Hi everyone,
I am currently working on establishing a co-culture system for NMJs. Unfortunately I have no experience how NMJ might look like in vitro. I performed some co-stainings with BTX and TUJ1 as well as synaptophysin, but am not sure if the co-localizations I see are true NMJs.
I hope someone can help me with this issue and share a few experience with NMJ stainings in vitro.
All the best,
Jessica
Hi Jessica,
You might take a look at the papers below from the lab of Joshua Sanes. They have a long history of excellent work in NMJ development and have used a wide range of preparations, including co-culture approaches. The imaging results are excellent, too, so you should be able to get a good look at what NMJs in a cocoulture should look like.
Nishimune H, Valdez G, Jarad G, Moulson CL, Müller U, Miner JH, Sanes JR. Laminins promote postsynaptic maturation by an autocrine mechanism at the neuromuscular junction. J Cell Biol. 2008 Sep 22;182(6):1201-15. doi: 10.1083/jcb.200805095.
PMID: 18794334
Burgess RW, Nguyen QT, Son YJ, Lichtman JW, Sanes JR. Alternatively spliced isoforms of nerve- and muscle-derived agrin: their roles at the neuromuscular junction.Neuron. 1999 May;23(1):33-44.
PMID:
10402191
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I do not know how NMJs in co-cultures looks like, but in vivo NMJs always have a set of synaptic myonuclei in the holes of the pretzel or ensheating the junctional folds of the neuromuscular end plate. So, you could stain for nuclei to see if the NMJs are associated and colocalized with synaptic myonuclei.
The literature on cultured NMJ morphology seems to vary from reports of small clusters of AChRs (often identified using alpha-BTX) along myotube membranes to pretzel-like structures qualitatively similar to those seen in mature animals. So the kinds of NMJs you'll see will depend on the kinds of muscle (i.e. mouse vs. chick, fast vs. slow muscle, etc) and motor neurons (primary cultures vs. stem cell-derived etc) you're using. Generally, the presynaptic terminal (identified with antibodies against synaptic vesicle proteins or by genetically labeling motor neurons with GFP) should follow the general shape of the postsynaptic structure, although with small variations. To conclusively demonstrate functional NMJs you should probably perform some kind of functional assessments at identified synapses (i.e. electrophysiological recordings and/or FM dye imaging).
See the attached paper for more details:
Article A Stem-Cell Based Bioassay to Critically Assess the Patholog...
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