Drosophila Neuromuscular Junction Formation in Culture

培养中果蝇神经肌肉接头的形成

• 批准号: 11694242
• 负责人: KIDOKORO Yoshiaki
• 金额: $ 4.1万
• 依托单位: Gunma University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694242/
• 关键词: Cultered neurons; Drosophila; mutants; synaptic transmission; shibire; neuromuscular junctions; vesicle recycling; synaptic vesicle pools

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.

i）胚胎果蝇神经元的培养物收集卵（受精后3至4小时）并用次氯酸盐消毒。将内容物吸入玻璃移液管中，并转移至补充有胎牛血清和胰岛素的Schneider培养基中。神经元在几天内发出处理，并形成相互联系。与肌肉细胞的共培养现在正在尝试。ii）囊泡池的大小，动员率，并在神经肌肉突触的果蝇突变体，shibire回收两个囊泡池，容易释放（RRP）和储备（RP）池，存在于果蝇神经肌肉接头。使用温度敏感的突变体，shibir^<ets>，我们研究了池的大小和囊泡动员率。在shibire^中<ts>，由于在非允许温度下缺乏内吞作用，突触电流在强直刺激期间持续下降，直到由于囊泡耗尽而停止。到那时，大约有84，000个量子被释放。细胞松弛素D可抑制RP中囊泡的动员，RRP中囊泡的大小约占总囊泡的14%~ 19%。在长时间强直刺激期间，囊泡再循环支持突触传递，最大再循环速率为1000个囊泡/s。

项目成果

Kidokoro,Y.: "Synapse Formation."Encyclopedia of Life Sciences 2000. 90. 1691-1697 (1999)

Kidokoro,Y.：“突触形成”。生命科学百科全书 2000. 90. 1691-1697 (1999)

Kuromi H,Kidokoro Y: "The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content"Journal of Neuroscience. 19. 1557-1565 (1999)

Kuromi H、Kidokoro Y：“光学确定的外/内循环囊泡池大小与量子含量相关”《神经科学杂志》。

Yoshihara,M.,Suzuki,K.,Kidokoro,Y.: "Two independent pathways mediated by cAMP/PKA enhance spontaneous transmitter release at Drosophila..."Journal of Neuroscience. 20. 8315-8322 (2000)

Yoshihara,M.、Suzuki,K.、Kidokoro,Y.：“cAMP/PKA 介导的两条独立途径增强果蝇的自发递质释放......”神经科学杂志。

Rheuben MB,Yoshihara M,,Kidokoro Y.: ""International Review of Neurobiology Volume 43-Neuromuscular Junctions in Drosophila""Academic Press. (1999)

Rheuben MB，Yoshihara M，，Kidokoro Y.：“《国际神经生物学评论第 43 卷-果蝇神经肌肉连接》”学术出版社。

Nishikawa K,Kidokoro Y.: "Octopamine inhibits synaptic transmission at the larval neuromuscular junction in Drosophila melanogaster."Brain Research. 837. 67-74 (1999)

Nishikawa K，Kidokoro Y.：“章鱼胺抑制果蝇幼虫神经肌肉接头处的突触传递。”大脑研究。