The Fly Neuromuscular Junction: Structure and Function

Description

The Drosophila larval neuromuscular junction (NMJ) has become one of the most powerful model systems to ask key neurobiological questions. This synapse is unparalleled by its accessibility, its simplicity, and the ability to manipulate genes important for synapse development and function. Its synapses have properties shared by many organisms including humans. The vast majority of genes that when mutated cause congenital disorders of the nervous system in humans, are present in the fruit fly genome, and fly models of human disorders are available. Thus, this preparation is a powerful tool to understand the normal function of these genes.
This book reviews outstanding work by recognized leaders in the fields of Drosophila cellular neurogenetics including developmental neurobiology, mechanisms of synaptic function, and experience dependent changes at synapses. The book also includes step-by-step protocols to study the cellular biology of the NMJ, making it a vital resource for researchers beginning their investigations with this system, for those who are training students and postdoctoral fellows in this area, or simply as a general reference material for neuroscientists and neuroscience professors in general.

* Provides a synthesis of the main topics in modern neurogenetics
* Includes step-by-step protocols for the use of the Drosophila NMJ system in neurobiology lab research
* Offers genetic approaches to study synapse development and function
*

Cover 2
Copyright page 6
Table of contents 7
Contributors 13
Preface 15
Chapter 1: Introduction on the Use of The Drosophila Embryonic/Larval Neuromuscular Junction As A Model System to Study Synapse 17
- I. Introduction 18
- II. Some History 19
- III. The Larval NMJ Is Highly Tractable 20
- IV. Methodology to Study NMJ Development 22
- V. Pathfinding at the Larval NMJ 26
  - A. The Motor Pathways 27
  - B. Axon Choice Points 28
  - C. Axon Fasciculation 31
- VI. Synaptic Target Recognition 32

Cover 2
Copyright page 6
Table of contents 7
Contributors 13
Preface 15
Chapter 1: Introduction on the Use of The Drosophila Embryonic/Larval Neuromuscular Junction As A Model System to Study Synapse 17
- I. Introduction 18
- II. Some History 19
- III. The Larval NMJ Is Highly Tractable 20
- IV. Methodology to Study NMJ Development 22
- V. Pathfinding at the Larval NMJ 26
  - A. The Motor Pathways 27
  - B. Axon Choice Points 28
  - C. Axon Fasciculation 31
- VI. Synaptic Target Recognition 32
  - A. Targeting Accuracy 33
  - B. Attractant Molecules During Synaptic Target Recognition 34
  - C. Repulsive Molecules During Synaptic Target Recognition 35
- VII. Dynamics of the Target Muscle 36
- VIII. Conclusions 37
- Acknowledgment 38
- References 38
Chapter 2: Development and Structure of Motoneurons 49
- I. Introduction 50
- II. Development of Motoneurons 50
  - A. Early Specification Programs 50
  - B. Motoneuron Subclasses: Specification of Ventrally Versus Dorsally Projecting Motoneurons 52
  - C. Ventrally Projecting Motoneurons 52
  - D. Dorsally Projecting Motoneurons 54
  - E. Motoneuron Specification Programs Are Conserved 54
  - F. Specification of Unique Motoneuron Identities 55
  - G. From Motoneuron Determinants to Axon Trajectories 56
- III. Class-Specific Patterns of Axonal Trajectories 56
  - A. Subsets Within the Class of Ventrally Projecting Motoneurons 57
  - B. A Hierarchy of Nerve Branching 58
- IV. Dendritic Development 59
  - A. Dendrites: Conservation Between Insects and Vertebrates 59
  - B. Organization of the Motor System: Coordinated Patterning of Pre-and Postsynaptic Terminals 61
