Neurons: Methods and Applications for the Cell Biologist

1. Auflage 2003
ISBN: 978-0-08-049663-4
Verlag: Elsevier Science & Techn.
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band Volume 71, 500 Seiten, Web PDF

Reihe: Methods in Cell Biology

Neurons: Methods and Applications for the Cell Biologist 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

Autoren/Hrsg.

Inhaltsverzeichnis

1;Cover;1
2;Contents;6
3;Contributors;12
4;Preface;16
5;Chapter 1. Comparing the Properties of Neuronal Culture Systems: A Shopping Guide for the Cell Biologist;18
5.1;I. Introduction;19
5.2;II. Approaching Experimental Questions Using Neurons;19
5.3;III. Choosing a Neuronal Culture System;25
5.4;IV. Conclusions;31
5.5;References;32
6;Chapter 2. Growing and Working with Peripheral Neurons;34
6.1;I. Introduction;35
6.2;II. Methods;36
6.3;III. Cultures;48
6.4;References;51
7;Chapter 3. Dissection and Culturing of Chick Ciliary Ganglion Neurons: A System Well Suited to Synaptic Study;54
7.1;I. General Introduction;55
7.2;II. Materials;59
7.3;III. Methods;60
7.4;IV. Variations on Dissection, Dissociation, and Culturing Themes;64
7.5;V. Concluding Comments;66
7.6;References;66
8;Chapter 4. The Culture of Chick Forebrain Neurons;68
8.1;I. Introduction;69
8.2;II. Growth and Development Characteristics;70
8.3;III. Isolation of Single Chick Forebrain Neurons;73
8.4;IV. Culture Conditions for Chick Forebrain Neurons;76
8.5;References;81
9;Chapter 5. Growing and Working with Spinal Motor Neurons;84
9.1;I. Introduction;85
9.2;II. Incubation of Fertilized Chicken Eggs;86
9.3;III. Preparation of Chick Embryo;88
9.4;IV. Dissection of Intact Spinal Cords and Isolation of Ventral Halves;90
9.5;V. Enzymatic and Mechanical Dissociation of Intact Spinal Cords or Ventral Halves;94
9.6;VI. Plating Motor Neurons Spinal Cord Neurons;95
9.7;VII. Motor Neuron Enrichment by Density Gradient Centrifugation;96
9.8;VIII. Experimental Use of Motor Neuron Cultures and Spinal Cord Cultures;97
9.9;IX. Preparation of Solutions, Culture Dishes, Media, and Media Supplements;99
9.10;References;102
10;Chapter 6. Avian Purkinje Neuronal Cultures: Extrinsic Control of Morphology by Cell Type and Glutamate;106
10.1;I. Introduction;107
10.2;II. Methods and Systems;109
10.3;III. Applications;117
10.4;References;122
11;Chapter 7. Culturing Hippocampal and Cortical Neurons;128
11.1;I. Introduction;129
11.2;II. Acquisition of Hippocampal and Cortical Neurons;130
11.3;III. Short-Term Culture Methods;134
11.4;IV. Long-Term Culture Methods;138
11.5;V. Summary;143
11.6;References;144
12;Chapter 8. Working with Xenopus Spinal Neurons in Live Cell Culture;146
12.1;I. Introduction;147
12.2;II. Neuronal Labeling;148
12.3;III. Culturing Xenopus Spinal Neurons;156
12.4;IV. Live Cell Imaging and Manipulations;165
12.5;V. Summary;171
12.6;References;171
13;Chapter 9. Culturing Neurons from the Snail Helisoma;174
13.1;I. Introduction;175
13.2;II. Maintaining Animals;176
13.3;III. Initial Dissection;177
13.4;IV. Culturing Nerve Cells;180
13.5;References;187
14;Chapter 10. The Tibial-1 Pioneer Pathway. An in Vivo Model for Neuronal Outgrowth and Guidance;188
14.1;I. Introduction;189
14.2;II. Development of the Tibial-1 (Ti1) Pathway;190
14.3;III. Guidance of Ti1 Axons;193
14.4;IV. Analysis of Axon Guidance Mechanisms in Grasshopper Procedures;200
