大众组织学量化在神经退行性变<em>果蝇</em
Summary
果蝇被广泛用作模型系统来研究神经变性。这个协议描述由退化，如由大脑液泡形成确定的，可量化的方法。它也最大限度地减少的效果因的实验程序由处理和切片对照和实验苍蝇，因为一个样本。
Abstract
进行性神经变性疾病如阿尔茨海默氏病（AD）或帕金森氏病（PD）是全世界人类健康日益严重的威胁。虽然哺乳动物模型提供了重要的见解致病的内在机制，在哺乳动物系统与他们的成本高一起的复杂性限制了其使用。因此，简单而完善的果蝇模型的系统提供了用于调查了在这些疾病影响的分子通路的替代方案。除了行为缺陷，神经变性疾病是由组织学表型，如神经元死亡和轴突病变表征。量化神经元变性，并确定它是如何由遗传和环境因素的影响，我们使用一个基于在成年果蝇大脑测量液泡组织学方法。为了尽量减少系统误差的影响，并直接比较从控制和记录部分在一个制备erimental苍蝇，我们使用石蜡切片的“衣领”方法。神经变性，然后通过测量已经在飞脑开发液泡的大小和/或数量的评估。这可以通过关注感兴趣的特定区域，或通过获得跨越完整头连续切片分析整个大脑来完成。因此，该方法允许人们测量不仅严重退化，而且在相对温和的表型是只在少数部分检测的，正常的老化期间发生。
Introduction
随着预期寿命的延长，神经退行性疾病如阿尔茨海默或帕金森已成为普通人群越来越健康威胁。根据美国国立卫生研究院，1.15亿人的全球预计将被老年痴呆症在2050年的影响虽然显著进展，在确定至少参与了一些这些疾病的基因和风险因素，其中许多已经取得，底层分子机制仍未知的或不充分的了解。
像线虫和果蝇简单无脊椎动物模式生物提供了多种实验的优势来研究神经退行性疾病，包括生命周期短，大量的后代，以及行之有效的，有时独特的遗传和分子生物学方法1的可用性机制-12。此外，这些生物是适合于偏互动屏幕，可识别通过神经退行性表型的加重或改善效果有助于这些疾病的因素。
分析这些基因的相互作用和评估老化的影响需要量化的协议，以检测神经功能障碍和衡量其严重程度。测量在果蝇行为方面，如嗅觉学习，负趋地性，或快趋光性，这提供了一个数值性能值13-21时，这种评估可以比较容易地完成。另外，也可以通过计数神经元，以确定对神经元存活的影响。然而，专注于特定人群，这显然是可识别的，像在PD的影响，即使这样，结果一直存在争议22-24多巴胺能神经元时，这是唯一可能的。
这里所描述的协议使用的衣领方法执行石蜡连续切片中，一种方法最初由海森堡和博尔，谁用它在果蝇 25分离解剖大脑突变体的发展。使用该套环方法的随后被改编，包括在冷冻切片，vibratome部分和塑料部分26-28。这里，采用这种方法，得到了整个飞头，然后可将其用于测量在苍蝇开发具有神经变性表型16,21,29-32液泡的连续切片。这些测量可以在特定脑区来完成，或者可以覆盖整个大脑;后一种方法可以让老化过程中观察到一个识别即使很弱的退行性表型。最后，使用套环时，最多20苍蝇可被处理为一个准备，这不仅耗时少，而且还允许用于控制和在相同的制备实验苍蝇的分析，最小化由于在微小变化的工件的制备方法。
Protocol
1.固定在项圈的头和石蜡包埋注：所有的在定影过程中的步骤应在通风橱中进行。甲基苯甲酸甲酯，而不是构成一个健康风险，具有高度不同的气味，这可以是压倒性如果在通风橱中不进行处理。 麻醉苍蝇之前，加入15毫升的氯仿和5毫升冰醋酸30毫升99％乙醇（不要混合的冰醋酸和氯仿）占50毫升卡诺的解决方案。在具有平坦底部，玻璃容器倒如一个晶化盘，以确保该环可以平放和由?…
Representative Results
使用在由眼睛颜料33包括整个飞头染色的连续切片所描述的方法的结果。这方面的一个部分示于图1B中 ，其中，来自个体头的部分被示出从上到下。从不同的苍蝇的部分被视为左至右在本实施例。为了便于取向和苍蝇的鉴定，无眼蝇（ 正弦oculis）被插入为在3位（箭头， 图1B）的标志物。 <p class="jove_content" fo:keep-together.within-page="1…
Discussion
所描述的方法提供了在果蝇的脑量化的神经变性的方法。而其它的方法，如计数特定的细胞类型，可用于鉴定神经变性，这种方法的优点是，它可被更一般地应用。细胞计数要求这些细胞可以使用一个特定的抗体或细胞特异性标记的表达，这并不总是可用可靠地识别。此外，已经表明，显着不同的结果可以与方法24中获得，这可能是由于用于标记和检测的条件。检测衰退的细胞的另…
Divulgations
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).
