Cell Culture in the Neurosciences

A fundamental problem in neuroscience is the elucidation of the cellular and molecular mechanisms underlying the development and function of the nervous system. The complexity of organization, the heteroge­ neity of cell types and their interactions, and the difficulty of controlling experimental variables in intact organisms make this a formidable task. Because of the ability that it affords to analyze smaller components of the nervous system (even single cells in some cases) and to better control experimental variables, cell culture has become an increasingly valuable tool for neuroscientists. Many aspects of neural development, such as proliferation, differentiation, synaptogenesis, and myelination, occur in culture with time courses remarkably similar to those in vivo. Thus, in vitro methods often provide excellent model systems for investigating neurobiological questions. Ross Harrison described the first culture of neural tissue in 1907 and used morphological methods to analyze the cultures. Since that time the technique has been progressively modified and used to address an ever widening range of developmental questions. In recent years a con­ vergence of new or improved cell culture, biochemical, electrophysiol­ ogical, and immunological methods has occurred and been brought to bear on neurobiological questions. This volume is intended not to be comprehensive but rather to highlight some of the latest findings, with a review of previous important work as well, in which combinations of these methods are used.

Contenu

I. Morphology and Biochemistry.- 1. Growth and Differentiation of Neural Cells in Defined Media.- 1. Introduction.- 2. Components of the Culture System.- 2.1. Synthetic Medium and Supplements.- 2.2. Substratum.- 2.3. Gaseous Environment.- 3. Serum-Free Defined Media for Neuronal Cells.- 3.1. Neuronal Cell Lines.- 3.2. Primary Neurons.- 4. Serum-Free Defined Media for Glial Cells.- 4.1. Glial Cell Lines.- 4.2. Primary Glia.- 5. Serum-Free Defined Media for Neural Precursor Cells.- 6. Conclusion.- 6.1. In Vivo vs. in Vitro Growth and Differentiation Requirements.- 6.2. Future Directions.- References.- 2. Neuronal and Gial Surface Antigens on Cells in Culture.- 1. Introduction.- 1.1. Scope.- 1.2. Toxins as Neuronal Markers.- 1.3. Monoclonal Antibodies That Bind to Neurons.- 1.4. Cross-Reactions of Monoclonal and Conventional Antibodies.- 2. Large Glycoproteins of the Neuronal Surface.- 2.1. NILE Glycoproteins.- 2.2. A Quartet of Related Glycoproteins: N-CAM, D2,NS-4, and BSP-2.- 3. Ganglioside Antigens on Neurons and Glia.- 3.1. GM4.- 3.2. GD3.- 3.3. GM1.- 3.4. GD1b and GT1b.- 3.5. GQ.- 3.6. Thyrotropin Receptor.- 3.7. Chol-1, a Polysialoganglioside Antigen.- 4. Neuronal Subset-Specific Monoclonal Antibodies.- 4.1. Retinal Cell Subpopulations.- 4.2. Cerebellar Neurons.- 4.3. Ciliary Ganglion Neurons.- 4.4. Sympathetic Ganglion Neurons.- 4.5. Cholinergic Neurons.- 5. Antibodies That Affect Cell Sorting Out or Neurite Outgrowth.- 6. Some Glial and Other Markers.- 6.1. Oligodendrocytes.- 6.2. Schwann Cells.- 6.3. Astrocytes.- 6.4. Ependymal Cells.- 6.5. Leptomeningeal Cells and Fibroblasts.- 7. Concluding Remarks.- References.- 3. Neuronotrophic Factors.- 1. Introduction.- 2. Cell Death, Neorite Pruning, and Synapse Maturation.- 3. Neuronotrophic Activity: In Vivo vs. in Vitro Aspects.- 4. Clonal Model Systems.- 4.1. Neuroblastoma.- 4.2. Rat Pheochromocytoma.- 5. Assay Systems.- 5.1. Cell Attachment.- 5.2. Cell Survival.- 5.3. Neurite Outgrowth.- 5.4. Neurotransmitter Synthesis.- 5.5. Radioassays.- 6. Factor Characterization.- 6.1. Sources of Activity.- 6.2. Purification and Characterization.- References.- 4. Hormonal Regulation of the Proliferation and Differentiation of Astrocytes and Oligodendrocytes in Primary Culture.- 1. Introduction.- 1.1. Glial Cell Classification.- 1.2. Primary Culture.- 1.3. Basic Modes of Hormone Action.- 2. Hormonal Influences on Proliferation.- 2.1. Oligodendrocytes.