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).

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。 <ol><li>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该溶液完全覆盖。 </li><li>麻醉与CO 2或乙醚苍蝇。 </li><li>螺纹蝇（最多20个与大多数环）可以通过颈部进入使用镊子的衣领。记对准所有磁头的相同的方向，如在图1A，并轻柔，保证不发生向头部或眼睛没有损害。 </li><li>包括随机位置，使苍蝇的顺序可以在部分很容易确定正弦oculis苍蝇（箭头， 图1A）。此外，如果实验苍蝇具有光或白色眼睛的颜色，螺纹一些红眼蝇，如野生型，在它们之间，以确保有足够的颜料是本染色的幻灯片。如果使用多个领衣领号码记录蝇量级的协议片一起。 </li><li>一旦领已经完成，将其放置在3.5准备的卡诺的解决方案 - 4小时。 </li><li> dump出来卡诺液到适当的处理罐，并开始清洗乙醇。请一定要慢慢倒入，以免扰乱容器中环的位置。 </li><li>洗套环在99％乙醇30分钟两次。 </li><li>洗在100％乙醇套环1小时。一定要改变时间的洗涤，以防overdehydration。 </li><li>放入甲基苯甲酸衣领O / N在室温。密封容器用石蜡膜以防止甲基苯甲酸甲酯的蒸发。 </li><li>倒入methylbenozate到通风柜适当一次性容器。加1的预先准备的混合物:1熔点低（56 - 57°C）石蜡和苯甲酸甲酯。从这点上来说，轴环需要在65℃下被保持在培养箱中以确保石蜡不硬化。 </li><li>倾出methylbenozate和石蜡混合物到适当的一次性容器，并倒入熔融的纯石蜡，保持在65℃，到套环。 </li><li>改变石蜡后30分钟，并重复此至少5次。至少6 - 8次洗涤应该执行。 </li><li>一旦洗涤完成后，放置套环与槽大约轴环的尺寸的橡胶冰块托盘。倒入熔化的石蜡过他们，直到完全覆盖，并允许它变硬O / N（尽量避免气泡）。 </li><li>取出石蜡块，包含3宁从冰块托盘的衣领。从使用刀片领子分开石蜡块，轻轻掰的衣领。该负责人将在蜡块，而机构会留在衣领。该块可以在室温下保持。 </li><li>清洁套环，在65℃下浸泡他们在一个deparafinization剂以除去石蜡，清洁光擦洗，并在重复使用之前在乙醇洗净。 </li></ol> 2.切割和安装<ol><li>暖加热板到50℃。将上盘对象持有人（或金属固定块）和刀片，让他们热身。 </li><li>取决于用于切片所需的方向，连接或者与朝向侧磁头（对于水平段）的石蜡块或朝上（对于正面部），以加热安装块（简要地熔融在接触侧的块）。从加热板上取下块并让其冷却至少10英里n至确保该石蜡硬化够适当的密封到安装块。保持平行，尽可能防止不均匀的部分中的块的表面对准头的行。 </li><li>取一个刀片并修剪多余的石蜡从蝇头远，使得只有一小行与嵌入式头保持（剃须刀刀片可以温热更容易修整）。确保不要修剪过多，使石蜡不切片（更多微调可以切片过程中进行）时断裂。 </li><li>安装块放入切片机的对象保持器，并确保磁头行的取向是尽可能平行于叶片的边缘。 </li><li>通过覆盖他们的聚-L-赖氨酸（PLL）的溶液的薄层准备显微镜载玻片并让它们干燥5分钟。覆盖它们与水之前不久使用。 </li><li>削减7微米的部分和部分的色带转移到幻灯片浮在水面上。&lt;BR /&gt;注:要获取水平段整个大脑，我们将收集从开始切入眼睛，直到头已经完全切断（从长鼻切入脑）当色带。可能需要对整个头部多个幻灯片。 </li><li>放置在37℃的热板的滑动，并允许色带展开约1分钟。 </li><li>去除多余的水（浇其关闭或使用组织）和干燥的幻灯片O / N。 </li><li>通过将玻片在填充有deparafinization剂（完全覆盖部分）一高，垂直滑动染色缸中删除从滑动的石蜡。执行的30分钟洗涤3次 - 每次60分钟。 </li><li>取下最后洗的幻灯片。将2滴嵌入媒体到幻灯片上，并用大盖玻片覆盖。 </li></ol> 3.拍摄和分析章节<ol><li>允许制备的玻片干燥1 - 2天。然后，检查它们放在荧光microsc在蓝光下单操作。 </li><li>使用较低的放大率确定苍蝇的方向，如果着眼于一个特定区域，以找到感兴趣的区域。 注:对于SWS苍蝇（ 见图2），我们发现，含有大合缝的部分，并拍摄图像（通常在40倍放大倍率）。当分析整个脑（ 如图3），我们通过从头部所有部分滚动，要么具有最严重的表型，或显示空泡所有节拍摄部分。 </li><li>为双盲分析，采取与数字图像，而不知道基因型并记录行中的套环数和头部的位置以后，以确定它们。 </li><li>一旦图像已被拍摄，使用成像软件进行分析。 </li><li>每计数或部分每头空泡的数量。为了测量液泡的大小，在一个软件程序中打开的图像，并选择与选择的空泡太湖确定在所选择的液泡的像素的量。 </li><li>转换为微米2，取在用于采集照片放大一个阶段微米校准幻灯片的照片。确定象素的数量在100微米2以计算换算因子。 </li><li>通过在上述步骤中计算出的换算因子除以象素数目转换的总像素数成微米2。 </li></ol>

