Activity-dependent processes play a central role in sculpting neuronal circuits, but the mechanisms that influence synapse stability at the neuromuscular junction have proven difficult to ascertain. In this proposal, the zebrafish model system is used to address the role of postsynaptic receptor activity in the establishment of presynaptic structure and function at the neuromuscular synapse. Unlike mammalian counterparts, zebrafish mutants lacking postsynaptic acetylcholine receptors survive well beyond synapse development. This offers a unique opportunity to study how mutations in the neuromuscular genetic program affect neuromuscular synapse development and function. To this end, zebrafish embryos have been experimentally altered to create a target field composed of synaptically active and inactive muscle. Primary motor neurons interact with this competitive target field, revealing a competition for synapse establishment with a bias towards active targets.
In Aim 1, these preliminary findings will be further investigated in order to determine how this competition occurs. Postsynaptic activity is either inactivated in wild type muscle using tethered 1-bungarotoxin, or the reciprocal manipulation is performed by restoring acetylcholine receptor expression in receptorless mutant lines.
In Aim 2, I will use the unique electrophysiological properties offered by the zebrafish neuromuscular synapse to determine the consequences of an altered target field size on development of presynaptic function. Zebrafish motility mutants have served as models for Brody's disease and episodic apnea as well as revealing a role for rapsyn in conferring congenital forms of human myasthenic syndrome. The research in this proposal when combined with existing mutant lines will help determine how activity-dependent signals can influence synapse stability during neuromuscular pathologies. From the standpoint of career development, the proposed studies fill a critical gap in training by providing experience in use of an in vivo animal model. Most notably, new skills in live imaging and the basics of electrophysiological measurements will be acquired. This builds on my long-term interest in synapse development. A synthesis of my graduate training in cell culture systems and use of zebrafish through postdoctoral training will greatly facilitate my move toward establishing independence. )
Zebrafish motility mutants have provided unexpected models for human neuromuscular disorders. Conservation of the genetic program instructing neuromuscular synapse development permits studies to determine how genetic alterations found in humans lead to neuromuscular pathologies. The proposed experiments will further capitalize on use of zebrafish for the purpose of training as well as identifying the mechanisms underlying synapse formation and function. )