  - C. Regulation of Dendritic Patterning 62
- References 64
- V. Conclusions 64
- Acknowledgment 64
Chapter 3: The Development of The Drosophila Larval Body Wall Muscles 71
- I. Introduction 71
- II. Muscle Specification 73
- III. Muscle Identity 75
- IV. Myoblast Fusion 77
- V. Muscle Attachment 81
- Acknowledgment 83
- References 83
- VI. Concluding Remarks 83
Chapter 4: Organization of the Efferent System and Structure of Neuromuscular Junctions In Drosophila 87
- I. Introduction 87
- II. General Organization of the Efferent System 89
- III. Neuromodulation? The Organization of Type-II and Type-III Terminals 91
- IV. Synaptic Junctions: The Development, Morphology, and Classification of Type-I Terminals 95
- V. Ultrastructure of Neuromuscular Synapses 98
- VI. Conclusions 100
- Acknowledgment 100
- References 100
Chapter 5: Development of Motoneuron Electrical Properties and Motor Output 107
- I. Introduction 108
- II. Development of Electrical Properties in Motoneurons 108
- III. Development of Synaptic Connectivity 110
- IV. Regulation of Membrane Excitability 111
- V. Regulation of Synaptic Connectivity 114
  - A. Role of the Cell Adhesion Molecule FasciclinII 114
  - B. TGF-beta Retrograde Signaling Strengthens Both Central and Peripheral Synapses 117
- VI. Summary 118
- Acknowledgment 118
- References 118
Chapter 6: Transmitter Release at the Neuromuscular Junction 121
- I. Introduction 122
- II. Physiological Properties of Transmitter Release 123
- III. Experimental Advantages and Limitations of the Fly NMJ 126
  - B. Dye Loading 126
  - A. Ca2+ Measurements 126
  - C. Alternative Measures of Exocytosis 127
- IV. How Do Vesicles Fuse: Full Fusion or Kiss-and-Run? 128
- V. Core Machinery of Exocytosis: Syntaxin, VAMP/Synaptobrevin, and SNAP-25 130
  - A. Synaptobrevins in the Fly 131
  - B. Tetanus Toxin as a Synapse-Silencing Reagent 133
  - C. Syntaxin1 134
  - D. SNAP-25 and SNAP-24 136
  - E. SNARE Proteins Act Late in the Vesicle Cycle 137
  - F. Summary of SNARE Proteins in Drosophila 137
- VI. Vesicular ATPase and Membrane Fusion 138
- VII. NSF and the Resetting of the SNARE Machinery 139
- VIII. Synaptotagmin and the Regulation of Transmitter Release 141
  - A. Synaptotagmin and Its Binding Properties 141
  - B. Synaptotagmin Phenotypes at the NMJ 142
  - C. Other Functions of Synaptotagmin 144
  - D. Multiple Synaptotagmins in the Fly 145
  - E. Summary of Synaptotagmin Function at the Fly NMJ 146
- IX. Exocyst at the NMJ 146
- X. Other Mutations of Proteins on the Target Membrane 148
  - A. Rop/Unc-18/n-Sec1 148
  - B. Unc-13 and Vesicle Priming 149
  - C. CAST/ERK at the Active Zone 149
- XI. Mutations in Peripheral Synaptic Vesicle Proteins 150
  - A. Synapsin 150
  - B. Cysteine String Protein and Hsc70 151
- References 152
- XII. Summary 152
Chapter 7: Vesicle Trafficking and Recycling at The Neuromuscular Junction: Two Pathways for Endocytosis 161
- I. Introduction 162
- II. Role of Ca2+ During Endocytosis 163
  - A. Ca2+ Is Required for Endocytosis 163
  - B. Voltage-Gated Ca2+ Channels Are Involved in Endocytosis 164
  - C. Presynaptic Terminals Contain Multiple Types of Ca2+ Channels 165
  - D. Distribution of Ca2+ Channels in the Presynaptic Terminal 168
- III. Distribution and Function of Different Synaptic Vesicles Pools 169
  - A. Exo/Endo Cycling (Readily Releasable) and Reserve Pools 169
  - B. Functional Roles of Two SV Pools 172
  - C. Cytochalasin D Inhibits and cAMP Facilitates Trafficking of SVs from the RP to the ECP 173
- IV. Mechanism of Endocytosis at Presynaptic Terminals 174
  - A. Existence of Two Pathways for Endocytosis 174
  - B. Are the Two Pathways for Endocytosis Linked to the Two SV Pools Separately? 176
