Guide to Research Techniques in Neuroscience

by Carter; Shieh

• ISBN13: 9780123748492
• ISBN10: 0123748496
• Edition: 1st
• Format: Paperback
• Copyright: 2009-09-22
• Publisher: Elsevier Science

Summary

Neuroscience is by definition a multidisciplinary field: some scientists study genes and proteins at the molecular level while others study neural circuitry. A single topic such as the auditory system can be studied using techniques from genetics, imaging, biochemistry, or electrophysiology. A young scientist must learn how to read the primary literature and then develop their own experiments. This book offers that scientist an overview of mainstream research techniques, provides guidelines on how to choose one technique over another, offers tips on analyzing data, and provides a list of references for additional detailed study. This book can also assist an experienced scientist understand published studies conducted outside their own subfield.*Written by Stanford University graduate students in neuroscience to provide a "hands-on" approach for other neuroscience graduate students *Techniques within one field will be compared so that user can select best technique for their experiment *Chapters include references (key articles, books, protocols) for additional detailed study *Data Analysis boxes in each chapter help with data interpretation and offer guidelines on how best to represent results *"Walk-through" boxes guide students through the experiment step-by-step

Table of Contents

Preface	p. xiii
Foreword	p. xvii
Introduction	p. xix
Whole Brain Imaging	p. 1
Structural Brain Imaging Techniques	p. 2
Cerebral Angiography	p. 3
Computerized Tomography (CT)	p. 4
Magnetic Resonance Imaging (MRI)	p. 6
Diffusion Magnetic Resonance imaging (diffusion MRI)	p. 12
Functional Brain Imaging Techniques	p. 13
Functional Magnetic Resonance Imaging (FMRI)	p. 14
Positron Emission Tomography (PET)	p. 18
Single-Proton Emission Computerized Tomography (SPECT)	p. 19
Electroencephalography (EEC)	p. 19
Magnetoencephalography (MEG)	p. 20
Optical Imaging	p. 21
Functional Imaging Experimental Design and Analysis	p. 23
Planning the Experiment	p. 24
Conducting the Experiment	p. 28
Manipulating Neural Activity during an Experiment	p. 30
Postexperimental Data Analysis	p. 33
Conclusion	p. 36
Suggested Reading and References	p. 36
Animal Behavior	p. 39
Considerations for Choosing and Performing a Behavioral Assay	p. 40
Choosing an Appropriate Model Organism	p. 40
Choosing an Appropriate Behavioral Paradigm	p. 41
Variability in Individuals	p. 43
Using Animal Behavior as a Model for Human Behavior	p. 43
Rodent Behavioral Paradigms	p. 44
Locomotor Activity	p. 44
Motor Coordination and Balance	p. 46
Sensory Function	p. 49
Nociception	p. 51
Spatial Learning and Memory	p. 52
Nonspatial Learning and Memory	p. 55
Social Behaviors	p. 56
Anxiety	p. 57
Depression	p. 59
Drosophila Behavioral Paradigms	p. 61
Locomotor Behavior	p. 61
Flight	p. 62
Sensory Function	p. 62
Learning and Memory	p. 65
Social Behaviors	p. 65
C. elegans Behavioral Paradigms	p. 66
Locomotor Behavior	p. 66
Sensory Behavior	p. 67
Nonhuman Primate Behavioral Paradigms	p. 69
Conclusion	p. 69
Suggested Reading and References	p. 69
Stereotaxic Surgeries and In Vivo Techniques	p. 73
Stereotaxic Surgeries	p. 74
Stereotaxic Surgeries in Rodents	p. 76
Stereotaxic Surgeries in Nonhuman Primates	p. 79
Implants for Long-Term Access to the Brain	p. 81
Sealable Chambers	p. 81
Cannulae	p. 81
Cranial Windows	p. 81
Measuring Neural Activity In Vivo	p. 82
Electrophysiology	p. 82
