title: The neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy.
abstract: The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.
authors.affiliation: Physiology and Bosch Institute, University of Sydney.
authors.forename: Nigel
authors.initials: N
authors.lastname: Tse
authors.affiliation: Motor Neuron Disease Research Group, Australian School of Advanced Medicine, Macquarie University.
authors.forename: Marco
authors.initials: M
authors.lastname: Morsch
authors.affiliation: Physiology and Bosch Institute, University of Sydney.
authors.forename: Nazanin
authors.initials: N
authors.lastname: Ghazanfari
authors.affiliation: Advanced Microscopy Facility, Bosch Institute, University of Sydney.
authors.forename: Louise
authors.initials: L
authors.lastname: Cole
authors.affiliation: Physiology and Bosch Institute, University of Sydney.
authors.forename: Archunan
authors.initials: A
authors.lastname: Visvanathan
authors.affiliation: Physiology and Bosch Institute, University of Sydney.
authors.forename: Catherine
authors.initials: C
authors.lastname: Leamey
authors.affiliation: Physiology and Bosch Institute, University of Sydney; billp@physiol.usyd.edu.au.
authors.forename: William D
authors.initials: WD
authors.lastname: Phillips
mesh_terms: D000818:Animals; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods*; D009045:Motor Endplate / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000033:anatomy & histology / Q000378:metabolism / Q000502:physiology*; D011950:Receptors, Cholinergic / Q000378:metabolism; D013569:Synapses / Q000378:metabolism / Q000502:physiology*
chemical_list: D009419:Nerve Tissue Proteins; D011950:Receptors, Cholinergic; C033652:postsynaptic density proteins