- V. Perspectives 177
- References 178
Chapter 8: Glutamate Receptors At The Drosophila Neuromuscular Junction 181
- I. Introduction 181
- II. Composition of Glutamate Receptors 183
- III. Receptor Subtypes at the NMJ: DGluRIIA Versus DGluRIIB 185
- IV. Clustering of Glutamate Receptors 187
- V. Glutamate Receptors and Synaptic Plasticity 190
- References 192
- VI. Conclusions 192
Chapter 9: Scaffolding Proteins at the Drosophila Neuromuscular Junction 197
- I. Introduction 198
- II. Structure of the Drosophila Larval Neuromuscular Junction 199
- IV. Dlg-Based Scaffold at the NMJ 200
  - A. Discs-Large 200
  - B. Synaptic Localization and Targeting of Dlg 202
  - C. Structure and Physiology of Dlg-Mutant NMJs 204
  - D. Dlg-Interacting Partners at the NMJ 205
- III. Multidomain Organization of Scaffolding Proteins 200
- V. Dystrophin 213
- VI. dGRIP 214
- VII. dX11/dMint/dLin-10 215
- VIII. Dliprin-alpha 218
  - A. Dliprin Regulates Active Zone Morphology and Synaptic Physiology 218
  - B. Dliprin-alpha Interacts with Drosophila Leukocyte Antigen-Related Receptor Tyrosine Phosphatases (DLAR) to Control Synaptic D 218
  - C. Dliprin-alpha as a Target for Regulated Degradation by the APC/C Complex 219
  - D. Not Just Cargo: Dliprin-alpha Promotes Proper Trafficking of Synaptic Vesicles 219
- IX. Bruchpilot: A Crash Pilot Targets the Active Zone 220
- X. Bazooka (Par-3)/Par-6/aPKC 221
- XI. Missing Prominents: Homer/Vesl, Shank/ProSAP, and GKAP/SAPAP 222
- XII. Perspectives 223
- References 224
Chapter 10: Synaptic Cytoskeleton At The Neuromuscular Junction 233
- I. Introduction 234
- II. Organization and Stability of Presynaptic Microtubules: A Role for Futsch 235
- III. Presynaptic MT Rearrangements: Straight Versus Looped MTs 238
- IV. Other Regulators of MT Stability at the NMJ 239
- V. Mechanisms of Signaling to the Presynaptic Cytoskeleton 241
- VI. Actin/Spectrin Cytoskeleton 245
- VII. Postsynaptic Cytoskeleton and Glutamate Receptor Anchoring 248
- VIII. Perspectives 249
- Acknowledgment 250
- References 250
Chapter 11: Plasticity and Second Messengers During Synapse Development 253
- I. Introduction 253
- II. Manifestations of Plasticity at the Larval Neuromuscular Junction 255
  - A. Functional Plasticity 255
  - B. Structural Plasticity 256
- III. Plasticity-Inducing Signals at the Larval Neuromuscular Junction 257
  - A. Synaptic Activity 257
  - B. Glutamate 258
  - C. Octopamine 259
  - D. Peptides and Secreted Proteins 259
  - E. Cell Adhesion Molecules 265
- IV. Major Intracellular Effectors of Plasticity at the Neuromuscular Junction 268
  - A. cAMP 268
  - B. cGMP 269
  - C. Calcium 270
  - D. Ras/MAPK 272
- V. Conclusions 274
- Acknowledgment 274
- References 274
Chapter 12: Retrograde Signaling That Regulates Synaptic Development and Function at the Drosophila Neuromuscular Junction 283
- I. Introduction 284
- II. Retrograde Signaling Revealed by Postsynaptic Manipulations 285
  - A. Glutamate Receptor-Dependent Retrograde Signaling 286
  - C. Postsynaptic Regulation of Local Translation 287
  - B. FasciclinII 287
  - D. Potassium Channels That Hyperpolarize Muscle Resting Potentials 288
  - F. Cytosolic Molecules: Amphiphysin and Dystrophin 289
  - E. Calcium Signaling Molecules: CaMKII and Synaptotagmin 4 289
- III. Known Retrograde Signals and Receptors 290
- IV. Postsynaptic Signal Integration with the BMP Pathway 292
- V. Presynaptic Signal Transduction for BMP Signaling 294
- VI. Perspectives 296
- Acknowledgment 297
- References 297
Chapter 13: Activity-Dependent Regulation of Transcription During Development of Synapses 303
- I. Introduction 304
