title: Development of the mouse neuromuscular junction in the absence of regulated secretion.
abstract: To investigate the role of neurotransmitter secretion in the development and stabilization of synapses, the innervation of the diaphragm and intercostal muscles was studied in munc18-1 null mutant mice, which lack regulated secretion. We found that this mutant is completely devoid of both spontaneous and evoked neuromuscular transmission throughout embryonic development. At embryonic day (E) 14, axonal targeting and main branching of the phrenic nerve were normal in this mutant, but tertiary branches were elongated and no terminal branches were observed at this stage, in contrast to control littermates. Acetylcholinesterase staining was observed in the endplate region of mutant muscle from E14 onwards, but not as dense and confined to spots as in controls. Acetylcholine receptor staining was also present in the endplate region of the mutant muscle. In this case, the staining density and the concentration in spots (clusters) were similar to controls, but the distribution of these clusters was less organized. Starting at E15, some receptor clusters co-localized with nerve terminal staining, suggesting synapses, but most clusters remained a-neural. Electron microscopical analysis confirmed the presence of synaptic structures in the mutant. Between E14 and birth, the characteristic staining pattern of nerve branches gradually disappeared in the mutant until, at E18, an elaborate meshwork of nerve fibers with no apparent organization remained. In the same period, most of the motor neuronal cell bodies in the spinal cord degenerated. In contrast, sensory ganglia in the dorsal root showed no obvious degeneration. These data suggest that regulated secretion is not essential for initial axon path finding, clustering of acetylcholine receptors, acetylcholinesterase or the formation of synapses. However, in the absence of regulated secretion, the maintenance of the motor neuronal system, organization of nerve terminal branches and stabilization of synapses is impaired and a-neural postsynaptic elements persist.
authors.affiliation: Department of Molecular Neuroscience, Rudolf Magnus Institute, University of Utrecht Medical Center, Utrecht, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Heeroma
authors.forename: J J
authors.initials: JJ
authors.lastname: Plomp
authors.forename: E W
authors.initials: EW
authors.lastname: Roubos
authors.forename: M
authors.initials: M
authors.lastname: Verhage
mesh_terms: D000110:Acetylcholinesterase / Q000032:analysis / Q000378:metabolism; D000818:Animals; D003964:Diaphragm / Q000294:innervation*; D004594:Electrophysiology; D019922:GAP-43 Protein / Q000032:analysis / Q000378:metabolism; D007366:Intercostal Muscles / Q000294:innervation*; D051379:Mice; D008817:Mice, Mutant Strains; D008854:Microscopy, Electron; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009469:Neuromuscular Junction / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism; D017933:Peripheral Nervous System / Q000196:embryology / Q000254:growth & development* / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D011978:Receptors, Nicotinic / Q000032:analysis / Q000378:metabolism; D013569:Synapses / Q000378:metabolism / Q000473:pathology; D016704:Synapsins / Q000032:analysis / Q000378:metabolism; D009435:Synaptic Transmission; D033921:Vesicular Transport Proteins
chemical_list: D019922:GAP-43 Protein; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D011978:Receptors, Nicotinic; C497938:Stxbp1 protein, mouse; D016704:Synapsins; D033921:Vesicular Transport Proteins; D000110:Acetylcholinesterase