<ol>
	<li>Alexander, A. G., Marfil, V., Li, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Caenorhabditis+elegans+as+a+model+to+study+Alzheimer's+disease+and+other+neurodegenerative+diseases.">Use of Caenorhabditis elegans as a model to study Alzheimer's disease and other neurodegenerative diseases.</a> Front Genet. 5, 279 (2014).</li><li>Bonini, N. M., Fortini, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neurodegenerative+disease+modeling+using+Drosophila.">Human neurodegenerative disease modeling using Drosophila.</a> Annu Rev Neurosci. 26, 627-656 (2003).</li><li>Calahorro, F., Ruiz-Rubio, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+as+an+experimental+tool+for+the+study+of+complex+neurological+diseases:+Parkinson's+disease,+Alzheimer's+disease+and+autism+spectrum+disorder.">Caenorhabditis elegans as an experimental tool for the study of complex neurological diseases: Parkinson's disease, Alzheimer's disease and autism spectrum disorder.</a> Invert Neurosci. 11, 73-83 (2011).</li><li>Chen, X., Barclay, J. W., Burgoyne, R. D., Morgan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+C.+elegans+to+discover+therapeutic+compounds+for+ageing-associated+neurodegenerative+diseases.">Using C. elegans to discover therapeutic compounds for ageing-associated neurodegenerative diseases.</a> Chemistry Central Journal. 9, 65 (2015).</li><li>Gama Sosa, M. A., De Gasperi, R., Elder, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+neurodegenerative+diseases+in+transgenic+systems.">Modeling human neurodegenerative diseases in transgenic systems.</a> Hum Genet. 131, 535-563 (2012).</li><li>Jaiswal, M., Sandoval, H., Zhang, K., Bayat, V., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+mechanisms+that+underlie+human+neurodegenerative+diseases+in+Drosophila.">Probing mechanisms that underlie human neurodegenerative diseases in Drosophila.</a> Annu Rev Genet. 46, 371-396 (2012).</li><li>Konsolaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fruitful+research:+drug+target+discovery+for+neurodegenerative+diseases+in+Drosophila.">Fruitful research: drug target discovery for neurodegenerative diseases in Drosophila.</a> Expert Opin Drug Discov. 8, 1503-1513 (2013).</li><li>Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+mutants+in+Drosophila:+a+means+to+identify+genes+and+mechanisms+involved+in+human+diseases.">Neurodegenerative mutants in Drosophila: a means to identify genes and mechanisms involved in human diseases.</a> Invert Neurosci. 5, 97-109 (2005).</li><li>Kretzschmar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swiss+cheese+et+allii,+some+of+the+first+neurodegenerative+mutants+isolated+in+Drosophila.">Swiss cheese et allii, some of the first neurodegenerative mutants isolated in Drosophila.</a> J Neurogenet. 23, 34-41 (2009).</li><li>Li, J., Le, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+neurodegenerative+diseases+in+Caenorhabditis+elegans.">Modeling neurodegenerative diseases in Caenorhabditis elegans.</a> Exp Neurol. 250, 94-103 (2013).</li><li>Prussing, K., Voigt, A., Schulz, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+as+a+model+organism+for+Alzheimer's+disease.">Drosophila melanogaster as a model organism for Alzheimer's disease.</a> Mol Neurodegener. 8, (2013).</li><li>Wentzell, J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+tauopathy+studies+in+flies+and+worms.">Alzheimer's disease and tauopathy studies in flies and worms.</a> Neurobiol Dis. 40, 21-28 (2010).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J Vis Exp. , (2011).</li><li>Barone, M. C., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+neurodegenerative+phenotypes+in+Drosophila+dopaminergic+neurons+by+climbing+assays+and+whole+brain+immunostaining.">Assessing neurodegenerative phenotypes in Drosophila dopaminergic neurons by climbing assays and whole brain immunostaining.</a> J Vis Exp. , e50339 (2013).</li><li>Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+expression+of+Swiss-cheese+(SWS),+the+Drosophila+orthologue+of+Neuropathy+Target+Esterase,+is+required+for+neuronal+ensheathment+and+function.">Glial expression of Swiss-cheese (SWS), the Drosophila orthologue of Neuropathy Target Esterase, is required for neuronal ensheathment and function.