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

Michael Shapira, Shlomo Seidman, Meira Sternfeld, Rina Timberg, Daniela Kaufer, James Patrick, Hermona Soreq*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos, Acetylthiocholine hydrolysis and α-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an ≃2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE- stained areas, which reached 0.17 μm2 in NMJs of control embryos raised at 21°C, increased up to 0.53 and 0.60 μm2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with 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 and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were >2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.

Original languageEnglish
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 - 13 Sep 1994

Keywords

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

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