Confocal Microendoscopy of Neuromuscular Synapses in Living Mice

Gonzalo Blanco1, Richard R. Ribchester2

1 Department of Biology, University of York, Heslington, York, United Kingdom, 2 Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, Edinburgh, United Kingdom
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110144
Online Posting Date:  March, 2012
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Abstract

Here we describe a step‐by‐step method for vital imaging of neuromuscular junctions (NMJ) and axons using fiber‐optic confocal microendoscopy (CME). A commercially available system, the Cellvizio Lab, can be applied to transgenic mouse lines expressing yellow fluorescent protein in all or pseudorandom sub‐subsets of motor neurons. Microscopic imaging in vivo is achieved by means of a flexible optical fiber probe that excites and collects the emitted light from fluorescently labeled structures. The hand‐held probe is introduced through small skin incisions to visualize nerves and neuromuscular junctions from superficial muscles. Interpolation software then reconstructs the images in real time. The images are of sufficient quality to permit screening of axonal and neuromuscular synaptic integrity and other aspects of their phenotype in live animals. Curr. Protoc. Mouse Biol. 2:1‐8 © 2012 by John Wiley & Sons, Inc.

Keywords: neuromuscular junction; imaging; microscopy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Surgical Procedure: Sciatic Nerve Lesion
  • Basic Protocol 2: Confocal Microendoscopy (CME) of Axons and Presynaptic Terminals
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Surgical Procedure: Sciatic Nerve Lesion

  Materials
  • Thy1.2‐YFP16 or thy1.2‐YFPH transgenic mice (Jackson Labs)
  • Vetergesic (buprenorphine 4.2 µg/ml)
  • 3% to 5% isoflurane/O2
  • Antiseptic
  • Fur clippers
  • Iris scissors and fine spring scissors (e.g., Fine Science Tools)
  • Watchmakers forceps (e.g., Fine Science Tools)
  • Wound clips or silk suture with integral suture needle (e.g., Ethicon, 7‐0)
  • Heating lamp

Basic Protocol 2: Confocal Microendoscopy (CME) of Axons and Presynaptic Terminals

  Materials
  • Thy1.2‐YFP16 or thy1.2‐YFPH transgenic mice (Jackson Labs)
  • Vetergesic (buprenorphine 4.2 µg/ml)
  • 3% to 5% isoflurane/O 2
  • Antiseptic
  • Cellvizio Lab (Mauna Kea Technologies)
  • Proflex S‐1500 probe (Mauna Kea Technologies)
  • Fur clippers
  • Iris scissors and fine spring scissors (e.g., Fine Science Tools)
  • Adhesive tape
  • Dissection board or homeothermic blanket
  • Watchmakers forceps (e.g., Fine Science Tools)
  • ImageCell software
  • Micromanipulator, optional
  • Wound clips or silk suture with integral suture needle (e.g., Ethicon, 7‐0)
  • Heating lamp
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Figures

Videos

Literature Cited

Literature Cited
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   Barretto, R.P., Messerschmidt, B., and Schnitzer, M.J. 2009. In vivo fluorescence imaging with high‐resolution microlenses. Nat. Methods 6:511‐512.
   Beirowski, B., Berek, L., Adalbert, R., Wagner, D., Grumme, D.S., Addicks, K., Ribchester, R.R., and Coleman, M.P. 2004. Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP‐H mice. J. Neurosci. Methods 134:23‐35.
   Brown, M.C. and Ironton, R. 1978. Sprouting and regression of neuromuscular synapses in partially denervated mammalian muscles. J. Physiol. 278:325‐348.
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   Rich, M.M. and Lichtman, J.W. 1989. In vivo visualization of pre‐ and postsynaptic changes during synapse elimination in reinnervated mouse muscle. J. Neurosci. 9:1781‐1805.
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   Wong, F., Fan, L., Wells, S., Hartley, R., Mackenzie, F.E., Oyebode, O., Brown, R., Thomson, D., Coleman, M.P., Blanco, G., and Ribchester, R.R. 2009. Axonal and neuromuscular synaptic phenotypes in Wld(S), SOD1(G93A) and ostes mutant mice identified by fiber‐optic confocal microendoscopy. Mol. Cell. Neurosci. 42:296‐307.
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