- II. Mechanisms of Transcriptional Activation During Long-Term Plasticity 305
- III. Experimental Paradigms of Protein Synthesis-Dependent Long-Term Plasticity in Drosophila 307
- IV. NMJ as a Model Synapse to Study Transcriptional Regulation of Developmental Plasticity 309
  - B. Transcriptional Regulation of Synaptic Development 310
  - A. Transcriptional Control of MN Cell Fate 310
  - C. Transcriptional Events in Postsynaptic Muscle That Regulate Plasticity 312
- V. Open Questions and Areas of Convergence 314
- Acknowledgment 316
- References 316
Chapter 14: Experience-Dependent Potentiation of Larval Neuromuscular Synapses 323
- I. Introduction 324
- II. Strategies to Alter the Activity of Motoneurons and NMJ Usage 324
- III. Experience-Dependent Synaptic Potentiation at NMJs 326
- IV. Mechanisms Involved in Experience-Dependent Potentiation 331
  - B. Phase-II: Presynaptic Potentiation with Basal Quantal Size and Without Bouton Outgrowth 332
  - A. Phase-I: Potentiation of NMJs Is Dependent on an Increase in Quantal Size 332
  - C. Phase-III and Phase-IV: Experience-Dependent Protein Synthesis and the Morphological Consolidation of Induced Functional Chan 333
- References 336
- V. Outlook 336
Chapter 15: Selected Methods for the Anatomical Study of Drosophila Embryonic and Larval Neuromuscular Junctions 339
- I. List of Protocols 339
- II. Protocols 340
  - Protocol 1: Dissection of the Larval Body Wall Muscles 340
    - A. Magnetic Chambers and Pins Assembly 340
    - B. Dissection of Larval Body Wall Muscles (First to Third Instars) 344
  - Protocol 2: Immunocytochemical Staining of Larval Body Wall Muscles 346
  - Protocol 3: Immunocytochemical Staining of Whole Embryos 348
    - A. Egg Laying 348
    - B. Dechorionation, Devitellinization, and Fixation 349
  - Protocol 4: Preparation of Early Embryonic Fillets (Flat Embryonic Preparations; Before Cuticle Deposition) 351
    - A. Construction of Chambers to Process Embryonic Fillets 352
    - B. Preparation of Embryos 353
  - Protocol 5: DiI Labeling of Embryonic NMJs and Their Innervating Motoneurons 355
    - B. Dissection of Late Embryonic Fillets 357
    - A. Sylgardreg-Coated Coverslips 357
  - Protocol 7: FM1–43 Labeling of Larval NMJs 361
    - A. Fixing Late Stage Embryos by Injecting Fixative Solution 367
  - III. Solutions 373
    - A. Jan's Saline 373
    - B. Ca2+-Free/EGTA Saline 374
    - C. HL-3 Saline (Haemolymph-Like Saline-3) 374
    - D. PB (0.1 M PB, pH 7.2) 374
    - G. TB (0.2 M Tris-HCl Buffer pH 7.2) 375
    - J. 5% GA 375
    - E. PBT (0.1 M PB, pH 7.2 with Triton X-100) 375
    - H. DAB Solution 375
    - K. 2% Uranyl Acetate Solution 375
    - F. PBS (Phosphate Buffer Saline) 375
    - I. 2.5% GA 375
    - L. 0.1 M CBo (0.1 M Osmotically Adjusted Cacodylate Buffer) 375
    - O. Lead Citrate Solution 376
    - M. 0.2 M CBo (0.2 M Osmotically Adjusted Cacodylate Buffer) 376
    - P. DiI Stock Solution 376
    - N. Toluidine Blue Stain 376
    - Q. DiI Working Solution 376
    - R. Modified Trump's Universal Fix (Make Fresh Before Using) 377
    - U. Fruit Juice Agar Plates for Egg Laying 377
    - S. Nonalcoholic Bouin's Fix 377
    - T. 4% Paraformaldehyde Fix (Discard if Older Than 5 Days; Keep at 4deg;C) 377
    - W. Live-Yeast Paste 378
    - X. Heptane Glue 378
    - V. Enriched Larval Medium (This Medium Is well Suited for Rearing Sickly Larvae) 378
    - Y. Dispersion Medium 378
    - Z. FM1-43 (N-(3-Triethylammoniumpropyl)-4-(p-di-butylaminostyryl)pyridinium-dibromide) 379
  - IV. Suppliers of Materials for Making Magnetic Chambers 379
  - References 380
- Appendix 383
- Index 391
- Contents of Recent Volumes 403

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