Paralytic Zebrafish Lacking Acetylcholine Receptors Fail to Localize Rapsyn Clusters to the Synapse

Abstract

Physiological analysis of two lines of paralytic mutant zebrafish, relaxed and sofa potato, reveals defects in distinct types of receptors in skeletal muscle. In sofa potato the paralysis results from failed synaptic transmission because of the absence of acetylcholine receptors, whereas relaxed mutants lack dihydropyridine receptor-mediated release of internal calcium in response to the muscle action potential. Synaptic structure and function appear normal in relaxed, showing that muscle paralysis per se does not impede proper synapse development. However, sofa potato mutants show incomplete development of the postsynaptic complex. Specifically, in the absence of ACh receptors, clusters of the receptor-aggregating protein rapsyn form in the extrasynaptic membrane but generally fail to localize to the subsynaptic region. Our results indicate that, although rapsyn molecules are capable of self-aggregation, interaction with ACh receptors is required for proper subsynaptic localization.

Nicotinic Receptors Mediate Changes in Spinal Motoneuron Development and Axonal Pathfinding in Embryonic Zebrafish Exposed to Nicotine

“…Moreover, in embryos continuously exposed to nicotine until 120 hpf, the axons of secondary motoneurons are also severely stunted (data not shown). These results are in stark contrast to the anatomy of motoneurons of zebrafish paralytic mutants, in which motoneuron axons appear normal, and to motoneurons of the developing chick, in which the axons are actually hyperbranched when muscle activity is blocked (Pittman and Oppenheim, 1979;Westerfield et al, 1990;Landmesser, 1992;Ono et al, 2001). This provides yet another line of evidence suggesting a neuronal mechanism for the action of nicotine in embryonic/ larval zebrafish.…”

“…In fact, these embryos behave somewhat like the nic-1 and sofa potato paralytic zebrafish mutants that have been well characterized (Westerfield et al, 1990;Ono et al, 2001). However, embryos exposed to nicotine perceive touch stimuli.…”

“…In the sofa potato paralytic mutant, synaptic transmission at the neuromuscular junction is abolished. However, the morphology of secondary motoneurons and their corresponding axonal trajectories and GFP expression patterns in 120 hpf sofa potato embryos appear to be normal (Ono et al, 2001); they are not stunted or retarded in their development. In the paralytic mutant known as nic-1, ACh receptor function in the muscle is completely blocked.…”

“…In embryos never exposed to nicotine, axons in the dorsal myotome have an elaborate GFP expression pattern (Fig. 4 B) [Ono et al (2001), their Fig. 1].…”

“…A fragment of the islet-1 promoter constitutively drives the GFP expression in these embryos (Higashijima et al, 2000). This transgenic line has been used recently to characterize the axonal branching patterns of motoneurons and also to characterize neuromuscular development in embryonic and larval zebrafish (Higashijima et al, 2000;Ono et al, 2001;Segawa et al, 2001). It is known that GFP expression is confined to a subclass of secondary motoneurons (Higashijima et al, 2000) in embryos older than 3 d postfertilization.…”