The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology (Frontiers in Neuroscience)

THE DYNAMIC SYNAPSE, MOLECULAR METHODS IN IONOTROPIC RECEPTOR BI0LOGY......Page 5
Table of Contents......Page 13
FRONTIERS IN NEUROSCIENCE......Page 3
Preface......Page 7
The Editors......Page 9
Contributors......Page 10
1.1 INTRODUCTION......Page 16
1.2.1 PROTEIN INTERACTION ASSAYS......Page 18
1.2.2.1 Microscopy......Page 22
1.2.2.2 Cell Biology Assays......Page 26
1.2.3.2 Phosphorylation......Page 27
1.2.4.2 Rectification Index......Page 28
1.2.4.3 Agonists and Toxins......Page 29
1.2.6.2 Knockout Mice......Page 30
1.3 SYNTHESIS, SUMMARY AND SPECULATION......Page 31
REFERENCES......Page 32
2.1 INTRODUCTION......Page 38
2.2.1 MINIMAL REQUIREMENTS AND CHARACTERISTICS......Page 39
2.2.2 EXPRESSION......Page 44
2.2.3 FUNCTIONAL RELEVANCE......Page 46
2.3 CONCLUSION......Page 47
REFERENCES......Page 48
3.1 INTRODUCTION......Page 52
3.1.1 WHY PROTEOMICS ?......Page 53
3.2 THE BRAIN AND THE PSD......Page 54
3.2.1 PROTEOMICS OF THE PSD......Page 55
3.3.1 2DE-LC-MS/MS......Page 56
3.3.2 1DE-LC-MS/MS......Page 57
3.3.4 RELATIVE QUANTIFICATION BY STABLE ISOTOPE LABELING AND MASS SPECTROMETRY......Page 58
3.3.5 IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)......Page 59
3.3.6 AFFINITY PURIFICATION......Page 60
3.4 A COMPARISON OF PSD PROTEOMICS STUDIES......Page 61
3.5 CHALLENGES......Page 62
3.6 CONCLUSIONS......Page 64
REFERENCES......Page 65
CONTENTS......Page 72
4.1 INTRODUCTION......Page 73
4.2 DESIGN, PRODUCTION AND CHARACTERIZATION OF GABAA RECEPTOR PHOSPHO-SITE SPECIFIC ANTIBODIES......Page 78
4.2.1 PHOSPHOPEPTIDE DESIGN AND PREPARATION......Page 79
4.2.2.1 Phosphopeptide Coupling Protocol Based on Terminal Cysteine Residue......Page 80
4.2.3 RABBIT IMMUNIZATION PROTOCOL......Page 83
4.2.4.1 First Screen: Dot-Immunoblotting of Synthetic Peptides......Page 84
4.2.4.2 Second Screen: Immunoblotting of Dephospho/Phospho Forms of the Holoprotein......Page 85
4.2.4.3 Third Screen: Expression of wt and Phospho-Site Mutants in Heterologous Cell Line Systems......Page 86
4.2.4.4 Fourth Screen: Dephospho/Phospho Peptide Block......Page 87
4.2.5.1 Purification of Total IgGs from the Phospho-Specific Antiserum......Page 90
4.2.5.2 Purification of Phospho-Specific Antibodies Using Affinity Chromatography with Peptide Columns......Page 91
REFERENCES......Page 93
5.1 INTRODUCTION......Page 98
5.2 OVERVIEW OF PAT SCREENING......Page 99
5.3.1 TRANSFECTION......Page 100
5.3.4 NOTES......Page 101
REFERENCES......Page 103
CONTENTS......Page 106
6.2.1 CLATHRIN AND NON-CLATHRIN MEDIATED ENDOCYTOSIS......Page 107
6.2.2 THE ENDOSOMAL SYSTEM AND THE RAB GTPASES......Page 109
6.2.2.1 Rab GTPases......Page 110
6.2.3 SIGNALS FOR ENDOCYTOSIS AND POST-ENDOCYTIC SORTING......Page 111
6.3.1 ANTIBODY FEEDING......Page 113
6.3.2 CELL SURFACE BIOTINYLATION......Page 118
6.3.2.1 Biotinylation Method......Page 119
6.3.2.3 Endocytosis......Page 121
