Limits to the development of fast neuromuscular transmission in zebrafish

Zebrafish embryos have small and slow miniature end-plate currents (mEPCs), whereas only a few days later larval mEPCs are an order of magnitude larger and faster, being among the fastest of all neuromuscular synapses. To identify the bases for these changes we compared, in embryos and larvae, the properties and distributions of acetylcholine (ACh) receptors (AChRs) and acetylcholinesterase (AChE) as well as the ultrastructure of the developing neuromuscular junctions (NMJs). To mimic synaptic release, patches of muscle membrane were exposed briefly (for 1 ms) to a saturating concentration (10 mM) of ACh. The AChR deactivation kinetics were twice as slow in embryos compared with larvae. In both embryos and larvae, AChRs demonstrated open channel block by millimolar ACh, and this was detected during mEPCs, indicating that a high concentration of ACh is released at immature and mature NMJs. AChR and AChE distributions were compared using the selective fluorescently conjugated labels alpha-bungarotoxin and fasciculin 2, respectively. In larvae, punctate AChR clusters were detected whereas junctional AChE staining was less intense than that found at adult NMJs. Transmission electron microscopy revealed immature nerve endings in embryos that were closely juxtaposed to the surrounding muscle cells, whereas mature larval NMJs had a wider synaptic cleft with a conspicuous basal lamina over a limited region of synaptic contact. Our results indicate that ACh is released at high concentrations at immature NMJs, but its clearance is prolonged and the AChRs are dispersed, resulting in a slow mEPC time course until a mature cleft appears with densely packed faster AChRs and abundant AChE.