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¥11,400,000 (Direct Cost: ¥11,400,000)
Fiscal Year 2006: ¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 2005: ¥5,900,000 (Direct Cost: ¥5,900,000)
1.Synaptic transmission in cacopholy(cac)-null mutant embryos
It has been demonstrated that Ca^<2+> influx through Drosophila N-type Ca^<2+> channels, encoded by the cacophony (cac) gene, triggers fast synaptic transmission. A remaining question is ; Is the cac Ca^<2+> channel the sole type of Ca^<2+> channel for fast synaptic transmission? Since the cac^<null> mutation is lethal, this question can only be answered by examining synaptic transmission in cac^<null> embryos. At the cac^<null> neuromuscular junction (NMJ), no fast synchronous synaptic transmission was detected upon nerve stimulation while infrequent delayed quantal synaptic events were observed. When the wild-type cac gene was introduced in the cac^<null> background, fast synaptic transmission recovered to 50% of heterozygous control embryos (cac^<null>/+). Thus, cac Ca^<2+> channels are necessary and sufficient for fast synaptic transmission. Unexpectedly, even in cac^<null> embryos nerve stimulation induced delayed releas
e of synaptic vesicles in the minority of cells in HL3 solution (1.5 mM Ca^<2+>) and all cells examined in 5 mM [Ca^<2+>]_e. These delayed events started to appear at around 8 ms after stimulation, quickly increased in frequency to a peak at around 13 ms and gradually decreased to the baseline after 〜80 ms. The delayed events were abolished by 10 nM PLTXII (a spider toxin analog) suggesting that voltage-gated Ca^<2+> channels, other than cac Ca^<2+> channels, are contributing to the delayed release, but were not affected by 50 μM La^<3+> that preferentially blocks one type of Ca^<2+> channel in the Drosophila presynaptic terminal. Taken together, the cac Ca^<2+> channel is the sole Ca^<2+> channel for fast synaptic transmission and another type of Ca^<2+> channel, non-cac, PLTXII-sensitive Ca^<2+> channel, is contributing to delayed transmitter release in cac^<null> embryos.
2.Distribution of cac Ca^<2+> channel clusters and the number of channels in each cluster at the presynaptic terminal.
Using a Drosophila transformant that expresses EGFP-tagged cac Ca^<2+> channels, we have visualized the distribution of channel clusters at the presynaptic terminal. The number of Ca^<2+> in each cluster was estimated by examining the bleaching process of fluorescence intensity. In the line scan mode of confocal laser microscopy, the fluorescence intensity of a cluster declined due to bleaching. During this process, we found that the intensity declines in steps, which indicates a contribution of each EGFP molecule. By counting the number of steps we should be able to estimate the number of Ca^<2+> channels in each cluster. It turned out that the number is relatively small, around 10, in agreement with previous results in other systems obtained using completely different methods. This information is indispensable for simulation of the process of exocytosis at each release site. Less