6.3.2.4 Further Trafficking Events......Page 122
6.3.2.5 Biotinylation of Slices......Page 123
6.3.2.6 Degradation Assays......Page 124
6.3.3 OTHER USEFUL TOOLS FOR STUDYING NEUROTRANSMITTER RECEPTOR ENDOCYTOSIS AND POST-ENDOCYTIC SORTING......Page 125
6.4 CONCLUSION......Page 127
REFERENCES......Page 128
CONTENTS......Page 134
7.1.1 AMPARS......Page 135
7.1.2 TRANSPORT AND TARGETING OF AMPARS WITHIN NEURONS......Page 136
7.2.1 GREEN FLUORESCENT PROTEINS......Page 137
7.2.2 CONSTRUCTION AND USE OF FLUORESCENTLY LABELED PROTEINS......Page 138
7.2.3 IMAGING HARDWARE......Page 139
7.2.5 GENERAL APPLICATIONS OF XFPS IN CONFOCAL MICROSCOPY......Page 140
7.3.1 VISUALIZING INTRACELLULAR MOVEMENT OF GFP-GLUR1 AND GLU R2......Page 141
7.3.2 PH-SENSITIVE GFP (PHLUORIN) TO MONITOR AMPAR SURFACE EXPRESSION AND DIFFUSION......Page 145
7.4.1 H SENSORS......Page 147
7.4.4 FLUORESCENT TIMER......Page 148
7.6 ACKNOWLEDGMENTS......Page 149
REFERENCES......Page 150
8.1 INTRODUCTION......Page 158
8.2.1 EXTRASYNAPTIC POOL OF RECEPTORS......Page 159
8.2.2 INTRACELLULAR POOL OF RECEPTORS......Page 160
8.3.1 IMPORTANCE OF THE DIFFUSION-TRAP MECHANISM IN SYNAPTIC RECEPTOR AGGREGATION......Page 161
8.3.3.1 Extrasynaptic Receptors......Page 162
8.3.4.1 Excitatory Glutamate Receptors......Page 164
8.3.4.2 Inhibitory Glycine Receptors......Page 165
REFERENCES......Page 166
9.1 INTRODUCTION......Page 170
9.2 PERSPECTIVE ON NONFUNCTIONAL RECEPTOR TAGGING TECHNIQUES......Page 171
9.3 EARLY STUDIES USING FUNCTIONAL TAGGING......Page 173
9.4 THE NATURE OF THE EPITOPE: CRITERIA FOR SELECTING A FUNCTIONAL TAG......Page 175
9.5 ADVANTAGES OF THE FUNCTIONAL TAG IN THE STUDY OF MOBILE RECEPTORS......Page 176
9.6 AMPA RECEPTOR RECTIFICATION AND SYNAPTIC “AMPAFICATION”......Page 177
9.7 MK801 CHANNEL BLOCK EXPOSES THE TRANSIENT NATURE OF SYNAPTIC NMDA RECEPTORS......Page 180
9.8 SYNAPTIC INHIBITION: THE MOBILITY OF EXTRASYNAPTIC GABA A RECEPTORS......Page 182
9.9 PHOTORECEPTIVE POTASSIUM CHANNELS AS SWITCHES OF NEURONAL EXCITABILITY......Page 184
9.10 DISADVANTAGES AND LIMITATIONS OF THE FUNCTIONAL TAG......Page 185
REFERENCES......Page 187
CONTENTS......Page 192
10.1.1 MIRNAS......Page 193
10.1.2 CHAPTER OVERVIEW......Page 194
10.2.1 EARLY DAYS—HISTORICAL PERSPECTIVE......Page 195
10.2.3 RISC COMPLEX ASSEMBLY AND ACTIVITY......Page 196
10.2.4 ORGANISM-SPECIFIC VARIATIONS IN THE RNAI MECHANISM......Page 197
10.3.2 HAIRPIN DSRNAS AND SHRNAS......Page 198
10.3.3 INDUCIBLE SYSTEMS......Page 199
10.4.1 C. ELEGANS......Page 200
10.4.3.1 Mammalian Cell Culture and Primary Culture......Page 201
10.4.3.2 Oocytes, Pre- and Post-Implantation Embryos, and Post-Natal Animals......Page 203
10.4.3.3 Adult Mice......Page 204
10.4.3.4 Stable Inheritable Genetic Knockdown — Transgenic Mice and Rats......Page 205
10.5.1 SIRNA/SHRNA DESIGN......Page 206
10.5.2 SPECIFICITY CONCERNS: OFF-TARGET EFFECTS AND THE INTERFERON RESPONSE......Page 207
