MICROSCOPY RESEARCH AND TECHNIQUE 49:1–2 (2000) Introduction to Organization of the Neuromuscular Junction: From Structure to Function EKATERINI KORDELI Biologie Cellulaire des Membranes, Institut Jacques Monod, CNRS, UMR 7592, Universités Paris 6/7, 75251 Paris-Cedex 05, France Progress in experimental science depends greatly on the tools of study that are available. The deciphering of the relationship between the structure and function of the neuromuscular junction (NMJ) offers a typical example. Almost a century ago, Ramon y Cajal and his coworkers applied silver impregnation techniques to neuromuscular preparations, techniques first developed for the study of neurons (Ramon y Cajal, 1928). This pioneering work set the basis for the concept of a discontinuity between neuronal and muscular cytoplasms at the neuromuscular synapse. Increasingly elaborate staining techniques were subsequently developed and used in conjunction with enzyme histochemistry and electrophysiology to unravel the structural bases of chemical synaptic transmission at the vertebrate NMJ. However, elucidation of the fine structure of the NMJ had to await the development of electron microscopy (EM) in the1950s. The higher resolution and rapid evolution of the various EM-associated techniques made it possible to establish the definite existence of a gap between the neuronal and muscular plasma membranes: the synaptic cleft. This structure comprises a specialized basal lamina where trophic factors, such as agrin and heregulin, involved in synaptogenesis and the enzyme acetylcholinesterase accumulate. Moreover, EM analysis revealed the two fundamental elements of the synaptic structure: the pre- and postsynaptic apparati (reviewed in Couteaux, 1973), which represent local differentiations of the motor nerve terminal and the sarcolemma, respectively. These two domains are characterized by the local accumulation of specialized organelles (presynaptic vesicles, postsynaptic golgi apparatus, fundamental nuclei, etc.) and membrane proteins (ion channels, cell adhesion molecules, cytoskeletal proteins). On the presynaptic side, the active zones contain the specialized molecular machinery for synaptic vesicle docking and neurotransmitter release that is responsible for the regulated secretion of acetylcholine by the nerve terminal. In front of the active zones of the nerve endings, the crests of the postsynaptic membrane folds exhibit extraordinarily high concentrations of a ligand-gated Na⫹/K⫹ channel, the nicotinic acetylcholine receptor (AChR), whereas voltage-sensitive Na ⫹ channels (NaChs) accumulate in the depths of the postsynaptic folds, in regions immediately adjacent to the AChRs. These two channels are responsible for the initiation of membrane depolarization and the generation of the endplate potential. A number of integral and cytoskeletal proteins accumulating at the postsynaptic membrane are believed to participate in the clustering and maintenance of AChRs and NaChs. © 2000 WILEY-LISS, INC. Synaptic transmission is a complex process requiring a high degree of coordination between pre- and postsynaptic structures. The first step in the study of this process aimed at unraveling the fine structure of the mature NMJ. The current challenge is to understand how such specializations develop and communicate at the molecular level. At this point, multiple experimental approaches are required. This topical issue presents recent advances in the study of the morphogenesis and functioning of the NMJ obtained by the combination of EM analysis and subcellular localization of synaptic proteins and transcripts, molecular biology, electrophysiology, genetics, and the use of several animal models ranging from Drosophila to mammals. Three articles in this issue deal with the morphogenesis of the NMJ. The first event in synaptogenesis is the synaptic target recognition. Rose and Chiba et al. (2000) present a summary of the current state of knowledge on the chemoaffinity hypothesis and the role of cell adhesion molecules in the initial recognition between neuronal processes and the sarcolemma leading to the future Drosophila NMJ. The relatively simple, well-defined pattern of synapses, the possibility of in vivo analysis, and the powerful molecular genetics make Drosophila a choice tool for the study of morphogenesis of the NMJ. Y. H. Koh et al. (2000) take advantage of the Drosophila NMJ model to study the molecular cascades and the role of synaptic signaling molecules, such as MAGUKs, involved in synaptic plasticity. The study of synaptogenesis in vertebrates is confronted with a high degree of tissue complexity. The current approach makes use of homologous recombination to generate mutant mice that lack the molecules involved in the synaptogenesis process (reviewed in Sanes and Lichtman, 1999). An alternative approach is the development of cellular model systems involving neuron-muscle cocultures to study in vitro synaptogenesis. M. Daniels et al. (2000) describe the establishment and characterization of a rodent neuron-muscle coculture, and provide examples of its application to the study of mammalian NMJ development. According to the vesicle hypothesis, quantal release of acetylcholine (ACh) from the synaptic vesicles into the synaptic cleft occurs upon fusion with the presynaptic membrane. This hypothesis has now become a wide consensus. However, a number of data do not fit into this model, instead they suggest that vesicle fusion This Topical Issue is dedicated to the memory of Professor René Couteaux, 1909 –1999. 2 and quantal ACh release might be two linked-but-distinct processes. Morel and M. Isräel (2000) propose an alternative model involving direct release of ACh through a membrane pore. Ultrastructural evidence in favor of this model is presented by Dunant (2000) who makes use of freeze-fracture EM to study rapid changes of presynaptic intramembrane particles with regard to ACh release and vesicle fusion. Both studies use the Torpedo electric organ, a source of abundant cholinergic and functionally active synaptosomes. A key enzyme in the control of cholinergic synaptic transmission at the NMJ is the acetylcholinesterase (AChE). Legay (2000) discusses recent data on the molecular structure and polymorphism, transcriptional regulation and synaptic localization mechanisms, and the implification of AChE in myasthenic syndromes. The morphogenesis and maintenance of the postsynaptic membrane domain is one of the most intensively studied aspects in the NMJ field. A major question addresses the mechanisms of specific local accumulation and clustering of postsynaptic membrane molecules. Cartaud et al. (2000) discuss the Torpedo electrocyte as a model system for the study of the supramolecular organization and clustering of AChRs, and membrane-cytoskeleton interactions, including the dystrophin complex and rapsyn, as one of the mechanisms responsible for AChR aggregation at the postsynaptic membrane. Caldwell (2000) discusses the current state of knowledge on the role of cytoskeletal and extracellular molecules in NaCh clustering at the troughs of the mammalian postsynaptic folds. Transcriptional regulation is another mechanism responsible for the local accumulation of synaptic proteins. RNAs encoding several synaptic integral and cytoskeletal proteins accumulate at the NMJ (reviewed in Sanes and Lichtman, 1999). Gramolini et al. (2000) present recent data on the compartmentalized expression and postsynaptic localization of the dystrophinrelated protein utrophin in skeletal muscle fibers. These studies also provide the basis for the development of a potential therapeutic strategy for Duchenne muscular dystrophy aimed at replacing dystrophin by extrasynaptically overexpressed utrophin. Finally, in my contribution, (Kordeli, 2000) I discuss the current state of knowledge of the spectrin-based skeleton that coexists with the dystrophin-associated protein complex at the postsynaptic membrane and is thought to participate in the organization of the postsynaptic folds and the clustering of synaptic proteins. REFERENCES Caldwell JH. 2000. Clustering of sodium channels at the neurocuscular junction. Microsc Res Tech 49:84 – 89. Cartaud J, Cartaud, Annie A, Kordeli, Ekaternini E, Ludosky MA, Marchard S, Stetzkowski-Marden F. 2000. Torpedo electrocyte: a model system to study membrane-cytoskeleton interactions at the postsynaptic membrane. Microsc Res Tech 49:73– 83. Couteaux R. 1973. Motor endplate structure. In Bourne GH, editor. Structure and function of muscle, vol 2. 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