Novel method for the labeling of distant neuromuscular junctions.
Essential to understanding the roles proteins and structural elements play at the synapse is to understand the development, remodeling and reinnervation of peripheral neuromuscular junctions. It has, however, been a challenging task to label and visualize neuromuscular junctions. In this paper we demonstrate how adenovirus technology can be combined with intraspinal microinjection techniques to follow both the development and the reinnervation of a distant peripheral neuromuscular junction in the rat. A recombinant adenovirus containing VAMP-2 (synaptobrevin-2) was fused to the green fluorescent protein (GFP) and microinjected into the region of the lumbar motor neurons. We were able to follow the neuronal incorporation, axonal transport and synaptic localization of the GFP-VAMP-2 using fluorescence microscopy. GFP-VAMP-2 was found in neuronal cell bodies, selected sciatic nerve axons and was concentrated in the presynaptic nerve terminal. During reinnervation of the neuromuscular junction, GFP-VAMP-2 allows us to follow the time course of junctional reinnervation. Thus, the microinjection of microliter amounts of labeled recombinant virus into locations far distant from target regions can be used to efficiently study the formation of neuromuscular junctions with a minimum of trauma to the animal.
Jacob
J M
JM
Department of Cell Biology, University of Oklahoma Health Sciences Center, College of Medicine, Oklahoma City, OK 73190, USA. jane-jacob@ouhsc.edu
Zhou
Q
Q
Liu
Y
Y
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001370:Axonal Transport / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009046:Motor Neurons / Q000737:chemistry / Q000502:physiology*; D009121:Muscle Denervation; D018482:Muscle, Skeletal / Q000294:innervation; D009409:Nerve Crush; D009469:Neuromuscular Junction / Q000737:chemistry / Q000502:physiology*; D009474:Neurons / Q000166:cytology; D017729:Presynaptic Terminals / Q000737:chemistry; D050683:R-SNARE Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050683:R-SNARE Proteins; D049452:Green Fluorescent Proteins