</a> Dis Model Mech. , (2015).</li><li>Iijima, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta42+mutants+with+different+aggregation+profiles+induce+distinct+pathologies+in+Drosophila.">Abeta42 mutants with different aggregation profiles induce distinct pathologies in Drosophila.</a> PLoS One. 3, e1703 (2008).</li><li>Krishnan, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+circadian+clock+accelerates+aging+in+neurodegeneration-prone+mutants.">Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.</a> Neurobiol Dis. 45, 1129-1135 (2012).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+rapid+iterative+negative+geotaxis+assay+and+its+use+in+a+genetic+screen+for+modifiers+of+Abeta(42)-induced+locomotor+decline+in+Drosophila.">Automated rapid iterative negative geotaxis assay and its use in a genetic screen for modifiers of Abeta(42)-induced locomotor decline in Drosophila.</a> Neurosci Bull. 31, 541-549 (2015).</li><li>Papanikolopoulou, K., Skoulakis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+distinct+phosphorylations+differentiate+Tau-dependent+learning+deficits+and+premature+mortality+in+Drosophila.">Temporally distinct phosphorylations differentiate Tau-dependent learning deficits and premature mortality in Drosophila.</a> Hum Mol Genet. 24, 2065-2077 (2015).</li><li>Ping, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+abeta42-induced+hyperexcitability+to+neurodegeneration,+learning+and+motor+deficits,+and+a+shorter+lifespan+in+an+Alzheimer's+model.">Linking abeta42-induced hyperexcitability to neurodegeneration, learning and motor deficits, and a shorter lifespan in an Alzheimer's model.</a> PLoS Genet. 11, e1005025 (2015).</li><li>Sujkowski, A., Rainier, S., Fink, J. K., Wessells, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+Induction+of+Human+NTE+(PNPLA6)+Rescues+Neurodegeneration+and+Mobility+Defects+of+Drosophila+swiss+cheese+(sws)+Mutants.">Delayed Induction of Human NTE (PNPLA6) Rescues Neurodegeneration and Mobility Defects of Drosophila swiss cheese (sws) Mutants.</a> PLoS One. 10, e0145356 (2015).</li><li>Barone, M. C., Sykiotis, G. P., Bohmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+activation+of+Nrf2+signaling+is+sufficient+to+ameliorate+neurodegenerative+phenotypes+in+a+Drosophila+model+of+Parkinson's+disease.">Genetic activation of Nrf2 signaling is sufficient to ameliorate neurodegenerative phenotypes in a Drosophila model of Parkinson's disease.</a> Dis Model Mech. 4, 701-707 (2011).</li><li>Coulom, H., Birman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+rotenone+models+sporadic+Parkinson's+disease+in+Drosophila+melanogaster.">Chronic exposure to rotenone models sporadic Parkinson's disease in Drosophila melanogaster.</a> J Neurosci. 24, 10993-10998 (2004).</li><li>Navarro, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+dopaminergic+neuronal+dysfunction+in+genetic+and+toxin-induced+models+of+Parkinson's+disease+in+Drosophila.">Analysis of dopaminergic neuronal dysfunction in genetic and toxin-induced models of Parkinson's disease in Drosophila.</a> J Neurochem. 131, 369-382 (2014).</li><li>Heisenberg, M., B&#246;hl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+anatomical+brain+mutants+of+Drosophila+by+histological+means.">Isolation of anatomical brain mutants of Drosophila by histological means.</a> Zeitschrift f&#252;r Naturforschung C. 34, 143 (1979).</li><li>Han, P. L., Meller, V., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+brain+revisited+by+enhancer+detection.">The Drosophila brain revisited by enhancer detection.</a> J Neurobiol. 31, 88-102 (1996).</li><li>Lin, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+in+situ+brain+imaging+for+mapping+the+Drosophila+connectome.">Automated in situ brain imaging for mapping the Drosophila connectome.</a> J Neurogenet. 29, 157-168 (2015).