Fluorescent Activity Indicators	p. 82
Measuring Neurochemistry in Vivo	p. 82
Microdialysis	p. 83
Voltammetry and Amperometry	p. 84
Manipulating the Brain In Vivo	p. 87
Physical Manipulation	p. 87
Pharmacological Manipulation	p. 87
Electrical Manipulation	p. 88
Genetic Manipulation	p. 89
Conclusion	p. 89
Suggested Reading and References	p. 89
Electrophysiology	p. 91
A Brief Review of the Electrical Properties of Neurons	p. 92
The Electrophysiology Rig	p. 95
Types of Electrophysiology Recordings	p. 99
Extracellular Recording	p. 103
Intracellular Recording	p. 107
Patch Clamp Techniques	p. 108
Electrophysiology Tissue Preparations	p. 110
In Vitro Recordings	p. 111
In Vivo Recordings	p. 115
Methods of Manipulating Neurons During Electrophysiology Experiments	p. 116
Conclusion	p. 117
Suggested Reading and References	p. 117
Microscopy	p. 119
Essential Principles of Microscopy	p. 120
Fundamental Parameters in Microscopy	p. 120
The Design of a Compound Microscope	p. 123
The Design of a Stereomicroscope	p. 127
Light Microscopy	p. 129
Fluorescence Microscopy	p. 129
Epifluorescent Microscopy	p. 132
Confocal Microscopy	p. 132
Two-Photon Microscopy	p. 133
Total Internal Reflection Fluorescence (TIRF) Microscopy	p. 134
Electron Microscopy	p. 135
Transmission Electron Microscopy (TEM)	p. 136
Scanning Electron Microscopy (SEM)	p. 136
Electron Tomography (ET)	p. 137
Preparing and Interpreting Microscopy Data	p. 141
Image Processing	p. 141
Interpreting Images	p. 142
Conclusion	p. 144
Suggested Reading and References	p. 144
Visualizing Nervous System Structure	p. 147
Tissue Preparation	p. 148
Fixation	p. 148
Embedding	p. 149
Sectioning	p. 149
Visualizing Morphology	p. 151
Cell Body Stains	p. 152
Fiber Stains	p. 153
Golgi Stain	p. 153
Intracellular and Juxtacellular Labeling	p. 154
Fluorescent Labels	p. 156
Chromogenic/Colorimetric Labels	p. 156
Radioactive Labels	p. 157
Cold Labels	p. 157
Visualizing Gene and Protein Expression	p. 157
In Situ Hybridization	p. 158
Immunohistochemistry	p. 160
Enzymatic Histochemistry	p. 161
Reporter Genes	p. 161
Visualizing Circuitry	p. 164
Anterograde and Retrograde Tracers	p. 164
Transsynaptic Labeling	p. 166
Conclusion	p. 167
Suggested Reading and References	p. 167
Visualizing Nervous System Function	p. 169
Static Markers of Activity	p. 170
Assaying Neural Activity in Fixed Tissue	p. 171
Assaying Cellular Function in Fixed Tissue	p. 172
Visualizing Neural Activity	p. 173
Imaging Voltage	p. 173
Imaging Calcium	p. 175
Imaging Synaptic Transmission	p. 178
Optically Manipulating Neural Activity	p. 178
Stimulation through Uncaging of Molecules	p. 179
Light-Activated Channels	p. 179
Visualizing Protein Function	p. 182
Time-Lapse Imaging with Reporter Genes	p. 182
Fluorescence Resonance Energy Transfer (FRET)	p. 182
Bimolecular Fluorescence Complementation (BiFC)	p. 184
Fluorescence Recovery after Photo bleaching (FRAP)	p. 184
Photoactivation and Photoconversion	p. 184
Optically Manipulating Protein Function	p. 185
Photoactivation/Photo-uncaging	p. 185
Photoinactivation	p. 185
Conclusion	p. 187
Suggested Reading and References	p. 188
Identifying Genes and Proteins of Interest	p. 191
A Brief Review of Genes and Proteins	p. 192
The Central Dogma of Molecular Biology	p. 192
DNA	p. 193
Transcription	p. 195
Translation	p. 195
Genetic Screens	p. 197
Forward Genetic Screen	p. 198
Reverse Genetic Screen	p. 202
In Silico Screens	p. 202
Blast	p. 203
Ensembl	p. 203
Molecular Screens	p. 203
cDNA Microarray Screen	p. 203
RNAi Screen	p. 205
Conclusion	p. 205
Suggested Reading and References	p. 205
