Confocal Microendoscopy of Neuromuscular Synapses in Living Mice

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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/O₂
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

Figure 1. (A ) Medial aspect of a mouse hind limb (cadaver), in which the tibial nerve (arrow) has been exposed through a small wound. The stainless‐steel tip of a Proflex S‐1500 probe is positioned in the wound over the exposed nerve. (B ) CME image of intact axons in the tibial nerve of an anesthetized thy1.2‐YFPH transgenic mouse. Only about 5% of the axons express YFP in this line and seven of them are visible in this image. (C ) CME image of intact axons in the tibial nerve of an anesthetized thy1.2‐YFP16 transgenic mouse. All the motor axons are labeled by YFP expression in this line. (D‐F ) CME images of a group of NMJs in axotomized flexor digitorum longus muscle of a Wld^S mutant mouse, obtained in imaging sessions conducted on successive days 3 (D), 4 (E), and 5 (F) after sciatic nerve section, following Basic Protocols 1 and 2. There is a delayed, progressive degeneration of motor axon terminals in this mutant strain following such nerve injury (arrows). Reprinted from Wong et al. () with permission

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Literature Cited

Literature Cited
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