</li><li>Strausfeld, N. J., Sinakevitch, I., Vilinsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mushroom+bodies+of+Drosophila+melanogaster:+an+immunocytological+and+golgi+study+of+Kenyon+cell+organization+in+the+calyces+and+lobes.">The mushroom bodies of Drosophila melanogaster: an immunocytological and golgi study of Kenyon cell organization in the calyces and lobes.</a> Microsc Res Tech. 62, 151-169 (2003).</li><li>Bettencourt da Cruz, A., Wentzell, J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swiss+Cheese,+a+protein+involved+in+progressive+neurodegeneration,+acts+as+a+noncanonical+regulatory+subunit+for+PKA-C3.">Swiss Cheese, a protein involved in progressive neurodegeneration, acts as a noncanonical regulatory subunit for PKA-C3.</a> J Neurosci. 28, 10885-10892 (2008).</li><li>Bolkan, B. J., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Tau+results+in+defects+in+photoreceptor+development+and+progressive+neuronal+degeneration+in+Drosophila.">Loss of Tau results in defects in photoreceptor development and progressive neuronal degeneration in Drosophila.</a> Dev Neurobiol. 74, 1210-1225 (2014).</li><li>Cook, M., Mani, P., Wentzell, J. S., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+RhoA+prenylation+in+the+loechrig+(loe)+mutant+leads+to+progressive+neurodegeneration.">Increased RhoA prenylation in the loechrig (loe) mutant leads to progressive neurodegeneration.</a> PLoS One. 7, e44440 (2012).</li><li>Wittmann, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tauopathy+in+Drosophila:+neurodegeneration+without+neurofibrillary+tangles.">Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.</a> Science. 293, 711-714 (2001).</li><li>Rasmuson, B., Green, M. M., Ewertson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QUALITATIVE+AND+QUANTITATIVE+ANALYSES+OF+EYE+PIGMENTS+AND+PTERIDINES+IN+BACK-MUTATIONS+OF+THE+MUTANT+wa+IN+DROSOPHILA+MELANOGASTER.">QUALITATIVE AND QUANTITATIVE ANALYSES OF EYE PIGMENTS AND PTERIDINES IN BACK-MUTATIONS OF THE MUTANT wa IN DROSOPHILA MELANOGASTER.</a> Hereditas. 46, 635-650 (1960).</li><li>Bettencourt da Cruz, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+the+MAP1B-related+protein+FUTSCH+leads+to+changes+in+the+neuronal+cytoskeleton,+axonal+transport+defects,+and+progressive+neurodegeneration+in+Drosophila.">Disruption of the MAP1B-related protein FUTSCH leads to changes in the neuronal cytoskeleton, axonal transport defects, and progressive neurodegeneration in Drosophila.</a> Mol Biol Cell. 16, 2433-2442 (2005).</li><li>Kretzschmar, D., Hasan, G., Sharma, S., Heisenberg, M., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+swiss+cheese+mutant+causes+glial+hyperwrapping+and+brain+degeneration+in+Drosophila.">The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila.</a> J Neurosci. 17, 7425-7432 (1997).</li><li>Dutta, S., Rieche, F., Eckl, N., Duch, C., Kretzschmar, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+expression+of+Swiss+cheese+(SWS),+the+Drosophila+orthologue+of+neuropathy+target+esterase+(NTE),+is+required+for+neuronal+ensheathment+and+function.">Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function.</a> Dis Model Mech. 9, 283-294 (2016).</li><li>Davis, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocerebrosidase+Deficiency+in+Drosophila+Results+in+alpha-Synuclein-Independent+Protein+Aggregation+and+Neurodegeneration.">Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration.</a> PLoS Genet. 12, e1005944 (2016).</li><li>Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+mediates+tau-induced+neurodegeneration+in+Drosophila.">Oxidative stress mediates tau-induced neurodegeneration in Drosophila.</a> J Clin Invest. 117, 236-245 (2007).</li><li>Kretzschmar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+and+neuronal+expression+of+polyglutamine+proteins+induce+behavioral+changes+and+aggregate+formation+in+Drosophila.">Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.</a> Glia. 49, 59-72 (2005).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+TDP-43+proteinopathy.">A Drosophila model for TDP-43 proteinopathy.</a> Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).</li></ol>