Molecular Cloning and Recombinant DNA Technology	p. 207
Isolating DNA Fragments	p. 208
Restriction Enzymes	p. 208
Polymerase Chain Reaction (PCR)	p. 210
Cloning DNA	p. 215
Vectors	p. 216
Ligation	p. 217
Transformation	p. 219
Purifying DNA	p. 219
Isolation and Characterization of DNA Fragments Using Gel Electrophoresis	p. 220
Purifying DNA from Host Cells	p. 221
Identifying DNA	p. 223
DNA Sequencing	p. 223
Nucleic Acid Hybridization Techniques	p. 224
Conclusion	p. 226
Suggested Reading and References	p. 227
Gene Delivery Strategies	p. 229
Physical Gene Delivery	p. 230
Microinjection	p. 230
Electroporation	p. 231
Biolistics	p. 234
Chemical Gene Delivery	p. 235
Calcium Phosphate Transfection	p. 235
Lipid Transfection	p. 236
Viral Gene Delivery	p. 237
Adenovirus	p. 239
Adeno-Associated Virus (AAV)	p. 240
Lentivirus	p. 240
Herpes Simplex Virus (HSV)	p. 240
Conclusion	p. 240
Suggested Reading and References	p. 241
Making and Using Transgenic Organisms	p. 243
Transgenes	p. 244
Reporter Genes	p. 244
Genes Used to Ablate Neurons	p. 246
Genes Used to Measure Neural Activity	p. 247
Genes Used to Manipulate Neural Activity	p. 247
Genes Used to Disrupt Endogenous Gene Function	p. 248
Overexpression or Misexpression of Endogenous Genes	p. 248
The Transgenic Construct	p. 248
Binary Transgenic Systems	p. 251
The Gal4/UAS System	p. 252
The Cre/Lox System	p. 253
The Flp/Frt System	p. 254
TheTet-off/Tet-on System	p. 254
Making Transgenic Organisms	p. 255
Making Transgenic Mice	p. 255
Making Transgenic Flies	p. 257
Making Transgenic Worms	p. 258
Making Other Transgenic Organisms	p. 260
Conclusion	p. 261
Suggested Reading and References	p. 261
Manipulating Endogenous Genes	p. 263
Genetic Targeting	p. 264
Knockout	p. 265
Knockin	p. 272
Conditional Knockout	p. 273
Disrupting Gene Products	p. 275
RNA Interference (RNAi)	p. 275
Morpholtnos	p. 277
Dominant Negatives	p. 277
Conclusion	p. 278
Suggested Reading and References	p. 279
Cell Culture Techniques	p. 281
Cell Culture Equipment and Reagents	p. 282
Equipment	p. 282
Culture Media	p. 284
Immortalized Cell Lines	p. 284
Primary Cell and Tissue Culture	p. 287
Dissociated Cell Cultures	p. 288
Slice Cultures	p. 289
Explant Cultures	p. 289
Stem Cell Cultures	p. 290
Embryonic Stem Cells	p. 290
Neural Stem Cells	p. 291
Induced Plurrpotent Stem Cells	p. 292
Manipulating Cells in Culture	p. 293
Transfection	p. 293
Coculture Systems	p. 293
Pharmacology	p. 294
Antibody Interference	p. 294
Conclusion	p. 295
Suggested Reading and References	p. 295
Biochemical Assays and Intracellular Signaling	p. 297
Introduction to Signal Transduction and Intracellular Signaling	p. 298
Fundamental Tools for Studying Proteins	p. 300
Making and Using Antibodies	p. 300
Purifying Proteins	p. 302
Immunoprecipitation (IP)	p. 304
Investigating Protein Expression	p. 306
Western Blot (WB)	p. 306
Enzyme-Linked Immunosorbent Assay (ELISA)	p. 308
Radioimmunoassay (RIA)	p. 310
Immunohistochemistry (IHC)	p. 312
Immunoelectron Microscopy (IEM)	p. 312
Reporter Proteins	p. 312
Investigating Protein-Protein Interactions	p. 313
Coimmunoprecipitation (Co-IP)	p. 313
Protein Affinity Chromatography	p. 313
Yeast Two-Hybrid Assay	p. 314
Investigating Posttranslational Modifications	p. 317
PTM-Specific Assays	p. 317
PTM-Specific Antibodies	p. 320
Investigating Protein-DNA Interactions	p. 321
Electrophoretic Mobility Shift Assay (EMSA)	p. 323
Chromatin Immunoprecipitation (ChIp)	p. 325
Luciferase Assay	p. 327
Conclusion	p. 328
Suggested Reading and References	p. 328
Glossary	p. 331
Index	p. 363
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