofTi1 Axons 176

IV. Analysis of Axon Guidance Mechanisms in Grasshopper Procedures 183

V. Laboratory Protocols 186 References 191

11. Techniques to Dissect Cellular and Subcellular Function in the Drosophila Nervous System

Heinrich J. G. Matthies and Kendal Broadie

I. Introduction 196

II. Background Sources of Information 196

III. Drosophila as a Genetic Model System for Molecular Neurobiology 199

IV. Biochemistry of the Drosophila Nervous System 203

V. Cell Biology Techniques in the Drosophila Nervous System 216 VI. Functional Analysis Techniques in the Drosophila Nervous System 224

VII. Drosophila Electrophysiology 228

References 238

12. PC12 Cells as a Model for Studies of Regulated Secretion in Neuronal and Endocrine Cells

I. Introduction 268

II. Propagation and Culture of PC12 Cells 270

III. Studying Secretion Using PC12 Cells 271

IV. Immunocytochemical Detection of Proteins in Intact and

Permeable PC12 Cells 283

References 285

13. B35 Neuroblastoma Cells: An Easily Transfected, Cultured Cell Model of Central Nervous System Neurons Carol A. Otey, Malika Boukhelifa, and Patricia Maness

I. Introduction 288

II. Applications Utilizing B35 and B50 Cells 289

III. Protocols 295

References 302

14. Live-Cell Imaging of Slow Axonal Transport in Cultured Neurons

Anthony Brown

I. Introduction 306

II. Culturing Neurons from Superior Cervical Ganglia 306

III. Transfecting Neurons by Nuclear Injection 310

IV. Injecting Neurons with Fluorescent Proteins 314

V. Observing Movement 314 VI. Conclusion 321

References 321

15. Making Proteins into Drugs: Assisted Delivery of Proteins and Peptides into

Living Neurons Gianluca Gallo

I. Introduction 326

II. Principles 326

III. Materials 330

IV. Methods 331

V. Conclusions and Perspectives 336 References 337

16. Tranfection of Primary Central and Peripheral Nervous System Neurons by Electroporation

Cecilia Y. Martinez and Peter J. Hollenbeck

I. Introduction 340

II. Electroporation Theory and Principles 341

III. Electroporation Protocol for Chick Primary Neurons 344

IV. Rationale for Setting Electroporation Parameters 345

V. Discussion 349 References 350

17. Biolistic Transfection

Paul C. Bridgman, Michael E. Brown, and Irina Balan

I. Introduction 354

II. Parameters that Affect Success of the Method 355

III. Factors that May Affect Performance but Have Not Been Tested 365

IV. Future Developments or Improvements 365

V. Conclusion 367 References 367

18. Expression of Transgenes in Primary Neurons from Chick Peripheral and Central Nervous Systems by Retroviral Infection of Early Embryos

Peter J. Hollenbeck and D. M. Fekete

I. Introduction 370

II. Building Retroviral Vectors and Growing Functional Virus 371

III. Infecting Early Chick Embryos by Injection 379

IV. Dissecting Embryos and Culturing Neurons 382

V. Conclusions 385 References 386

19. Production and Use of Replication-Deficient Adenovirus for Transgene Expression in Neurons

L. S. Minamide, A. E. Shaw, P. D. Sarmiere, O. Wiggan, M. T. Maloney, Barbara W. Bernstein, J. M. Sneider,J. A. Gonzalez, and James R. Bamburg

I. Introduction 388

II. Preparing Adenoviral Expression Vectors 391

III. Production of Adenovirus in HEK293 Cells 397

IV. Purification and Storage of Adenovirus Stocks 400

V. Titer Assays 402

VI. Safety Issues

VII. Infection of Primary Neurons VIII. Conclusions and Perspectives References

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