作者什么都没有透露。

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一方法是利用细胞死亡标记物，如抗活化的caspase 3。但是，这只是确定细胞主动经历细胞死亡，一旦细胞已经死亡，它们不再可检测。这里所提出的方法的另一个优点是，没有染色，需要由于autofluorescence由眼睛色素，从而节省了时间和最小化所引起的染色条件的变化的工件而引起的。要注意的是，使用这种方法时，果蝇的眼睛有足够颜料染色的滑动是非常重要的。如果眼睛颜色太浅，加入一些野生型飞往衣领将是可取的，以确保足够的和整个幻灯片，甚至染色。这种方法的一个额外的优点是，多个区域可以被检查，即使在相同的头部。虽然我们不显示的数据，我们已经使用这些部分来检查在片材皮层30,36视网膜和神经胶质细胞的损失的神经变性。如这里描述的，该方法提供水平段，但通过熔化到物体保持器，当它以90°转动石蜡块，还可以得到正面部。因此，该方法是一种多用途的过程，允许神经变性的在飞大脑的及时和有效的测量。由于真空形成uoles已在人类神经变性疾病的许多飞模型，包括模型的AD中观察到，PD，肌萎缩性侧索硬化症（ALS），和由多聚谷氨酰胺重复序列16,37-40疾病，这种方法可用于定量神经变性表型多种疾病模型。 总体来说，这个协议很简单，一旦设备的初始设置进行轻松完成。一些注意事项要牢记要仔细时候乙醇清洗，以避免过度脱水的头，仔细修剪石蜡块，以免失去部分或头部，并没有过度扩张对水功能区时，幻灯片在热块。如果色带被允许太远扩张，撕裂飞头可导致在功能区中的头的顺序可能会丢失。此外，作为盲基因型，而这样做的分析是必不可少的，以避免偏见。这最好是由具有一个人制备SLI来实现DES和保存记录而另一个人走的是图片和做测量。这种方法的局限性是，只有少数细胞变性将非常难以检测。在这种情况下，受影响的细胞群的特异性染色。将更多的信息。此外，这种方法不允许一个不同类型的细胞死亡，这需要更具体的方法，例如TUNEL染色，以确定凋亡细胞死亡的区分。最后，这种方法不能细胞死亡和轴突变性，这也将是如在神经纤维液泡检测区分。 如在我们的结果显示，该方法可用于处理在特定脑区或整个大脑退化。根据我们的经验，当表型是相当强的也仅仅是分析一个特定的区域是有用的，即使所有的大脑区域受到影响。这显著降低了工作负荷和不影响结果。我们最初分析几个领域中的SWS突变和比较只有一个区域时观察到的表型的进展非常相似的结果和分析所有区域时（数据未显示）。然而，应该指出的是，明确地可识别区域应选择以避免工件由于不同地区或不同层次的分析。 与此相反，在表型是相对温和的情况下，最好是分析整个大脑，因为在一个特定的地区发现的空泡的可能性是低的。例如，这种情况下确定与年龄相关的神经变性时，如图3，这导致仅4 - 5液泡在60日龄蝇整个大脑。当在整个大脑计数空泡，人们必须考虑到小空泡会仅在一个部分中显示，而较大的将延伸超过几个部分。关于后者，可调节的接近acent部分使用这种方法时（ 图1）提供了另一优点，因为它是比较容易地确定相同液泡是否存在于几个部分。 总之，这个协议可以证明用于研究不同的神经变性疾病的许多果蝇模型是有用的。鉴定改善或加重退行性表型可以提供有关导致或修改的疾病如阿尔茨海默氏症和帕金森氏基本机制关键见解相互作用的蛋白质。在本公开中，我们使用这种方法来检测变性是渐进的，其中动物正常发育，但显示在老化过程中增加变性。此外，此方法也可以适于确定是受发育缺陷，这应该已经存在于新eclosed苍蝇引起变性。