14.5;V. Laboratory Protocols;203
14.6;References;208
15;Chapter 11. Techniques to Dissect Cellular and Subcellular Function in the Drosophila Nervous System;212
15.1;I. Introduction;213
15.2;II. Background Sources of Information;213
15.3;III. Drosophila as a Genetic Model System for Molecular Neurobiology;216
15.4;IV. Biochemistry of the Drosophila Nervous System;220
15.5;V. Cell Biology Techniques in the Drosophila Nervous System;233
15.6;VI. Functional Analysis Techniques in the Drosophila Nervous System;241
15.7;VII. Drosophila Electrophysiology;245
15.8;References;255
16;Chapter 12. PC12 Cells as a Model for Studies of Regulated Secretion in Neuronal and Endocrine Cells;284
16.1;I. Introduction;285
16.2;II. Propagation and Culture of PC12 Cells;287
16.3;III. Studying Secretion Using PC12 Cells;288
16.4;IV. Immunocytochemical Detection of Proteins in Intact and Permeable PC12 Cells;300
16.5;References;302
17;Chapter 13. B35 Neuroblastoma Cells: An Easily Transfected, Cultured Cell Model of Central Nervous System Neurons;304
17.1;I. Introduction;305
17.2;II. Applications Utilizing B35 and B50 Cells;306
17.3;III. Protocols;312
17.4;References;319
18;Chapter 14. Live-Cell Imaging of Slow Axonal Transport in Cultured Neurons;322
18.1;I. Introduction;323
18.2;II. Culturing Neurons from Superior Cervical Ganglia;323
18.3;III. Transfecting Neurons by Nuclear Injection;327
18.4;IV. Injecting Neurons with Fluorescent Proteins;331
18.5;V. Observing Movement;331
18.6;VI. Conclusion;338
18.7;References;338
19;Chapter 15. Making Proteins into Drugs: Assisted Delivery of Proteins and Peptides into Living Neurons;342
19.1;I. Introduction;343
19.2;II. Principles;343
19.3;III. Materials;347
19.4;IV. Methods;348
19.5;V. Conclusions and Perspectives;353
19.6;References;354
20;Chapter 16. Tranfection of Primary Central and Peripheral Nervous System Neurons by Electroporation;356
20.1;I. Introduction;357
20.2;II. Electroporation Theory and Principles;358
20.3;III. Electroporation Protocol for Chick Primary Neurons;361
20.4;IV. Rationale for Setting Electroporation Parameters;362
20.5;V. Discussion;366
20.6;References;367
21;Chapter 17. Biolistic Transfection;370
21.1;I. Introduction;371
21.2;II. Parameters that Affect Success of the Method;372
21.3;III. Factors that May Affect Performance but Have Not Been Tested;382
21.4;IV. Future Developments or Improvements;382
21.5;V. Conclusion;384
21.6;References;384
22;Chapter 18. Expression of Transgenes in Primary Neurons from Chick Peripheral and Central Nervous Systems by Retroviral Infection of Early Embryos;386
22.1;I. Introduction;387
22.2;II. Building Retroviral Vectors and Growing Functional Virus;388
22.3;III. Infecting Early Chick Embryos by Injection;396
22.4;IV. Dissecting Embryos and Culturing Neurons;399
22.5;V. Conclusion;402
22.6;References;403
23;Chapter 19. Production and Use of Replication-Deficient Adenovirus for Transgene Expression in Neurons;404
23.1;I. Introduction;405
23.2;II. Preparing Adenoviral Expression Vectors;408
23.3;III. Production of Adenovirus in HEK293 Cells;414
23.4;IV. Purification and Storage of Adenovirus Stocks;417
23.5;V. Titer Assays;419
23.6;VI. Safety Issues;422
23.7;VII. Infection of Primary Neurons;423
23.8;VIII. Conclusions and Perspectives;430
23.9;References;431
24;Index;434
25;Volume in Series;452