Gene Control of Synaptic Transmission

半胱氨酸串蛋白(CSPs)是一种突触蛋白，在果蝇神经肌肉接头的神经递质释放中起重要作用。据报道，在一个温度敏感的CSP突变体中，神经诱发的突触电位在不允许的温度下没有观察到，而自发突触电位仍然存在。因此推测CSP蛋白与神经末梢钙离子通道相互作用，导致神经末梢钙离子内流。在这个项目中，我们用一种名为深红AM的染料测量了3龄幼虫突触前神经末梢在动作电位过程中钙离子浓度的变化。在RCom温度下，CSP突变体幼虫在重复神经刺激时可以清楚地检测到钙信号。然而，在不允许的温度下(32ﾟC)，在相同的刺激下没有观察到信号。由于CSP蛋白位于突触小泡膜上，突触小泡在动作电位1到达时必须处于正确的位置。如果没有突触小泡，我们预计不会有钙离子内流。为了验证这一假设，我们使用了另一个突变体shibire，在该突变体中，囊泡回收在不允许的温度下受到损害。在这个突变体中，已经显示出当神经在不允许的温度下被重复刺激时，囊泡被耗尽，并且没有观察到神经诱发突触电位。在这种耗竭状态下，在重复刺激神经时，神经末梢没有观察到钙信号。因此，利用两个完全独立的果蝇突变体，我们证明了突触小泡和CSPs对突触前钙通道的功能是至关重要的。

Cysteine string proteins (csps) are synaptic proteins that are important for evoked neurotransmitter release at Drosophila neuromuscular junctions. In a temperature-sensitive csp mutant it has been reported that nerve-evoked synaptic potentials are not observed in non-permissive temperature while spontaneous synaptic potentials remain. When csp proteins were expressed together with Ca^<2+>channel proteins in Xenopus oocytes Ca^<2+>inward currents were greater than when Ca^<2+>channel proteins alone were expressed. Thus it has been speculated that csp proteins are interacting Ca^<2+>channels for Ca^<2+>influx at the nerve terminal. In this project we measured changes of Ca^<2+>concentration in the presynaptic nerve terminal during action potentials in 3rd instar larvae using a dye, Ca^<2+>Crimson AM.Upon repetitive nerve stimulation in csp mutant larvae Ca^<2+>signals were clearly detected at rcom temperature. However, at non-permissive temperature (32ﾟC) no signals were observed upon identical stimulation. In wild-type larvae Ca^<2+>signals were detected even at 32ﾟC.Therefore, we suggest that the normal functin of csp proteins is necessary for Ca^<2+>influx. Since csp proteins are on the synaptic vesicle membrane, synaptic vesicles have to be in the right position at the time of action potentia1 arrival. If there are no synaptic vesicles we expect no Ca^<2+>influx. To test this hypothesis we used another mutant, shibire, in which vesicle recycling is impaired at non-permissive temperature. In this mutant it has been shown that vesicles are depleted and no nerve evoked synaptic potentials are observed when the nerve was stimulated repetitively at non-permissive temperature. In this depleted condition no Ca^<2+>signals were observed at the nerve terminal upon repetitive nerve-stimulation. Thus, using two completely independent Drosophila mutants, we demonstrated that synaptic vesicles and csps are vital for the function of presynaptic Ca^<2+>channels.