TRANSGENIC ENGINEERING OF NEUROMUSCULAR-JUNCTIONS IN XENOPUS-LAEVIS EMBRYOS TRANSIENTLY OVEREXPRESSING KEY CHOLINERGIC PROTEINS

To examine the role of key cholinergic proteins in the formation of ne uromuscular junctions (NMJs), we expressed DNAs encoding the mouse mus cle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Ac etylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenat es of transgenic embryos revealed transient overexpression of the resp ective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximate to 2-fold increase in endogenous Xen opus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 mu m(2) in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 mu m(2) in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJ s with average postsynaptic lengths up to 1.8-fold greater than contro ls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals i n addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were >2-fo ld larger in hAChE-expressing NMJs labeled in their nerve terminals th an in those labeled in muscle alone, reflecting a hAChE-induced increa se in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve- muscle connections.