Drosophila Neuromuscular Junction Formation in Culture

i)收集胚胎果蝇神经元细胞（受精后3 - 4小时）的培养物，用次氯酸盐消毒。将内容物吸入玻璃移液管中，转移到添加了胎牛血清和胰岛素的施耐德培养基中。神经元在几天内发出经过处理的信息，并形成相互联系。现在正在尝试与肌肉细胞共培养。果蝇神经肌肉连接处存在两个囊泡池，即易释放囊泡池（RRP）和储备囊泡池（RP）。使用温度敏感突变体shibir^<ets>，我们研究了池大小和囊泡动员率。在shibire^<ts>中，由于在非允许温度下缺乏内吞作用，突触电流在破伤风刺激期间持续下降，直到由于囊泡耗竭而停止。到那时，大约有84,000量子被释放。以RRP的1/7-1/10的速率从RP中动员囊泡。细胞松弛素D抑制RP的囊泡动员，使我们估计RRP的大小占所有囊泡的14%-19%。在长时间强直刺激下，囊泡循环支持突触传递，最大循环速率为1000个囊泡/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.