Materials
| Name of the Reagent/Equipment | Company | Catalog Number | |
| Collar | Genesee Scientific | TS 48-100 | We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html |
| Rubber ice cube tray for embedding | Household store | The size can be made to fit by glueing in additional walls | |
| Crystallizing dish | Fisher Scientific company | 08-762-3 | |
| Ether | Fisher Scientific Company | E138-1 | |
| Ethanol | Decon Laboratories Inc. | 2701 | |
| Choloroform | Fisher Scientific Company | C298-500 | |
| Glacial Acetic Acid | Fisher Scientific Company | A38-212 | |
| Methylbenzoate | Fisher Scientific Company | M205-500 | Distinct Odor |
| Use in fume hood | |||
| Low Melting Point Paraffin Wax | Fisher Scientific Company | T565 | Make sure to keep extra melted in a 65°C waterbath |
| Microtome | Leica Biosystems | Reichert Jung 2040 Autocut | |
| Microscope Slide | Fisher Scientific Company | 12-550D | |
| Microscope Cover Glass | Fisher Scientific Company | 12-545-M | |
| SafeClear | Fisher Scientific Company | 314-629 | Three different containers for washes |
| Vertical Staining Jar with Cover | Ted Pella Inc. | 432-1 | |
| Permount | Fisher Scientific Company | SP15-500 | |
| Poly-L-lysine Solution | Sigma Life Science | P8290-500 |
References
- Alexander, A. G., Marfil, V., Li, C. Use of Caenorhabditis elegans as a model to study Alzheimer’s disease and other neurodegenerative diseases. Front Genet. 5, 279 (2014).
- Bonini, N. M., Fortini, M. E. Human neurodegenerative disease modeling using Drosophila. Annu Rev Neurosci. 26, 627-656 (2003).
- Calahorro, F., Ruiz-Rubio, M. Caenorhabditis elegans as an experimental tool for the study of complex neurological diseases: Parkinson’s disease, Alzheimer’s disease and autism spectrum disorder. Invert Neurosci. 11, 73-83 (2011).
- Chen, X., Barclay, J. W., Burgoyne, R. D., Morgan, A. Using C. elegans to discover therapeutic compounds for ageing-associated neurodegenerative diseases. Chemistry Central Journal. 9, 65 (2015).
- Gama Sosa, M. A., De Gasperi, R., Elder, G. A. Modeling human neurodegenerative diseases in transgenic systems. Hum Genet. 131, 535-563 (2012).
- Jaiswal, M., Sandoval, H., Zhang, K., Bayat, V., Bellen, H. J. Probing mechanisms that underlie human neurodegenerative diseases in Drosophila. Annu Rev Genet. 46, 371-396 (2012).
- Konsolaki, M. Fruitful research: drug target discovery for neurodegenerative diseases in Drosophila. Expert Opin Drug Discov. 8, 1503-1513 (2013).
- Kretzschmar, D. Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases. Invert Neurosci. 5, 97-109 (2005).
- Kretzschmar, D., et al. Swiss cheese et allii, some of the first neurodegenerative mutants isolated in Drosophila. J Neurogenet. 23, 34-41 (2009).
- Li, J., Le, W. Modeling neurodegenerative diseases in Caenorhabditis elegans. Exp Neurol. 250, 94-103 (2013).
- Prussing, K., Voigt, A., Schulz, J. B. Drosophila melanogaster as a model organism for Alzheimer’s disease. Mol Neurodegener. 8, (2013).
- Wentzell, J., Kretzschmar, D. Alzheimer’s disease and tauopathy studies in flies and worms. Neurobiol Dis. 40, 21-28 (2010).
- Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration. J Vis Exp. , (2011).