- 2.2. Astrocytes.- 3. Hormonal Influences on Differentiation.- 3.1. Oligodendrocytes.- 3.2. Astrocytes.- 4. Conclusion.- References.- 5. Environmental Influences on the Development of Sympathetic Neurons.- 1. Development of Sympathetic Neurons in Culture.- 1.1. Early Noradrenergic Phenotype.- 1.2. Conditions That Promote Noradregenic Development.- 1.3. Conditions That Promote Cholinergic Development.- 1.4. Effects of Factor "Cholinergic Factor".- 2. Comparisons with the Development of Cholinergic Sympathetic Neurons in Vivo.- 2.1. Cholinergic Sympathetic Innervation of Sweat Glands.- 2.2. Developmental Changes in the Transmitter-Related Phenotype of Sweat Gland Innervation.- References.- 6. In Vitro Analysis of Quail Neural Crest Cell Differentiation.- 1. Derivatives of the Neural Crest.- 2. Origin and Migration of Neural Crest Cells.- 3. Analysis of Neural Crest Cell Differentiation in Vivo.- 4. In Vitro Analysis of Neural Crest Cell Differentiation.- 4.1. Primary Cultures.- 4.2. Secondary Cultures.- 5. Regulation of in Vitro Neural Crest Cell Development by Humoral Factors.- 5.1. Heart Cell-Conditioned Medium.- 5.2. Tumor-Promoting Phorbol Esters.- 6. In Vitro Development of Neural Crest Cells on Artificial Substrata and Extracellular Matrix Components.- 6.1. Basic Polyamino Acids.- 6.2. Extracellular Matrix Components.- 7. Lineage Formation by Neural Crest Cells.- 7.1. Membrane Heterogeneity among Early Neural Crest Cells.- 7.2. Competence for Terminal Differentiation.- References.- 7. Biochemical Differentiation in Serum-Free Aggregating Brain Cell Cultures.- 1. Introduction.- 2. Methodology.- 2.1. Mechanical Dissociation of Brain Tissue.- 2.2. Culture Medium.- 2.3. Cell Aggregation.- 2.4. Maintenance of Aggregating Brain Cell Cultures.- 2.5. Culture Handling.- 3. Cellular Growth and Differentiation.- 3.1. Synthesis of DNA Synthesis and Protein.- 3.2. Biochemical Differentiation.- 4. Evaluation of the Culture System.- References.- 8. PC12 Cells as a Model of Neuronal Differentiation.- 1. Development of PC12 Cells.- 2. General Characteristics.- 2.1. Transmitter Content.- 2.2. Transmitter-Synthesizing Enzymes.- 2.3. Receptors.- 2.4. Electrophysiology.- 3. Actions of NGF.- 3.1. NGF Receptors.- 3.2. Rapid, Membrane-Associated Actions.- 3.3. Changes in Phosphorylation.- 3.4. Long-Term, Transcription-Dependent Alterations.- 3.5. Neurite Outgrowth.- 3.6. Synapse Formation.- 3.7. Antigenic Alterations.- 3.8. Effect of Dexamethasone.- 4. Subclones.- 5. Comparison with Adrenal Chromaffin Cells in Culture.- 6. Comparison with Sympathetic Neurons in Culture.- 7. PC12 Cells as a Model for the Early Events in Neuronal Differentiation.- 8. Conclusion.- References.- 9. Neural Differentiation of Pluripotent Embryonal Carcinoma Cells.- 1. Description of the Cellular Systems.- 1.1. PCC7-S AzaRl, Clone 1009.- 1.2. P19 Cell Line.- 1.3. Cl7Sl, Clone 1003.- 1.4. F9 Cell Line.- 2. Biochemical Studies.- 2.1. Changes in Intermediate Filament Proteins during 1003 Differentiation.- 2.2. Specific Changes in Protein Patterns of 1003 and 1009 Cells during Neural Differentiation.- 3. Conclusion.- References.- II. Electrophysiology.- 10. Neuronal Development in Culture: Role of Electrical Activity.- 1. Introduction.- 2. Methods Overview for Developmental Studies.- 2.1. Cell Culture Techniques.- 2.2. Analytical Probes.- 2.3. Neurochemical-Specific Assays.- 3. Description of Development.- 3.1. Morphology.- 3.2. Electrophysiological Development.- 3.3. Biochemical Development.- 4. Electrical Activity and Neuronal Development.- 4.1. Overview.- 4.2. Electrical Blockade and Neuronal Survival.- 4.3. Critical Developmental Periods.- 4.4. Trophic Factors.- 5. Summary.- References.- 11. Electrophysiological Studies of Cultured Mammalian CNS Neurons.- 1. Introduction.- 2. Methods Overview.- 2.1. Mechanical Requirements.- 2.2. Electronic Requirements.- 3. Membrane Excitability.- 3.1. Chemical Excitability.- 3.2. Electrical Excitability.- 4. Conclusion.- References.- 12.…