Walogorsky et al. published in PNAS studied nicotinic receptors in the neuromuscular junction, which continues to be the main focus of the lab. A mutant zebrafish with a dominant mutation in the αsubunit of acetylcholine receptors (AChRs) in the neuromuscular junction was studied (twister mutant;twi+/-). The locomotion of this mutant displayed improvement with development. The embryonic AChRs containing the γsubunit produced slowly decaying synaptic currents suggestive of Slow-Channel Syndrome, a human disease resulting from mutations of AChR subunits. However, the current displayed a much faster decay upon the appearance of adult ε, despite the existence of twister mutation in the αsubunit. When γsubunit expression was suppressed with antisense, the synaptic decay became significantly faster. Thus, the continued expression of αharboring the twister mutation into adulthood is tolerated because of the εexpression and associated recovery, raising the likelihood of unappreciated myasthenic cases that benefit from the γ−εswitch. Another paper studying the sensitivity of zebrafish neurons to ethanol was published in PLoS ONE. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear.We examined whether intrinsic properties of neurons play any roles in their resistance. In order to better control the internal ethanol concentration of larvae, we sectioned the larvae and the cranial half was soaked in ethanol solution. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular Ca2+ response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Because hindbrain neurons examined with calcium indicators are implicated in the startle response, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment selectively reduced long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets. In 2012-2013 we focused our efforts on two projects using acetylcholine receptor mutants. In the first project, we manipulated the timing of synapse formation and studied its effect on the functional development of synapse. In the second project, we analyzed distinct mechanisms of synapse formation in slow and fast muscles. Fumi Ono gave several invited talks at Universities (University of Miami, University of Tokyo, University of Niigata, University of Kagoshima) and an international meeting (International Drug Abuse Research Society Meeting, Mexico City).
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