10.6 CONCLUSION......Page 208
REFERENCES......Page 209
CONTENTS......Page 220
11.1 INTRODUCTION......Page 221
11.3.1 THE SINDBIS VIRUS SYSTEM......Page 222
11.3.1.2 Viral Infection of Neurons in Intact Tissues: The Acute Slice Method......Page 223
11.3.2 OTHER VIRAL SYSTEMS......Page 224
11.4.1 MICROINJECTION AND BIOLISTICS......Page 226
11.4.2 CHEMICAL APPROACHES......Page 227
11.5 ELECTRICAL APPROACHES: ELECTROPORATION AND NUCLEOFECTION......Page 228
11.6.1 FUNCTION BLOCKING AND PROTEIN–PROTEIN INTERACTION BLOCKING PEPTIDES, PROTEINS AND ANTIBODIES......Page 230
11.6.2 IDENTIFICATION OF PROTEIN–PROTEIN BINDING DOMAINS AND DESIGN OF PROTEIN–PROTEIN INTERACTION BLOCKING PEPTIDES......Page 233
11.7.1 INTRODUCING PEPTIDES AND PROTEINS INTO NEURONS VIA THE ELECTROPHYSIOLOGICAL RECORDING PIPETTE......Page 236
11.7.1.1 Some Considerations......Page 238
11.8 TRANSDUCING NEURONS WITH MEMBRANEPERMEANT PEPTIDES AND PROTEINS......Page 239
11.9 CONCLUSION......Page 240
11.10 ACKNOWLEDGMENTS......Page 243
REFERENCES......Page 244
12.1 INTRODUCTION......Page 256
12.2 GENERATION OF SINDBIS VIRAL PARTICLES......Page 257
12.3 IN VIVO INJECTION OF VIRAL PARTICLES......Page 258
12.4 USE OF ACUTE IN VIVO EXPRESSION OF RECOMBINANT PROTEINS FOR CELL-BASED ASSAYS......Page 259
REFERENCES......Page 262
13.1 INTRODUCTION......Page 264
13.3 LENTIVIRUS-BASED HETEROLOGOUS EXPRESSION......Page 265
13.4 LENTIVIRUS-BASED GENETIC MANIPULATIONS......Page 267
13.5 DELIVERY OF LENTIVIRAL PARTICLES INTO THE BRAIN......Page 268
13.6 EXPERIMENTAL APPLICATIONS......Page 269
REFERENCES......Page 271
14.1 INTRODUCTION......Page 276
14.2 AMPA RECEPTORS AND CHANGES IN PHOSPHORYLATION DURING LTP AND LTD......Page 277
14.3 CHANGES IN GLUR1 WITH LTP AND LTD......Page 278
14.4 GLUR1 PHOSPHORYLATION: EARLY EXPRESSION VERSUS LATE MAINTENANCE OF LTP?......Page 279
14.5 DIFFERENT MECHANISMS OF LTP IN YOUNG VERSUS OLD......Page 281
14.6 ROLE OF PKA IN LTP......Page 282
14.8 POTENTIAL INTERACTION BETWEEN GLUR1 AND GLUR2 PHOSPHORYLATION SITES DURING LTD......Page 283
14.9 CONCLUSION......Page 285
REFERENCES......Page 286
15.1 INTRODUCTION......Page 294
15.2 ORFEOME PROJECTS: THE MAMMALIAN GENE COLLECTION AND OTHER PROVIDERS OF FULLLENGTH cDNA COLLECTIONS......Page 295
15.3 INFERRING NEW GENE FUNCTION FROM PROTEIN-PROTEIN INTERACTION DATASETS......Page 298
15.4 WHOLE GENOME shRNAi LIBRARIES......Page 301
15.5 THE MOUSE AS A CORNERSTONE FOR INTEGRATING GENOMIC AND POSTGENOMIC RESOURCES......Page 303
15.5.1 A MOUSE KNOCKOUT FOR EVERY GENE......Page 304
15.5.2.1 Spontaneous Mouse Mutants......Page 305
15.5.2.2 Stargazer, Lurcher and Hotfoot Mutants: Insights into Glutamate Receptor Gating, Assembly and Trafficking......Page 306
15.5.2.3 Large-Scale Neurological Random Mutagenesis Screens......Page 308
15.5.2.4 New Strategies for the Generation and Identification of Mouse Mutants......Page 309
REFERENCES......Page 310
Colorplates......Page 322