Bringing together the latest information into one easily accessible resource, The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology explores the diverse tools and technologies used to study synaptic processes. Written by world-renowned leaders in the field, the book delineates newly developed techniques, methods, and conceptual advances used for studying neurotransmitter receptors and other synaptic proteins.

A broad array of molecular, biochemical, imaging, and electrophysiological approaches for studying the biology of synapses are described. Specific topics include the use of proteomics to study synaptic protein complexes, the development of phosphorylation state-specific antibodies, post-genomic tools applied to the study of synapses, and RNA interference in neurons. In addition, several chapters focus on methods for gene and protein delivery into neuronal tissue. The use of biochemical, electrophysiological, and optical tagging techniques to study the movement and membrane trafficking of neurotransmitter receptors in the membranes of live nerve cells are also discussed. To complement these approaches, the application of state-of-the-art approaches for achieving long-term alterations in the genetic complement of neurons in vivo using viral vectors or homologous recombinations of ES cells is also described.

Contents

• Preface
• The Editors
• Contributors
• 1. Methods for Uncovering the Mechanisms of AMPA Receptor Trafficking
  Sophie Restituito and Edward B. Ziff.
  • 1.1 INTRODUCTION
  • 1.2 APPROACHES USED IN THE STUDY OF AMPA RECEPTOR TRAFFICKING
  • 1.3 SYNTHESIS, SUMMARY AND SPECULATION
  • REFERENCES
• 2. Long-Term Plasticity at Inhibitory Synapses: A Phenomenon That Has Been Overlooked
  Jean-Luc Gaiarsa and Yehezkel Ben-Ari.
  • 2.1 INTRODUCTION
  • 2.2 LONG-TERM CHANGES IN THE STRENGTH OF INHIBITORY SYNAPSES
  • 2.3 CONCLUSION
  • REFERENCES
• 3. New Tricks for an Old Dog: Proteomics of the PSD
  Bryen A. Jordan, Brian D. Fernholz, Thomas A. Neubert, and Edward B. Ziff.
  • 3.1 INTRODUCTION
  • 3.2 THE BRAIN AND THE PSD
  • 3.3 METHODS
  • 3.4 A COMPARISON OF PSD PROTEOMICS STUDIES
  • 3.5 CHALLENGES
  • 3.6 CONCLUSIONS
  • REFERENCES
• 4. Phosphorylation Site-Specific Antibodies as Research Tools in Studies of Native GABA_A Receptors
  Jasmina N. Jovanovic.
  • 4.1 INTRODUCTION
  • 4.2 DESIGN, PRODUCTION AND CHARACTERIZATION OF GABA_A RECEPTOR PHOSPHO-SITE SPECIFIC ANTIBODIES
  • 4.3 CONCLUDING REMARKS
  • REFERENCES
• 5. Protein Palmitoylation by DHHC Protein Family
  Yuko Fukata, David S. Bredt, and Masaki Fukata.
  • 5.1 INTRODUCTION
  • 5.2 OVERVIEW OF PAT SCREENING
  • 5.3 PROTOCOLS
  • REFERENCES
• 6. Studying the Localization, Surface Stability and Endocytosis of Neurotransmitter Receptors by Antibody Labeling and Biotinylation Approaches
  I. Lorena Arancibia-Cárcamo, Benjamin P. Fairfax, Stephen J. Moss, and Josef T. Kittler.
  • 6.1 INTRODUCTION
  • 6.2 ENDOCYTOSIS
  • 6.3 METHODS FOR STUDYING ENDOCYTOSIS
  • 6.4 CONCLUSION
  • REFERENCES
• 7. Visualization of AMPAR Trafficking and Surface Expression
  Pavel V. Perestenko and Jeremy M. Henley.
  • 7.1 INTRODUCTION
  • 7.2 FLUORESCENT PROTEIN TOOLS
  • 7.3 APPLICATION OF XFP IMAGING TO AMPARS
  • 7.4 FUTURE IMAGING POSSIBILITIES
  • 7.5 CONCLUSION
  • REFERENCES
• 8. Neurotransmitter Dynamics
  Sabine Lévi and Antoine Triller.
  • 8.1 INTRODUCTION
  • 8.2 RECEPTOR TRAFFICKING
  • 8.3 RECEPTOR DYNAMICS
  • REFERENCES
• 9. Receptor Dynamics at the Cell Surface Studied Using Functional Tagging
  Philip Thomas and Trevor G. Smart.
  • 9.1 INTRODUCTION
