Drosophila Neuromuscular Junction Formation in Culture

收集受精后3~4小时的果蝇胚胎神经元培养物，用次氯酸盐消毒。内容物被吸入玻璃吸管，并转移到添加胎牛血清和胰岛素的施耐德培养液中。神经元在几天内发出经过处理的信号，并形成相互联系。与肌肉细胞的共培养正在进行中。ii)果蝇突变体Shibia神经肌肉突触的囊泡池的大小、动员速率和循环利用在果蝇的神经肌肉接头上存在两个囊泡池，即易释放(RRP)和储备(RP)池。利用对温度敏感的突变体Shibir^&lt；ETS&gt；，我们研究了池的大小和囊泡的动员速率。在Shibire^&lt；ts&gt；，由于在不允许的温度下缺乏内吞作用，突触电流在强直刺激过程中不断下降，直到由于囊泡耗尽而停止。到那时，大约有84,000个量子被租赁。囊泡是以1/7~1/10的速率从RP中动员出来的，细胞松弛素D抑制了从RP中动员囊泡的作用，使我们能够估计RRP的大小为所有囊泡的14%~19%。小泡回收支持强直刺激持续期间的突触传递，最大回收速率为1000个小泡/S。

i)Cultures of embryonic Drosophila neuronsEggs(3 to 4 hours after fertilization)were collected and sterilized with hypochlorate. The content was sucked into a glass pipette and transferred into Schneider's medium supplemented with fetal calf serum and insulin. Neurons send out processed within a few days and form contacts each other. Co-cultures with muscle cells are now being tried.ii)Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibireTwo vesicle pools, readily releasable(RRP)and reserve(RP)pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibir^<ets>, we studied pool sizes and vesicle mobilization rates. In shibire^<ts>, due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were relased. Vesicles were mobilized from RP at a rate of 1/7-1/10 of RRP.Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.