Levitan Edwin S, Lanni Frederick, Shakiryanova Dinara
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Nat Protoc. 2007;2(5):1117-25. doi: 10.1038/nprot.2007.142.
Recently, it has become possible to directly detect changes in neuropeptide vesicle dynamics in nerve terminals in vivo and to measure the release of neuropeptides induced experimentally or evoked by normal behavior. These results were obtained with the use of transgenic fruit flies that express a neuropeptide tagged with green fluorescent protein. Here, we describe how vesicle movement and neuropeptide release can be studied in the larval Drosophila neuromuscular junction using fluorescence microscopy. Analysis methods are described for quantifying movement based on time lapse and fluorescence recovery after photobleaching data. Specific approaches that can be applied to nerve terminals include single particle tracking, correlation and Fourier analysis. Utilization of these methods led to the first detection of vesicle mobilization in nerve terminals and the discoveries of activity-dependent capture of transiting vesicles and post-tetanic potentiation of neuropeptide release. Overall, this protocol can be carried out in an hour with ready Drosophila.
最近，已经能够直接检测体内神经末梢中神经肽囊泡动力学的变化，并测量由实验诱导或正常行为诱发的神经肽释放。这些结果是通过使用表达带有绿色荧光蛋白标记的神经肽的转基因果蝇获得的。在这里，我们描述了如何使用荧光显微镜在幼虫果蝇神经肌肉接头中研究囊泡运动和神经肽释放。描述了基于时间推移和光漂白数据后的荧光恢复来量化运动的分析方法。可应用于神经末梢的具体方法包括单粒子跟踪、相关性分析和傅里叶分析。这些方法的应用首次检测到神经末梢中的囊泡动员，并发现了转运囊泡的活性依赖性捕获和神经肽释放的强直后增强。总体而言，使用现成的果蝇，该实验方案可以在一小时内完成。