- Barone, M. C., Bohmann, D. Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining. J Vis Exp. , e50339 (2013).
- Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. Glial expression of Swiss-cheese (SWS), the Drosophila orthologue of Neuropathy Target Esterase, is required for neuronal ensheathment and function. Dis Model Mech. , (2015).
- Iijima, K., et al. Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila. PLoS One. 3, e1703 (2008).
- Krishnan, N., et al. Loss of circadian clock accelerates aging in neurodegeneration-prone mutants. Neurobiol Dis. 45, 1129-1135 (2012).
- Liu, H., et al. Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila. Neurosci Bull. 31, 541-549 (2015).
- Papanikolopoulou, K., Skoulakis, E. M. Temporally distinct phosphorylations differentiate Tau-dependent learning deficits and premature mortality in Drosophila. Hum Mol Genet. 24, 2065-2077 (2015).
- Ping, Y., et al. Linking abeta42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer’s model. PLoS Genet. 11, e1005025 (2015).
- Sujkowski, A., Rainier, S., Fink, J. K., Wessells, R. J. Delayed Induction of Human NTE (PNPLA6) Rescues Neurodegeneration and Mobility Defects of Drosophila swiss cheese (sws) Mutants. PLoS One. 10, e0145356 (2015).
- Barone, M. C., Sykiotis, G. P., Bohmann, D. Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson’s disease. Dis Model Mech. 4, 701-707 (2011).
- Coulom, H., Birman, S. Chronic exposure to rotenone models sporadic Parkinson’s disease in Drosophila melanogaster. J Neurosci. 24, 10993-10998 (2004).
- Navarro, J. A., et al. Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson’s disease in Drosophila. J Neurochem. 131, 369-382 (2014).
- Heisenberg, M., Böhl, K. Isolation of anatomical brain mutants of Drosophila by histological means. Zeitschrift für Naturforschung C. 34, 143 (1979).
- Han, P. L., Meller, V., Davis, R. L. The Drosophila brain revisited by enhancer detection. J Neurobiol. 31, 88-102 (1996).
- Lin, C. W., et al. Automated in situ brain imaging for mapping the Drosophila connectome. J Neurogenet. 29, 157-168 (2015).
- Strausfeld, N. J., Sinakevitch, I., Vilinsky, I. The mushroom bodies of Drosophila melanogaster: an immunocytological and golgi study of Kenyon cell organization in the calyces and lobes. Microsc Res Tech. 62, 151-169 (2003).
- Bettencourt da Cruz, A., Wentzell, J., Kretzschmar, D. Swiss Cheese, a protein involved in progressive neurodegeneration, acts as a noncanonical regulatory subunit for PKA-C3. J Neurosci. 28, 10885-10892 (2008).
- Bolkan, B. J., Kretzschmar, D. Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila. Dev Neurobiol. 74, 1210-1225 (2014).
- Cook, M., Mani, P., Wentzell, J. S., Kretzschmar, D. Increased RhoA prenylation in the loechrig (loe) mutant leads to progressive neurodegeneration. PLoS One. 7, e44440 (2012).
- Wittmann, C. W., et al. Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science. 293, 711-714 (2001).
- Rasmuson, B., Green, M. M., Ewertson, G. QUALITATIVE AND QUANTITATIVE ANALYSES OF EYE PIGMENTS AND PTERIDINES IN BACK-MUTATIONS OF THE MUTANT wa IN DROSOPHILA MELANOGASTER. Hereditas. 46, 635-650 (1960).
- Bettencourt da Cruz, A., et al. Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila. Mol Biol Cell. 16, 2433-2442 (2005).
- Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., Benzer, S. The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila. J Neurosci. 17, 7425-7432 (1997).
- Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function. Dis Model Mech. 9, 283-294 (2016).
- Davis, M. Y., et al. Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration. PLoS Genet. 12, e1005944 (2016).
- Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. Oxidative stress mediates tau-induced neurodegeneration in Drosophila. J Clin Invest. 117, 236-245 (2007).
- Kretzschmar, D., et al. Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila. Glia. 49, 59-72 (2005).
- Li, Y., et al. A Drosophila model for TDP-43 proteinopathy. Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).
Citer Cet Article
Sunderhaus, E. R., Kretzschmar, D. Mass Histology to Quantify Neurodegeneration in Drosophila. J. Vis. Exp. (118), e54809, doi:10.3791/54809 (2016).