Kelly Seth M, Elchert Alexandra, Kahl Michael
Program in Neuroscience, The College of Wooster; Department of Biology, The College of Wooster;
Program in Biochemistry, Cellular, and Molecular Biology, The College of Wooster.
J Vis Exp. 2017 Nov 6(129):56174. doi: 10.3791/56174.
Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.
神经系统发育涉及一系列由多个信号通路和调控网络协调的连续事件。这些通路中涉及的许多蛋白质在哺乳动物和其他真核生物（例如果蝇黑腹果蝇）之间具有进化保守性，这表明在这些生物体的发育过程中存在相似的组织原则。重要的是，果蝇已被广泛用于识别调节哺乳动物所需过程（包括神经发生、分化、轴突导向和突触发生）的细胞和分子机制。果蝇也已成功用于模拟多种人类神经发育疾病。在这里，我们描述了一种对成年果蝇大脑中的蛋白质进行逐步显微解剖、固定和免疫荧光定位的方案。该方案重点关注两个示例神经元群体，即蘑菇体神经元和视网膜光感受器，并包括使用带有可抑制细胞标记的镶嵌分析（MARCM）技术追踪单个蘑菇体神经元的可选步骤。展示了来自野生型和突变型大脑的示例数据，以及轴突导向缺陷评分标准的简要描述。虽然该方案重点介绍了两种用于研究蘑菇体和光感受器神经元形态的成熟抗体，但使用该方案也可以研究果蝇大脑的其他区域以及其他大脑区域内蛋白质的定位。