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>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&#160;</td>
		</tr>
		<tr>
			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
			<td>C298-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
		<tr>
			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
		</tr>
		<tr>
			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass histology to quantify neurodegeneration in drosophila

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。

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。 量化神经变性，我们测量，可以在这些部分被检测到液泡的形成。空泡被定义为圆形，黑斑是绿色荧光神经纤维内（ 图2中箭头和3）或皮质和是在果蝇大脑至少连续2个部分可见。通过量化神经退行性疾病测量液泡可以通过集中在一个特定的脑区域或者通过分析整个大脑来完成。限制性分析到大脑的特定区域是在情况下，一个突变只影响特定区域，例如在OLK突变体34中的futsch&#39;嗅觉裂片是有用的，但它也可以当在所有或许多严重退化用于大脑的区域。后者的一个例子是瑞士奶酪（SWS）的突变体（ 图2），其中，测量所有空泡会消耗过多的时间。因此，我们只用了一个图像和，以确保该测量是在相同的水平总是做，我们采取了所有图像在很大的合缝（GC， 图2A），其仅包含在一个或两个部分的电平。虽然我们没有在1日龄SWS"1苍蝇检测空泡形成（数据未显示），失功能的等位基因35，有些液泡检测我ñ7日龄SWS 1苍蝇（箭头， 图2A）。老化的苍蝇14天（ 图2B）和21天（ 图2C）进一步增加这种表型，显示出其进步性。计数中使用所描述的方法的deutocerebral神经毡（DN）空泡数证实了随着年龄液泡的数目显著增加。另外，通过液泡包围的组合区与老化（ 图2D）中的显著增加。 然而，并非所有的突变体表现出这样的严重的表型作为SWS，而在这种情况下，在变性的差异是难以确定集中于小面积时。同样，在老化过程中发生变性是相当温和的（ 图3A - C），因此，我们量化这种表型分析时，整个大脑。确定所有空泡的总和揭示大脑随着年龄增长显著，这也如测量这些空泡（ 图3D）的组合区域时。 <img alt="图1" src="/files/ftp_upload/54809/54809fig1.jpg" /> 图1. 石蜡连续切片。 A）使用方法衣领，实验组和对照苍蝇可以通过线程他们到一个衣领被处理成一个样本。无眼正弦oculis蝇插入方向（箭头）。 B）的示意图示出在套环飞头的方向。 C）在此图像中，从不同的飞头部分是面向从左到右的幻灯片。从顶部在滑动底部，从同一个飞头连续切片中可以看出。在这种情况下，一个正弦oculis苍蝇插入在位置三（箭头）。的部分由荧光眼色素该洗涤过的染色<html>切割后的部分。比例尺在A = 5毫米，C = 0.5mm左右。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54809/54809fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54809/54809fig2.jpg" /> 图2. 渐进神经退行性疾病中的 瑞士奶酪 突变。从7 -日（A），14 -日- （B）和21 -日- （C）老SWS 1苍蝇石蜡头部分。该箭头指向那些与衰老开发空泡。年龄相关变性通过计数空泡的数量和测量它们的组合面积（D）的定量。扫描电镜分析苍蝇数量表示。比例尺= 25微米。 *** P &lt;0.001。csource.jove.com/files/ftp_upload/54809/54809fig2large.jpg"目标="_空白"&gt; 点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/54809/54809fig3.jpg" /> 图3. 神经退行性疾病发生与年龄无关。从10 -日- （A），30 -日- （B）和60 -日- （C）老野生型果蝇石蜡头部分。该箭头指向了老年果蝇研制空泡。年龄相关变性通过计数空泡的数量和测量它们的组合面积（D）的定量。扫描电镜分析苍蝇数量表示。比例尺= 25微米。 *** P &lt;0.001。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54809/54809fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。 &lt;</a>/ A&gt;

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

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。

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

mass-histology-to-quantify-neurodegeneration-in-drosophila

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

10.1K 次观看.  Oregon Health & Sciences University.  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。 

视频: Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

突触量化:基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

科研

神经科学

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

对检测方法果蝇行为

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

利用3D打印技术来合并MRI与组织学:一个大脑切片协议

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

慢性创伤性脑病模型的一种新方法<em&gt;果蝇</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

大众组织学量化在神经退行性变

摘要

探索更多视频

此视频中的章节