Quantitative analysis of synaptic boutons in Drosophila primary neuronal cultures
Quantitative analysis of synaptic boutons in Drosophila primary neuronal cultures
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DOI：
10.1016/j.brainres.2009.05.026
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发表时间：
2009-07-14
期刊：
影响因子：
2.9
通讯作者：
Lee, Daewoo
中科院分区：
文献类型：
--
作者：
Darya, Kauroon;Ganguly, Archan;Lee, Daewoo
由于很难在果蝇的小大脑中获取这些突触，目前关于果蝇中枢突触的结构和定量方面的信息很少。在这里，我们开发了一种新的方法来定量分析中央突触使用果蝇原代神经元培养。使用两种不同的标记来鉴定突触钮扣：用synaptotagmin （Syt）标记GFP；转基因eGFP和抗Syt抗体。这些标记清楚地识别了点状突触钮扣，并且两种信号很好地重叠。此外，这些点信号在Syt零突变体Syt（AD4）衍生的神经元培养物中完全不存在，有力地证明了抗Syt（+）点是突触前终末。由于抗syt信号较强且广泛，因此选择它来量化神经元培养中的突触钮扣。利用图像分析软件image J，根据anti-Syt（+）信号的大小和强度对突触钮扣进行量化。野生型神经元突触钮扣的数量在培养3 ~ 9天增加了27%。在FasII（e86）突变体的神经元培养物中，这种增加要大得多（142%），该突变体显示突触生长和稳定的改变。在野生型和FasII（e86）神经元中也观察到神经突长度平行增加。有趣的是，gaba能突触钮扣的数量在这段时间内并没有增加，这表明特定类型钮扣的发展和维持的独特机制。我们的研究结果成功地表明，果蝇突触钮扣可以被量化，从而我们可以研究调节果蝇中央突触结构特性的基因和信号通路。(C) 2009 Elsevier B.V.版权所有
Little information is currently available for structural and quantitative aspects of Drosophila central synapses due to difficulties in accessing those synapses in the tiny fly brain. Here, we developed a new approach to quantitatively analyze central synapses using Drosophila primary neuronal cultures. Two different markers were used to identify synaptic boutons: GFP marking with a synaptotagmin (Syt)::eGFP transgene and anti-Syt antibody. These markers clearly recognized puncta-like synaptic boutons and both signals were well overlapped. In addition, these puncta signals were completely absent in neuronal cultures derived from a Syt null mutant Syt(AD4), firmly demonstrating that anti-Syt(+) puncta are presynaptic terminals. Since anti-Syt signals were stronger and extensive, it was chosen to quantify synaptic boutons in the neuronal culture. Using an image analysis software Image J, synaptic boutons were quantified on the basis of the size and intensity of anti-Syt(+) signals. The number of synaptic boutons in wild type neurons increased by 27% between 3 and 9 days in culture. This increase was much greater (142%) in neuronal cultures derived from a FasII(e86) mutant known to show alterations in synapse growth and stabilization. A parallel increase in neurite length was also observed in both wild type and FasII(e86) neurons. Interestingly, the number of GABAergic synaptic boutons did not increase during this time, indicating distinctive mechanisms underlying development and maintenance of specific types of boutons. Our results successfully showed that Drosophila synaptic boutons can be quantified and thus we can examine genes and signaling pathways regulating structural properties of central synapses in Drosophila. (C) 2009 Elsevier B.V. All rights reserved.