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 large r and faster, being among the fastest of all neuromuscular synapses. To ide ntify the bases for these changes we compared, in embryos and larvae, the p roperties and distributions of acetylcholine (ACh) receptors (AChRs) and ac etylcholinesterase (AChE) as well as the ultrastructure of the developing n euromuscular 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 c ompared with larvae. In both embryos and larvae, AChRs demonstrated open ch annel block by millimolar ACh, and this was detected during mEPCs, indicati ng that a high concentration of ACh is released at immature and mature NMJs . AChR and AChE distributions were compared using the selective fluorescent ly conjugated labels alpha -bungarotoxin and fasciculin 2, respectively. In larvae, punctate AChR clusters were detected whereas junctional AChE stain ing was less intense than that found at adult NMJs. Transmission electron m icroscopy revealed immature nerve endings in embryos that were closely juxt aposed to the surrounding muscle cells, whereas mature larval NMJs had a wi der synaptic cleft with a conspicuous basal lamina over a limited region of synaptic contact. Our results indicate that ACh is released at high concen trations at immature NMJs, but its clearance is prolonged and the AChRs are dispersed, resulting in a slow mEPC time course until a mature cleft appea rs with densely packed faster AChRs and abundant AChE.