Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.

M. Shapira, S. Seidman, M. Sternfeld, R. Timberg, D. Kaufer, J. Patrick, H. Soreq

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

To examine the role of key cholinergic proteins in the formation ofneuromuscular junctions (NMJs), we expressed DNAs encoding the mousemuscle nicotinic acetylcholine receptor (nAChR) or human brain andmuscle acetylcholinesterase (hAChE) in developing Xenopus laevisembryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding inhomogenates of transgenic embryos revealed transient overexpression ofthe respective proteins for at least 4 days postfertilization. Moreover,hAChE injection induced an approximately 2-fold increase in endogenousXenopus nAChR. Electron microscopy coupled with cytochemical stainingfor AChE activity revealed that AChE-stained areas, which reached 0.17microns2 in NMJs of control embryos raised at 21 degrees C, increased upto 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively.These increases coincided with the appearance of a class of large NMJswith average postsynaptic lengths up to 1.8-fold greater than controls.As much as 57% and 34% of the NMJs in animals transgenic for nAChR andhAChE, respectively, displayed AChE activity in nerve terminals inaddition to muscle labeling, as compared with 10% nerve-labeled NMJs incontrol animals. Moreover, area, but not length values, were > 2-foldlarger in hAChE-expressing NMJs labeled in their nerve terminals than inthose labeled in muscle alone, reflecting a hAChE-induced increase insynaptic cleft width. These findings indicate that modulation ofcholinergic neurotransmission in NMJs modifies the features ofnerve-muscle connections.
Original languageEnglish GB
Pages (from-to)9072-9076
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume91
Issue number19
DOIs
StatePublished - 1 Sep 1994
Externally publishedYes

Keywords

  • acetylcholinesterase cytochemistry
  • electron microscopy
  • nicotinic muscle acetylcholine receptor
  • posttranscriptional regulation
  • synaptic targeting

ASJC Scopus subject areas

  • General

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