  • 9.2 PERSPECTIVE ON NONFUNCTIONAL RECEPTOR TAGGING TECHNIQUES
  • 9.3 EARLY STUDIES USING FUNCTIONAL TAGGING
  • 9.4 THE NATURE OF THE EPITOPE: CRITERIA FOR SELECTING A FUNCTIONAL TAG
  • 9.5 ADVANTAGES OF THE FUNCTIONAL TAG IN THE STUDY OF MOBILE RECEPTORS
  • 9.6 AMPA RECEPTOR RECTIFICATION AND SYNAPTIC “AMPAFICATION”
  • 9.7 MK801 CHANNEL BLOCK EXPOSES THE TRANSIENT NATURE OF SYNAPTIC NMDA RECEPTORS
  • 9.8 SYNAPTIC INHIBITION: THE MOBILITY OF EXTRASYNAPTIC GABA_A RECEPTORS
  • 9.9 PHOTORECEPTIVE POTASSIUM CHANNELS AS SWITCHES OF NEURONAL EXCITABILITY
  • 9.10 DISADVANTAGES AND LIMITATIONS OF THE FUNCTIONAL TAG
  • 9.11 CONCLUSION
  • REFERENCES
• 10. RNAi and Applications in Neurobiology
  Tija C. Jacob.
  • 10.1 INTRODUCTION TO RNAi
  • 10.2 RNAi MECHANISM
  • 10.3 GENERATING RNAi — TYPES OF RNAi
  • 10.4 RNAi DELIVERY — SPECIFIC METHODS FOR EMPLOYING RNAi IN VARIOUS EXPERIMENTAL SYSTEMS
  • 10.5 RNAi EXPERIMENTAL DESIGN STRATEGY
  • 10.6 CONCLUSION
  • REFERENCES
• 11. Transfecting and Transducing Neurons with Synthetic Nucleic Acids and Biologically Active Macromolecules
  Josef T. Kittler, Jonathan G. Hanley, and John T. R. Isaac.
  • 11.1 INTRODUCTION
  • 11.2 METHODS FOR NEURONAL GENE TRANSFER
  • 11.3 VIRAL APPROACHES
  • 11.4 CHEMICAL AND PHYSICAL TRANSFECTION APPROACHES
  • 11.5 ELECTRICAL APPROACHES: ELECTROPORATION AND NUCLEOFECTION
  • 11.6 TRANSDUCING NEURONS WITH BIOLOGICALLY ACTIVE PEPTIDES, PROTEINS AND ANTIBODIES
  • 11.7 FUNCTIONAL STUDIES USING FUNCTION BLOCKING AND PROTEIN–PROTEIN INTERACTION-BLOCKING PEPTIDES, PROTEINS AND ANTIBODIES
  • 11.8 TRANSDUCING NEURONS WITH MEMBRANE-PERMEANT PEPTIDES AND PROTEINS
  • 11.9 CONCLUSION
  • REFERENCES
• 12. Acute In Vivo Expression of Recombinant Proteins in Rat Brain Using Sindbis Virus
  Hélène Marie and Robert C. Malenka.
  • 12.1 INTRODUCTION
  • 12.2 GENERATION OF SINDBIS VIRAL PARTICLES
  • 12.3 IN VIVO INJECTION OF VIRAL PARTICLES
  • 12.4 USE OF ACUTE IN VIVO EXPRESSION OF RECOMBINANT PROTEINS FOR CELL-BASED ASSAYS
  • 12.5 CONCLUSIONS
  • REFERENCES
• 13. Lentivirus-Based Genetic Manipulations in Neurons In Vivo
  Pavel Osten, Tanjew Dittgen, and Pawel Licznerski.
  • 13.1 INTRODUCTION
  • 13.2 LENTIVIRUS-BASED VECTOR SYSTEMS
  • 13.3 LENTIVIRUS-BASED HETEROLOGOUS EXPRESSION
  • 13.4 LENTIVIRUS-BASED GENETIC MANIPULATIONS
  • 13.5 DELIVERY OF LENTIVIRAL PARTICLES INTO THE BRAIN
  • 13.6 EXPERIMENTAL APPLICATIONS
  • 13.7 CONCLUSIONS
  • REFERENCES
• 14. AMPA Receptor Phosphorylation in Synaptic Plasticity: Insights from Knockin Mice
  Hey-Kyoung Lee.
  • 14.1 INTRODUCTION
  • 14.2 AMPA RECEPTORS AND CHANGES IN PHOSPHORYLATION DURING LTP AND LTD
  • 14.3 CHANGES IN GLUR1 WITH LTP AND LTD
  • 14.4 GLUR1 PHOSPHORYLATION: EARLY EXPRESSION VERSUS LATE MAINTENANCE OF LTP?
  • 14.5 DIFFERENT MECHANISMS OF LTP IN YOUNG VERSUS OLD
  • 14.6 ROLE OF PKA IN LTP
  • 14.7 GLUR1 PHOSPHORYLATION SITES IN LTD AND RECEPTOR TRAFFICKING
  • 14.8 POTENTIAL INTERACTION BETWEEN GLUR1 AND GLUR2 PHOSPHORYLATION SITES DURING LTD
  • 14.9 CONCLUSION
  • REFERENCES
• 15. Genomic and Post-Genomic Tools for Studying Synapse Biology
  Josef T. Kittler and Peter L. Oliver.
  • 15.1 INTRODUCTION
  • 15.2 ORFEOME PROJECTS: THE MAMMALIAN GENE COLLECTION AND OTHER PROVIDERS OF FULL-LENGTH cDNA COLLECTIONS
  • 15.3 INFERRING NEW GENE FUNCTION FROM PROTEIN-PROTEIN INTERACTION DATASETS
  • 15.4 WHOLE GENOME shRNAi LIBRARIES
  • 15.5 THE MOUSE AS A CORNERSTONE FOR INTEGRATING GENOMIC AND POSTGENOMIC RESOURCES
  • 15.6 CONCLUSION
  • REFERENCES

Series editors: Sidney A Simon and Miguel AL Nicolelis

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use.