Our studies of mutant zebrafish continue to advance understanding of synaptic transmission and inheritable forms of human neuromuscular disorders such as myasthenic syndrome, slow channel syndrome and Brody disease. The proposed studies now capitalize on mutant lines which we have shown either lack synaptic transmission altogether, or lack the ability of receptors to localize subsynaptically, the ability of muscle to release calcium, the ability of muscle to sequester calcium, or possess abnormal levels of synaptic drive, suggesting miscommunication between presynaptic nerve and postsynaptic muscle. Exploiting the advantages provided by these lines, we will determine the role of synaptic activity in shaping both presynaptic and postsynaptic development. These studies will be greatly facilitated by use of our recently developed tethered toxin methodology that silences synaptic transmission in a cell autonomous fashion. Thus, we can determine the consequences of adjusting synaptic drive and/or excitability of individual cells within the motoneuron target field. Rescue of individual postsynaptic cells in mutant lines will provide a complementary approach wherein a single cell is rendered active in an otherwise silent field of target muscle. This combination of approaches will be used to determine the involvement of postsynaptic activity in the developmental loss of electrical coupling, acquisition of presynaptic transmitter release properties and establishment of postsynaptic receptor kinetics. The study is possible by virtue of our ability, for the first time in any vertebrate preparation, to simultaneously record from a motoneuron and target muscle in vivo. This is due to electrically compact muscle that allows whole cell voltage clamp and transparency, facilitating identification and patch clamp of motoneurons deep within the spinal cord. The combined advantages of new approaches and preparation now provide unique opportunities or examining long standing questions of synaptic function.
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